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Role of oxygen and the OxyR protein in the response to iron limitation in Rhodobacter sphaeroides
(2014)
Background: High intracellular levels of unbound iron can contribute to the production of reactive oxygen species (ROS) via the Fenton reaction, while depletion of iron limits the availability of iron-containing proteins, some of which have important functions in defence against oxidative stress. Vice versa increased ROS levels lead to the damage of proteins with iron sulphur centres. Thus, organisms have to coordinate and balance their responses to oxidative stress and iron availability. Our knowledge of the molecular mechanisms underlying the co-regulation of these responses remains limited. To discriminate between a direct cellular response to iron limitation and indirect responses, which are the consequence of increased levels of ROS, we compared the response of the alpha-proteobacterium Rhodobacter sphaeroides to iron limitation in the presence or absence of oxygen. Results: One third of all genes with altered expression under iron limitation showed a response that was independent of oxygen availability. The other iron-regulated genes showed different responses in oxic or anoxic conditions and were grouped into six clusters based on the different expression profiles. For two of these clusters, induction in response to iron limitation under oxic conditions was dependent on the OxyR regulatory protein. An OxyR mutant showed increased ROS production and impaired growth under iron limitation. Conclusion: Some R. sphaeroides genes respond to iron limitation irrespective of oxygen availability. These genes therefore reflect a "core iron response" that is independent of potential ROS production under oxic, iron-limiting conditions. However, the regulation of most of the iron-responsive genes was biased by oxygen availability. Most strikingly, the OxyR-dependent activation of a subset of genes upon iron limitation under oxic conditions, including many genes with a role in iron metabolism, revealed that elevated ROS levels were an important trigger for this response. OxyR thus provides a regulatory link between the responses to oxidative stress and to iron limitation in R. sphaeroides.
Microcin C (McC) is a peptide-nucleotide antibiotic produced by Escherichia coli cells harboring a plasmid-borne operon mccABCDE. The heptapeptide MccA is converted into McC by adenylation catalyzed by the MccB enzyme. Since MccA is a substrate for MccB, a mechanism that regulates the MccA/MccB ratio likely exists. Here, we show that transcription from a promoter located upstream of mccA directs the synthesis of two transcripts: a short highly abundant transcript containing the mccA ORF and a longer minor transcript containing mccA and downstream ORFs. The short transcript is generated when RNA polymerase terminates transcription at an intrinsic terminator located in the intergenic region between the mccA and mccB genes. The function of this terminator is strongly attenuated by upstream mcc sequences. Attenuation is relieved and transcription termination is induced when ribosome binds to the mccA ORF. Ribosome binding also makes the mccA RNA exceptionally stable. Together, these two effects-ribosome induced transcription termination and stabilization of the message-account for very high abundance of the mccA transcript that is essential for McC production. The general scheme appears to be evolutionary conserved as ribosome-induced transcription termination also occurs in a homologous operon from Helicobacter pylori.
Noncoding RNAs are integral to a wide range of biological processes, including translation, gene regulation, host-pathogen interactions and environmental sensing. While genomics is now a mature field, our capacity to identify noncoding RNA elements in bacterial and archaeal genomes is hampered by the difficulty of de novo identification. The emergence of new technologies for characterizing transcriptome outputs, notably RNA-seq, are improving noncoding RNA identification and expression quantification. However, a major challenge is to robustly distinguish functional outputs from transcriptional noise. To establish whether annotation of existing transcriptome data has effectively captured all functional outputs, we analysed over 400 publicly available RNA-seq datasets spanning 37 different Archaea and Bacteria. Using comparative tools, we identify close to a thousand highly-expressed candidate noncoding RNAs. However, our analyses reveal that capacity to identify noncoding RNA outputs is strongly dependent on phylogenetic sampling. Surprisingly, and in stark contrast to protein-coding genes, the phylogenetic window for effective use of comparative methods is perversely narrow: aggregating public datasets only produced one phylogenetic cluster where these tools could be used to robustly separate unannotated noncoding RNAs from a null hypothesis of transcriptional noise. Our results show that for the full potential of transcriptomics data to be realized, a change in experimental design is paramount: effective transcriptomics requires phylogeny-aware sampling.
Bacterial small RNAs (sRNAs) are key elements of regulatory networks that modulate gene expression. The sRNA RydC of Salmonella sp. and Escherichia coli is an example of this class of riboregulators. Like many other sRNAs, RydC bears a 'seed' region that recognises specific transcripts through base-pairing, and its activities are facilitated by the RNA chaperone Hfq. The crystal structure of RydC in complex with E. coli Hfq at 3.48 angstrom resolution illuminates how the protein interacts with and presents the sRNA for target recognition. Consolidating the protein-RNA complex is a host of distributed interactions mediated by the natively unstructured termini of Hfq. Based on the structure and other data, we propose a model for a dynamic effector complex comprising Hfq, small RNA, and the cognate mRNA target.
Phenotypically identical cells can dramatically vary with respect to behavior during their lifespan and this variation is reflected in their molecular composition such as the transcriptomic landscape. Singlecell transcriptomics using next-generation transcript sequencing (RNA-seq) is now emerging as a powerful tool to profile cell-to-cell variability on a genomic scale. Its application has already greatly impacted our conceptual understanding of diverse biological processes with broad implications for both basic and clinical research. Different single-cell RNAseq protocols have been introduced and are reviewed here – each one with its own strengths and current limitations. We further provide an overview of the biological questions single-cell RNA-seq has been used to address, the major findings obtained from such studies, and current challenges and expected future developments in this booming field.
Bioassay-guided fractionation of a chloroform extract of Valeriana wallichii (V. wallichii) rhizomes lead to the isolation and identification of caffeic acid bornyl ester (1) as the active component against Leishmania major (L. major) promastigotes (IC50 = 48.8 µM). To investigate the structure-activity relationship (SAR), a library of compounds based on 1 was synthesized and tested in vitro against L. major and L. donovani promastigotes, and L. major amastigotes. Cytotoxicity was determined using a murine J774.1 cell line and bone marrow derived macrophages (BMDM). Some compounds showed antileishmanial activity in the concentration range of pentamidine and miltefosine which are the standard drugs in use. In the L. major amastigote assay compounds 15, 19 and 20 showed good activity with relatively low cytotoxicity against BMDM, resulting in acceptable selectivity indices. Molecules with adjacent phenolic hydroxyl groups exhibited elevated cytotoxicity against murine cell lines J774.1 and BMDM. The Michael system seems not to be essential for antileishmanial activity. Based on the results compound 27 can be regarded as new lead structure for further structure optimization
Background
The capacity of the recombinant Vaccinia virus GLV-1h68 as a single agent to efficiently treat different human or canine cancers has been shown in several preclinical studies. Currently, its human safety and efficacy are investigated in phase I/II clinical trials. In this study we set out to evaluate the oncolytic activity of GLV-1h68 in the human lung adenocarcinoma cell line PC14PE6-RFP in cell cultures and analyzed the antitumor potency of a combined treatment strategy consisting of GLV-1h68 and cyclophosphamide (CPA) in a mouse model of PC14PE6-RFP lung adenocarcinoma.
Methods
PC14PE6-RFP cells were treated in cell culture with GLV-1h68. Viral replication and cell survival were determined by plaque assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. Subcutaneously implanted PC14PE6-RFP xenografts were treated by systemic injection of GLV-1h68, CPA or a combination of both. Tumor growth and viral biodistribution were monitored and immune-related antigen profiling of tumor lysates was performed.
Results
GLV-1h68 efficiently infected, replicated in and lysed human PC14PE6-RFP cells in cell cultures. PC14PE6-RFP tumors were efficiently colonized by GLV-1h68 leading to much delayed tumor growth in PC14PE6-RFP tumor-bearing nude mice. Combination treatment with GLV-1h68 and CPA significantly improved the antitumor efficacy of GLV-1h68 and led to an increased viral distribution within the tumors. Pro-inflammatory cytokines and chemokines were distinctly elevated in tumors of GLV-1h68-treated mice. Factors expressed by endothelial cells or present in the blood were decreased after combination treatment. A complete loss in the hemorrhagic phenotype of the PC14PE6-RFP tumors and a decrease in the number of blood vessels after combination treatment could be observed.
Conclusions
CPA and GLV-1h68 have synergistic antitumor effects on PC14PE6-RFP xenografts. We strongly suppose that in the PC14PE6-RFP model the enhanced tumor growth inhibition achieved by combining GLV-1h68 with CPA is due to an effect on the vasculature rather than an immunosuppressive action of CPA. These results provide evidence to support further preclinical studies of combining GLV-1h68 and CPA in other highly angiogenic tumor models. Moreover, data presented here demonstrate that CPA can be combined successfully with GLV-1h68 based oncolytic virus therapy and therefore might be promising as combination therapy in human clinical trials.
Although the DNA methyltransferase 2 family is highly conserved during evolution and recent reports suggested a dual specificity with stronger activity on transfer RNA (tRNA) than DNA substrates, the biological function is still obscure. We show that the Dictyostelium discoideum Dnmt2-homologue DnmA is an active tRNA methyltransferase that modifies C38 in \(tRNA^{Asp(GUC)}\) in vitro and in vivo. By an ultraviolet-crosslinking and immunoprecipitation approach, we identified further DnmA targets. This revealed specific tRNA fragments bound by the enzyme and identified \(tRNA^{Glu(CUC/UUC)}\) and \(tRNA^{Gly(GCC)}\) as new but weaker substrates for both human Dnmt2 and DnmA in vitro but apparently not in vivo. Dnmt2 enzymes form transient covalent complexes with their substrates. The dynamics of complex formation and complex resolution reflect methylation efficiency in vitro. Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress. However, dnmA expression only partially correlated with tRNA methylation in vivo. Strikingly, dnmA expression in the laboratory strain AX2 was significantly lower than in the NC4 parent strain. As expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation.
Integrative "Omics"-Approach Discovers Dynamic and Regulatory Features of Bacterial Stress Responses
(2013)
Bacteria constantly face stress conditions and therefore mount specific responses to ensure adaptation and survival. Stress responses were believed to be predominantly regulated at the transcriptional level. In the phototrophic bacterium Rhodobacter sphaeroides the response to singlet oxygen is initiated by alternative sigma factors. Further adaptive mechanisms include post-transcriptional and post-translational events, which have to be considered to gain a deeper understanding of how sophisticated regulation networks operate. To address this issue, we integrated three layers of regulation: (1) total mRNA levels at different time-points revealed dynamics of the transcriptome, (2) mRNAs in polysome fractions reported on translational regulation (translatome), and (3) SILAC-based mass spectrometry was used to quantify protein abundances (proteome). The singlet oxygen stress response exhibited highly dynamic features regarding short-term effects and late adaptation, which could in part be assigned to the sigma factors RpoE and RpoH2 generating distinct expression kinetics of corresponding regulons. The occurrence of polar expression patterns of genes within stress-inducible operons pointed to an alternative of dynamic fine-tuning upon stress. In addition to transcriptional activation, we observed significant induction of genes at the post-transcriptional level (translatome), which identified new putative regulators and assigned genes of quorum sensing to the singlet oxygen stress response. Intriguingly, the SILAC approach explored the stress-dependent decline of photosynthetic proteins, but also identified 19 new open reading frames, which were partly validated by RNA-seq. We propose that comparative approaches as presented here will help to create multi-layered expression maps on the system level ("expressome"). Finally, intense mass spectrometry combined with RNA-seq might be the future tool of choice to re-annotate genomes in various organisms and will help to understand how they adapt to alternating conditions.
Pf38 is a surface protein of the malarial parasite Plasmodium falciparum. In this study, we produced and purified recombinant Pf38 and a fusion protein composed of red fluorescent protein and Pf38 (RFP-Pf38) using a transient expression system in the plant Nicotiana benthamiana. To our knowledge, this is the first description of the production of recombinant Pf38. To verify the quality of the recombinant Pf38, plasma from semi-immune African donors was used to confirm specific binding to Pf38. ELISA measurements revealed that immune responses to Pf38 in this African subset were comparable to reactivities to AMA-1 and \(MSP1_{19}\). Pf38 and RFP-Pf38 were successfully used to immunise mice, although titres from these mice were low (on average 1:11.000 and 1:39.000, respectively). In immune fluorescence assays, the purified IgG fraction from the sera of immunised mice recognised Pf38 on the surface of schizonts, gametocytes, macrogametes and zygotes, but not sporozoites. Growth inhibition assays using \(\alpha Pf38\) antibodies demonstrated strong inhibition \((\geq 60 \% ) \) of the growth of blood-stage P. falciparum. The development of zygotes was also effectively inhibited by \(\alpha Pf38\) antibodies, as determined by the zygote development assay. Collectively, these results suggest that Pf38 is an interesting candidate for the development of a malaria vaccine.
The Vpr protein from type 1 and type 2 Human Immunodeficiency Viruses (HIV-1 and HIV-2) is thought to inactivate several host proteins through the hijacking of the DCAF1 adaptor of the Cul4A ubiquitin ligase. Here, we identified two transcriptional regulators, ZIP and sZIP, as Vpr-binding proteins degraded in the presence of Vpr. ZIP and sZIP have been shown to act through the recruitment of the NuRD chromatin remodeling complex. Strikingly, chromatin is the only cellular fraction where Vpr is present together with Cul4A ubiquitin ligase subunits. Components of the NuRD complex and exogenous ZIP and sZIP were also associated with this fraction. Several lines of evidence indicate that Vpr induces ZIP and sZIP degradation by hijacking DCAF1: (i) Vpr induced a drastic decrease of exogenously expressed ZIP and sZIP in a dose-dependent manner, (ii) this decrease relied on the proteasome activity, (iii) ZIP or sZIP degradation was impaired in the presence of a DCAF1-binding deficient Vpr mutant or when DCAF1 expression was silenced. Vpr-mediated ZIP and sZIP degradation did not correlate with the growth-related Vpr activities, namely G2 arrest and G2 arrest-independent cytotoxicity. Nonetheless, infection with HIV-1 viruses expressing Vpr led to the degradation of the two proteins. Altogether our results highlight the existence of two host transcription factors inactivated by Vpr. The role of Vpr-mediated ZIP and sZIP degradation in the HIV-1 replication cycle remains to be deciphered.
Introduction: Surgery is currently the definitive treatment for early-stage breast cancer. However, the rate of positive surgical margins remains unacceptably high. The human sodium iodide symporter (hNIS) is a naturally occurring protein in human thyroid tissue, which enables cells to concentrate radionuclides. The hNIS has been exploited to image and treat thyroid cancer. We therefore investigated the potential of a novel oncolytic vaccinia virus GLV1h-153 engineered to express the hNIS gene for identifying positive surgical margins after tumor resection via positron emission tomography (PET). Furthermore, we studied its role as an adjuvant therapeutic agent in achieving local control of remaining tumors in an orthotopic breast cancer model.
Methods: GLV-1h153, a replication-competent vaccinia virus, was tested against breast cancer cell lines at various multiplicities of infection (MOIs). Cytotoxicity and viral replication were determined. Mammary fat pad tumors were generated in athymic nude mice. To determine the utility of GLV-1h153 in identifying positive surgical margins, 90% of the mammary fat pad tumors were surgically resected and subsequently injected with GLV-1h153 or phosphate buffered saline (PBS) in the surgical wound. Serial Focus 120 microPET images were obtained six hours post-tail vein injection of approximately 600 mu Ci of I-124-iodide.
Results: Viral infectivity, measured by green fluorescent protein (GFP) expression, was time-and concentrationdependent. All cell lines showed less than 10% of cell survival five days after treatment at an MOI of 5. GLV-1h153 replicated efficiently in all cell lines with a peak titer of 27 million viral plaque forming units (PFU) ( < 10,000-fold increase from the initial viral dose) by Day 4. Administration of GLV-1h153 into the surgical wound allowed positive surgical margins to be identified via PET scanning. In vivo, mean volume of infected surgically resected residual tumors four weeks after treatment was 14 mm(3) versus 168 mm(3) in untreated controls (P < 0.05).
Conclusions: This is the first study to our knowledge to demonstrate a novel vaccinia virus carrying hNIS as an imaging tool in identifying positive surgical margins of breast cancers in an orthotopic murine model. Moreover, our results suggest that GLV-1h153 is a promising therapeutic agent in achieving local control for positive surgical margins in resected breast tumors.
Background
Oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. Unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. Here, we evaluate \(^{19}\)F magnetic resonance imaging (MRI) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (PFC) for monitoring of oncolytic virotherapy.
Methodology/Principal Findings
The Vaccinia virus strain GLV-1h68 was used as an oncolytic agent for the treatment of different tumor models. Systemic application of PFC emulsions followed by \(^1H\)/\(^{19}\)F MRI of mock-infected and GLV-1h68-infected tumor-bearing mice revealed a significant accumulation of the \(^{19}\)F signal in the tumor rim of virus-treated mice. Histological examination of tumors confirmed a similar spatial distribution of the \(^{19}\)F signal hot spots and \(CD68^+\)-macrophages. Thereby, the \(CD68^+\)-macrophages encapsulate the GFP-positive viral infection foci. In multiple tumor models, we specifically visualized early inflammatory cell recruitment in Vaccinia virus colonized tumors. Furthermore, we documented that the \(^{19}\)F signal correlated with the extent of viral spreading within tumors.
Conclusions/Significance
These results suggest \(^{19}\)F MRI as a non-invasive methodology to document the tumor-associated host immune response as well as the extent of intratumoral viral replication. Thus, \(^{19}\)F MRI represents a new platform to non-invasively investigate the role of the host immune response for therapeutic outcome of oncolytic virotherapy and individual patient response.
Abstract
In line with the key role of methionine in protein biosynthesis initiation and many cellular processes most microorganisms have evolved mechanisms to synthesize methionine de novo. Here we demonstrate that, in the bacterial pathogen Staphylococcus aureus, a rare combination of stringent response-controlled CodY activity, T-box riboswitch and mRNA decay mechanisms regulate the synthesis and stability of methionine biosynthesis metICFE-mdh mRNA. In contrast to other Bacillales which employ S-box riboswitches to control methionine biosynthesis, the S. aureus metICFE-mdh mRNA is preceded by a 5′-untranslated met leader RNA harboring a T-box riboswitch. Interestingly, this T-box riboswitch is revealed to specifically interact with uncharged initiator formylmethionyl-tRNA \((tRNA_i^{fMet})\)while binding of elongator \(tRNA^{Met}\) proved to be weak, suggesting a putative additional function of the system in translation initiation control. met leader RNA/metICFE-mdh operon expression is under the control of the repressor CodY which binds upstream of the met leader RNA promoter. As part of the metabolic emergency circuit of the stringent response, methionine depletion activates RelA-dependent (p)ppGpp alarmone synthesis, releasing CodY from its binding site and thereby activating the met leader promoter. Our data further suggest that subsequent steps in metICFE-mdh transcription are tightly controlled by the 5′ met leader-associated T-box riboswitch which mediates premature transcription termination when methionine is present. If methionine supply is limited, and hence \((tRNA_i^{fMet})\) becomes uncharged, full-length met leader/metICFE-mdh mRNA is transcribed which is rapidly degraded by nucleases involving RNase J2. Together, the data demonstrate that staphylococci have evolved special mechanisms to prevent the accumulation of excess methionine. We hypothesize that this strict control might reflect the limited metabolic capacities of staphylococci to reuse methionine as, other than Bacillus, staphylococci lack both the methionine salvage and polyamine synthesis pathways. Thus, methionine metabolism might represent a metabolic Achilles' heel making the pathway an interesting target for future anti-staphylococcal drug development.
Author Summary
Prokaryote metabolism is key for our understanding of bacterial virulence and pathogenesis and it is also an area with huge opportunity to identify novel targets for antibiotic drugs. Here, we have addressed the so far poorly characterized regulation of methionine biosynthesis in S. aureus. We demonstrate that methionine biosynthesis control in staphylococci significantly differs from that predicted for other Bacillales. Notably, involvement of a T-box instead of an S-box riboswitch separates staphylococci from other bacteria in the order. We provide, for the first time, direct experimental proof for an interaction of a methionyl-tRNA-specific T-box with its cognate tRNA, and the identification of initiator \((tRNA_i^{fMet})\) as the specific binding partner is an unexpected finding whose exact function in Staphylococcus metabolism remains to be established. The data further suggest that in staphylococci a range of regulatory elements are integrated to form a hierarchical network that elegantly limits costly (excess) methionine biosynthesis and, at the same time, reliably ensures production of the amino acid in a highly selective manner. Our findings open a perspective to exploit methionine biosynthesis and especially its T-box-mediated control as putative target(s) for the development of future anti-staphylococcal therapeutics.
Abstract
Sulphur is an essential element that all pathogens have to absorb from their surroundings in order to grow inside their infected host. Despite its importance, the relevance of sulphur assimilation in fungal virulence is largely unexplored. Here we report a role of the bZIP transcription factor MetR in sulphur assimilation and virulence of the human pathogen Aspergillus fumigatus. The MetR regulator is essential for growth on a variety of sulphur sources; remarkably, it is fundamental for assimilation of inorganic S-sources but dispensable for utilization of methionine. Accordingly, it strongly supports expression of genes directly related to inorganic sulphur assimilation but not of genes connected to methionine metabolism. On a broader scale, MetR orchestrates the comprehensive transcriptional adaptation to sulphur-starving conditions as demonstrated by digital gene expression analysis. Surprisingly, A. fumigatus is able to utilize volatile sulphur compounds produced by its methionine catabolism, a process that has not been described before and that is MetR-dependent. The A. fumigatus MetR transcriptional activator is important for virulence in both leukopenic mice and an alternative mini-host model of aspergillosis, as it was essential for the development of pulmonary aspergillosis and supported the systemic dissemination of the fungus. MetR action under sulphur-starving conditions is further required for proper iron regulation, which links regulation of sulphur metabolism to iron homeostasis and demonstrates an unprecedented regulatory crosstalk. Taken together, this study provides evidence that regulation of sulphur assimilation is not only crucial for A. fumigatus virulence but also affects the balance of iron in this prime opportunistic pathogen.
Author Summary
Invasive pulmonary aspergillosis (IPA) is a life-threatening disease that affects primarily immunosuppressed patients. During the last decades the incidence of this disease that is accompanied by high mortality rates has increased. Since opportunistic pathogenic fungi, unlike other pathogens, do not express specific virulence factors, it is becoming more and more clear that the elucidation of fungal metabolism is an essential task to understand fungal pathogenicity and to identify novel antifungal targets. In this work we report genetic inactivation of the sulphur transcription regulator MetR in Aspergillus fumigatus and subsequent study of the resulting phenotypes and transcriptional deregulation of the mutant. Here we show that regulation of sulphur assimilation is an essential process for the manifestation of IPA. Moreover, a regulatory connection between sulphur metabolism and iron homeostasis, a further essential virulence determinant of A. fumigatus, is demonstrated in this study for the first time. A deeper knowledge of sulphur metabolism holds the promise of increasing our understanding of fungal virulence and might lead to improved antifungal therapy.
Abstract
In the murine model of Leishmania major infection, resistance or susceptibility to the parasite has been associated with the development of a Th1 or Th2 type of immune response. Recently, however, the immunosuppressive effects of IL-10 have been ascribed a crucial role in the development of the different clinical correlates of Leishmania infection in humans. Since T cells and professional APC are important cellular sources of IL-10, we compared leishmaniasis disease progression in T cell-specific, macrophage/neutrophil-specific and complete IL-10-deficient C57BL/6 as well as T cell-specific and complete IL-10-deficient BALB/c mice. As early as two weeks after infection of these mice with L. major, T cell-specific and complete IL-10-deficient animals showed significantly increased lesion development accompanied by a markedly elevated secretion of IFN-γ or IFN-γ and IL-4 in the lymph nodes draining the lesions of the C57BL/6 or BALB/c mutants, respectively. In contrast, macrophage/neutrophil-specific IL-10-deficient C57BL/6 mice did not show any altered phenotype. During the further course of disease, the T cell-specific as well as the complete IL-10-deficient BALB/c mice were able to control the infection. Furthermore, a dendritic cell-based vaccination against leishmaniasis efficiently suppresses the early secretion of IL-10, thus contributing to the control of parasite spread. Taken together, IL-10 secretion by T cells has an influence on immune activation early after infection and is sufficient to render BALB/c mice susceptible to an uncontrolled Leishmania major infection.
Author Summary
The clinical symptoms caused by infections with Leishmania parasites range from self-healing cutaneous to uncontrolled visceral disease and depend not only on the parasite species but also on the type of the host's immune response. It is estimated that 350 million people worldwide are at risk, with a global incidence of 1–1.5 million cases of cutaneous and 500,000 cases of visceral leishmaniasis. Murine leishmaniasis is the best-characterized model to elucidate the mechanisms underlying resistance or susceptibility to Leishmania major parasites in vivo. Using T cell-specific and macrophage-specific mutant mice, we demonstrate that abrogating the secretion of the immunosuppressive cytokine IL-10 by T cells is sufficient to render otherwise susceptible mice resistant to an infection with the pathogen. The healing phenotype is accompanied by an elevated specific inflammatory immune response very early after infection. We further show that dendritic cell-based vaccination against leishmaniasis suppresses the early secretion of IL-10 following challenge infection. Thus, our study unravels a molecular mechanism critical for host immune defense, aiding in the development of an effective vaccine against leishmaniasis.
Candida albicans and Candida dubliniensis are pathogenic fungi that are highly related but differ in virulence and in some phenotypic traits. During in vitro growth on certain nutrient-poor media, C. albicans and C. dubliniensis are the only yeast species which are able to produce chlamydospores, large thick-walled cells of unknown function. Interestingly, only C. dubliniensis forms pseudohyphae with abundant chlamydospores when grown on Staib medium, while C. albicans grows exclusively as a budding yeast. In order to further our understanding of chlamydospore development and assembly, we compared the global transcriptional profile of both species during growth in liquid Staib medium by RNA sequencing. We also included a C. albicans mutant in our study which lacks the morphogenetic transcriptional repressor Nrg1. This strain, which is characterized by its constitutive pseudohyphal growth, specifically produces masses of chlamydospores in Staib medium, similar to C. dubliniensis. This comparative approach identified a set of putatively chlamydospore-related genes. Two of the homologous C. albicans and C. dubliniensis genes (CSP1 and CSP2) which were most strongly upregulated during chlamydospore development were analysed in more detail. By use of the green fluorescent protein as a reporter, the encoded putative cell wall related proteins were found to exclusively localize to C. albicans and C. dubliniensis chlamydospores. Our findings uncover the first chlamydospore specific markers in Candida species and provide novel insights in the complex morphogenetic development of these important fungal pathogens.
Bacteria Regulate Intestinal Epithelial Cell Differentiation Factors Both In Vitro and In Vivo
(2013)
Background: The human colon harbours a plethora of bacteria known to broadly impact on mucosal metabolism and function and thought to be involved in inflammatory bowel disease pathogenesis and colon cancer development. In this report, we investigated the effect of colonic bacteria on epithelial cell differentiation factors in vitro and in vivo. As key transcription factors we focused on Hes1, known to direct towards an absorptive cell fate, Hath1 and KLF4, which govern goblet cell.
Methods: Expression of the transcription factors Hes1, Hath1 and KLF4, the mucins Muc1 and Muc2 and the defensin HBD2 were measured by real-time PCR in LS174T cells following incubation with several heat-inactivated E. coli strains, including the probiotic E. coli Nissle 1917+/- flagellin, Lactobacilli and Bifidobacteria. For protein detection Western blot experiments and chamber-slide immunostaining were performed. Finally, mRNA and protein expression of these factors was evaluated in the colon of germfree vs. specific pathogen free vs. conventionalized mice and colonic goblet cells were counted.
Results: Expression of Hes1 and Hath1, and to a minor degree also of KLF4, was reduced by E. coli K-12 and E. coli Nissle 1917. In contrast, Muc1 and HBD2 expression were significantly enhanced, independent of the Notch signalling pathway. Probiotic E. coli Nissle 1917 regulated Hes1, Hath1, Muc1 and HBD2 through flagellin. In vivo experiments confirmed the observed in vitro effects of bacteria by a diminished colonic expression of Hath1 and KLF4 in specific pathogen free and conventionalized mice as compared to germ free mice whereas the number of goblet cells was unchanged in these mice.
Conclusions: Intestinal bacteria influence the intestinal epithelial differentiation factors Hes1, Hath1 and KLF4, as well as Muc1 and HBD2, in vitro and in vivo. The induction of Muc1 and HBD2 seems to be triggered directly by bacteria and not by Notch.
Background: Despite availability of efficient treatment regimens for early stage colorectal cancer, treatment regimens for late stage colorectal cancer are generally not effective and thus need improvement. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) strains is a promising new strategy for therapy of a variety of human cancers.
Methods: Oncolytic efficacy of replication-competent vaccinia virus GLV-1h68 was analyzed in both, cell cultures and subcutaneous xenograft tumor models.
Results: In this study we demonstrated for the first time that the replication-competent recombinant VACV GLV-1h68 efficiently infected, replicated in, and subsequently lysed various human colorectal cancer lines (Colo 205, HCT-15, HCT-116, HT-29, and SW-620) derived from patients at all four stages of disease. Additionally, in tumor xenograft models in athymic nude mice, a single injection of intravenously administered GLV-1h68 significantly inhibited tumor growth of two different human colorectal cell line tumors (Duke’s type A-stage HCT-116 and Duke’s type C-stage SW-620), significantly improving survival compared to untreated mice. Expression of the viral marker gene ruc-gfp allowed for real-time analysis of the virus infection in cell cultures and in mice. GLV-1h68 treatment was well-tolerated in all animals and viral replication was confined to the tumor. GLV-1h68 treatment elicited a significant up-regulation of murine immune-related antigens like IFN-γ, IP-10, MCP-1, MCP-3, MCP-5, RANTES and TNF-γ and a greater infiltration of macrophages and NK cells in tumors as compared to untreated controls.
Conclusion: The anti-tumor activity observed against colorectal cancer cells in these studies was a result of direct viral oncolysis by GLV-1h68 and inflammation-mediated innate immune responses. The therapeutic effects occurred in tumors regardless of the stage of disease from which the cells were derived. Thus, the recombinant vaccinia virus GLV-1h68 has the potential to treat colorectal cancers independently of the stage of progression.
ABSTRACT Bacteria organize many membrane-related signaling processes in functional microdomains that are structurally and functionally similar to the lipid rafts of eukaryotic cells. An important structural component of these microdomains is the protein flotillin, which seems to act as a chaperone in recruiting other proteins to lipid rafts to facilitate their interaction. In eukaryotic cells, the occurrence of severe diseases is often observed in combination with an overproduction of flotillin, but a functional link between these two phenomena is yet to be demonstrated. In this work, we used the bacterial model Bacillus subtilis as a tractable system to study the physiological alterations that occur in cells that overproduce flotillin. We discovered that an excess of flotillin altered specific signal transduction pathways that are associated with the membrane microdomains of bacteria. As a consequence of this, we detected significant defects in cell division and cell differentiation. These physiological alterations were in part caused by an unusual stabilization of the raft-associated protease FtsH. This report opens the possibility of using bacteria as a working model to better understand fundamental questions related to the functionality of lipid rafts.
IMPORTANCE The identification of signaling platforms in the membrane of bacteria that are functionally and structurally equivalent to eukaryotic lipid rafts reveals a level of sophistication in signal transduction and membrane organization unexpected in bacteria. It opens new and promising venues to address intricate questions related to the functionality of lipid rafts by using bacteria as a more tractable system. This is the first report that uses bacteria as a working model to investigate a fundamental question that was previously raised while studying the role of eukaryotic lipid rafts. It also provides evidence of the critical role of these signaling platforms in orchestrating diverse physiological processes in prokaryotic cells.
The Staphylococcus aureus regulatory saePQRS system controls the expression of numerous virulence factors, including extracellular adherence protein (Eap), which amongst others facilitates invasion of host cells. The saePQRS operon codes for 4 proteins: the histidine kinase SaeS, the response regulator SaeR, the lipoprotein SaeP and the transmembrane protein SaeQ. S. aureus strain Newman has a single amino acid substitution in the transmembrane domain of SaeS (L18P) which results in constitutive kinase activity. SDS was shown to be one of the signals interfering with SaeS activity leading to inhibition of the sae target gene eap in strains with SaeS(L) but causing activation in strains containing SaeS(P). Here, we analyzed the possible involvement of the SaeP protein and saePQ region in SDS-mediated sae/eap expression. We found that SaePQ is not needed for SDS-mediated SaeS signaling. Furthermore, we could show that SaeS activity is closely linked to the expression of Eap and the capacity to invade host cells in a number of clinical isolates. This suggests that SaeS activity might be directly modulated by structurally non-complex environmental signals, as SDS, which possibly altering its kinase/phosphatase activity.
Background
The emergence of antibiotic resistant bacteria in recent decades has highlighted the importance of developing new drugs to treat infections. However, in addition to the design of new drugs, the development of accurate preclinical testing methods is essential. In vivo imaging technologies such as bioluminescence imaging (BLI) or magnetic resonance imaging (MRI) are promising approaches. In a previous study, we showed the effectiveness of \(^{19}\)F MRI using perfluorocarbon (PFC) emulsions for detecting the site of Staphylococcus aureus infection. In the present follow-up study, we investigated the use of this method for in vivo visualization of the effects of antibiotic therapy.
Methods/Principal findings
Mice were infected with S. aureus Xen29 and treated with 0.9% NaCl solution, vancomycin or linezolid. Mock treatment led to the highest bioluminescence values during infection followed by vancomycin treatment. Counting the number of colony-forming units (cfu) at 7 days post-infection (p.i.) showed the highest bacterial burden for the mock group and the lowest for the linezolid group. Administration of PFCs at day 2 p.i. led to the accumulation of \(^{19}\)F at the rim of the abscess in all mice (in the shape of a hollow sphere), and antibiotic treatment decreased the \(^{19}\)F signal intensity and volume. Linezolid showed the strongest effect. The BLI, cfu, and MRI results were comparable.
Conclusions
\(^{19}\)F-MRI with PFCs is an effective non-invasive method for assessing the effects of antibiotic therapy in vivo. This method does not depend on pathogen specific markers and can therefore be used to estimate the efficacy of antibacterial therapy against a broad range of clinically relevant pathogens, and to localize sites of infection.
Many microRNAs (miRNAs) are co-regulated during the same physiological process but the underlying cellular logic is often little understood. The conserved, immunomodulatory miRNAs miR-146 and miR-155, for instance, are co-induced in many cell types in response to microbial lipopolysaccharide (LPS) to feedback-repress LPS signalling through Toll-like receptor TLR4. Here, we report that these seemingly co-induced regulatory RNAs dramatically differ in their induction behaviour under various stimuli strengths and act non-redundantly through functional specialization; although miR-146 expression saturates at sub-inflammatory doses of LPS that do not trigger the messengers of inflammation markers, miR-155 remains tightly associated with the pro-inflammatory transcriptional programmes. Consequently, we found that both miRNAs control distinct mRNA target profiles; although miR-146 targets the messengers of LPS signal transduction components and thus downregulates cellular LPS sensitivity, miR-155 targets the mRNAs of genes pervasively involved in pro-inflammatory transcriptional programmes. Thus, miR-155 acts as a broad limiter of pro-inflammatory gene expression once the miR-146 dependent barrier to LPS triggered inflammation has been breached. Importantly, we also report alternative miR-155 activation by the sensing of bacterial peptidoglycan through cytoplasmic NOD-like receptor, NOD2. We predict that dosedependent responses to environmental stimuli may involve functional specialization of seemingly coinduced miRNAs in other cellular circuitries as well.
Depending on the environmental conditions, the pathogenic yeast Candida albicans can undergo different developmental programs, which are controlled by dedicated transcription factors and upstream signaling pathways. C. albicans strains that are homozygous at the mating type locus can switch from the normal yeast form (white) to an elongated cell type (opaque), which is the mating-competent form of this fungus. Both white and opaque cells use the Ste11-Hst7-Cek1/Cek2 MAP kinase signaling pathway to react to the presence of mating pheromone. However, while opaque cells employ the transcription factor Cph1 to induce the mating response, white cells recruit a different downstream transcription factor, Tec1, to promote the formation of a biofilm that facilitates mating of opaque cells in the population. The switch from the white to the opaque cell form is itself induced by environmental signals that result in the upregulation of the transcription factor Wor1, the master regulator of white-opaque switching. To get insight into the upstream signaling pathways controlling the switch, we expressed all C. albicans protein kinases from a tetracycline-inducible promoter in a switching-competent strain. Screening of this library of strains showed that a hyperactive form of Ste11 lacking its N-terminal domain (Ste11ΔN467) efficiently stimulated white cells to switch to the opaque phase, a behavior that did not occur in response to pheromone. Ste11ΔN467-induced switching specifically required the downstream MAP kinase Cek1 and its target transcription factor Cph1, but not Cek2 and Tec1, and forced expression of Cph1 also promoted white-opaque switching in a Wor1-dependent manner. Therefore, depending on the activation mechanism, components of the pheromone-responsive MAP kinase pathway can be reconnected to stimulate an alternative developmental program, switching of white cells to the mating-competent opaque phase.
Campylobacter jejuni is currently the leading cause of bacterial gastroenteritis in humans. Comparison of multiple Campylobacter strains revealed a high genetic and phenotypic diversity. However, little is known about differences in transcriptome organization, gene expression, and small RNA (sRNA) repertoires. Here we present the first comparative primary transcriptome analysis based on the differential RNA–seq (dRNA–seq) of four C. jejuni isolates. Our approach includes a novel, generic method for the automated annotation of transcriptional start sites (TSS), which allowed us to provide genome-wide promoter maps in the analyzed strains. These global TSS maps are refined through the integration of a SuperGenome approach that allows for a comparative TSS annotation by mapping RNA–seq data of multiple strains into a common coordinate system derived from a whole-genome alignment. Considering the steadily increasing amount of RNA–seq studies, our automated TSS annotation will not only facilitate transcriptome annotation for a wider range of pro- and eukaryotes but can also be adapted for the analysis among different growth or stress conditions. Our comparative dRNA–seq analysis revealed conservation of most TSS, but also single-nucleotide-polymorphisms (SNP) in promoter regions, which lead to strain-specific transcriptional output. Furthermore, we identified strain-specific sRNA repertoires that could contribute to differential gene regulation among strains. In addition, we identified a novel minimal CRISPR-system in Campylobacter of the type-II CRISPR subtype, which relies on the host factor RNase III and a trans-encoded sRNA for maturation of crRNAs. This minimal system of Campylobacter, which seems active in only some strains, employs a unique maturation pathway, since the crRNAs are transcribed from individual promoters in the upstream repeats and thereby minimize the requirements for the maturation machinery. Overall, our study provides new insights into strain-specific transcriptome organization and sRNAs, and reveals genes that could modulate phenotypic variation among strains despite high conservation at the DNA level.
Der Hefepilz Candida albicans gehört zu den opportunistischen Infektionserregern. Er ist Teil der natürlichen Mikroflora der Schleimhäute des Gastrointestinal- und Urogenitaltraktes des Menschen. Bei Störungen des natürlichen Gleichgewichts dieser Flora kann es zu oberflächlichen Mykosen, z. B. der oropharyngealen Candidiasis (Mundsoor), kommen. Besonders immunsupprimierte Patienten, wie AIDS-Patienten, leiden häufig unter immer wiederkehrenden Infektionen, die mitunter auch zu schwerwiegenden Infektionsverläufen, bis hin zu lebensbedrohlichen systemischen Mykosen führen können. Zur Therapie solcher Erkrankungen werden oft Ergosterolbiosyntheseinhibitoren, wie Fluconazol, eingesetzt. Besonders bei wiederkehrenden Infektionen und wiederholender Therapie ist C. albicans in der Lage, gegen diese häufig verabreichten Antimykotika Resistenzen zu entwickeln. Hierbei spielen Zink-Cluster-Transkriptionsfaktoren eine zentrale Rolle. Zink-Cluster-Proteine gehören zu einer pilzspezifischen Familie von Transkriptionsfaktoren, die ein großes Spektrum an zellulären Prozessen regulieren. Die gut charakterisierten Regulatoren Upc2, Tac1 und Mrr1 gehören zu den Zink-Cluster-Transkriptionsfaktoren, die maßgeblich zur Resistenzentwicklung von C. albicans beitragen. Upc2 kontrolliert die Expression vieler Ergosterolbiosynthesegene, besonders die von ERG11, welches für die Zielstruktur des gängigen Antimykotikums Fluconazol kodiert. Tac1 und Mrr1 hingegen regulieren die Expression von Multidrug-Effluxpumpen, den ABC-Transportern CDR1 und CDR2 bzw. dem Major Facilitator MDR1. Gain-of-function-Mutationen in diesen Transkriptionsfaktoren resultieren in einer konstitutiven Überexpression ihrer Zielgene und sind verantwortlich für die Resistenz vieler klinischer Isolate. In dieser Arbeit wurde gezeigt, dass die Fusion von Mrr1 mit der Gal4-Aktivierungsdomäne von Saccharomyces cerevisiae zu einem konstitutiv aktiven Hybridtranskriptionsfaktor führte, der eine MDR1-Überexpression bewirkte und Fluconazolresistenz vermittelte. Dieses Hybridprotein vermittelte sogar eine höhere Resistenz als ein Mrr1 mit natürlich vorkommenden gain-of-function-Mutationen. Analoge Fusionen mit Tac1 und Upc2 resultierten ebenfalls in einer konstitutiven Aktivierung dieser Transkriptionsfaktoren, die einen starken Anstieg der Fluconazolresistenz zur Folge hatte. Daraus ergab sich die Schlussfolgerung, dass dies eine generelle Methode sein könnte, die Zink-Cluster-Transkriptionsfaktoren künstlich zu aktivieren und so ihre biologischen Funktionen zu offenbaren, ohne die genauen Bedingungen für ihre Aktivität zu kennen. Deshalb wurde auf der Basis dieser Strategie eine Bibliothek von C.-albicans-Stämmen konstruiert, in der alle 82 putativen Zink-Cluster-Transkriptionsfaktoren in dieser möglicherweise hyperaktiven Form exprimiert werden. Untersuchungen dieser Bibliothek offenbarten neue Transkriptionsfaktoren, die Fluconazolresistenz vermittelten, aber auch noch unbekannte Regulatoren der Morphogenese und andere Phänotypen konnten beobachtet werden. Um einen tieferen Einblick in die Funktionsweise zu bekommen, wurden die Transkriptionsprofile der vier Transkriptionsfaktoren ermittelt, die in ihrer hyperaktiven Form die höchste Fluconazolresistenz bewirkten. Dabei stellte sich heraus, dass die zwei künstlich aktivierten (*) Regulatoren ZCF34* und ZNC1* die Expression der wichtigsten Multidrug-Effluxpumpe CDR1 stark hochregulierten. Der Transkriptionsfaktor mit dem vorläufigen Namen ZCF34 konnte im Verlauf dieser Arbeit als ein wichtiger Regulator für die CDR1-Expression identifiziert werden. Er ist sowohl an der Aktivierung der Expression von CDR1 beteiligt als auch für die basale CDR1-Promotoraktivität notwendig. Aus diesem Grund wurde er in MRR2 (multidrug resistance regulator 2) umbenannt. Mit der Entdeckung eines neuen Regulators der wichtigsten Multidrug-Effluxpumpe von C. albicans wurde ein wichtiger Beitrag zum Verständnis der Regulation solcher Transporter geleistet. Die Überexpression dieser Pumpen ist einer der häufigsten Resistenzmechanismen in C. albicans. Auf diesem Wege kann Resistenz gegen strukturell völlig unterschiedliche Antimykotika bewirkt werden. Somit stellen sowohl diese Effluxpumpen, als auch deren Regulatoren mögliche Angriffsziele für die Entwicklung neuer oder Weiterentwicklung bereits vorhandener Antimykotika dar.
Malaria is a vector-borne disease caused by the protozoan parasite of the genus Plasmodium and it is transmitted from human to human by female Anopheles mosquitoes during a blood meal. For malaria transmission to occur, the malaria parasite must undergo a crucial developmental sexual phase inside the mosquito midgut. In this study, we sought to investigate the interplay of the malaria parasite in the mosquito midgut with regard to the identification of novel types of transmission blocking intervention strategies. These strategies are aimed at reducing the spread of malaria by blocking the development of the mosquito midgut-specific stages of Plasmodium. We focused on three aspects. The first aspect was to investigate the interplay between mosquito midgut bacteria and malaria parasites in order to determine the potential influence of malaria parasites on the composition of the mosquito gut microbiota and also determine midgut bacteria which could be exploited as vehicles for the generation of paratransgenic Anopheles mosquitoes. We analyzed the microbial diversity of gut bacteria of the Asian malaria vector Anopheles stephensi during development and under different feeding regimes, including feeds on malaria parasite-infected blood, using the human pathogenic P. falciparum as well as the rodent malaria model P. berghei. 16S rRNA and DGGE analyses demonstrated a reduction in the microbial diversity during mosquito development from egg to adult and identified the gram-negative bacterium Elizabethkingia meningoseptica as the dominant species in the midgut of laboratory-reared male and female mosquitoes. E. meningoseptica is transmitted between generations and its predominance in the mosquito midgut was not altered by diet, when the gut microbiota was compared between sugar-fed and blood-fed female mosquitoes. Furthermore, feeds on blood infected with malaria parasites did not impact the presence of E. men-ingoseptica in the gut. Interestingly, extracts from E. meningoseptica exhibited antibacterial, antifungal and antiplasmodial activities, which may account for its dominance in the midgut of the malaria vector. Isolates of E. meningoseptica were cultivable, making the bacterium a potential candidate vehicle for the generation of paratransgenic Anopheles mosquitoes. The second aspect of this thesis was to determine transcriptome changes that occur during the first half hour following transmission of P. falciparum to the mosquito vector in order to better understand gene regulation mechanisms important for the change of hosts and determine novel proteins which could be exploited in malaria transmission blocking interventions. We initially used suppression subtractive hybridization (SSH) to compare mRNA levels of P. falciparum gametocytes before and 30 min fol-lowing activation. We identified a total of 126 genes for which transcript expression changed during gametogenesis. Among these, 17.5% had putative functions in signaling, 14.3% were assigned to cell cycle and gene expression, 8.7% were linked to the cytoskeleton or motor complex, 7.9% were involved in proteostasis and 6.4% in metabolism, 12.7% were genes encoding for cell surface associated proteins, 11.9% were assigned to other functions, and 20.6% represented genes of unknown function. For 40% of the identified genes there has as yet not been any protein evidence. We further selected a subset of 34 genes from all the above ontology groups and analyzed the transcript changes during gametogenesis in detail by quantitative realtime RT-PCR. Of these, 29 genes were expressed in gametocytes, and for 20 genes transcript expres-sion in gametocytes was increased compared to asexual blood stage parasites. Transcript levels of eight genes were particularly high in activated gametocytes, pointing at functions downstream of gametocyte transmission to the mosquito which could be exploited in malaria transmission blocking strategies. The last aspect of this thesis was to determine the transmission blocking effect of a range of antimicrobial molecules as transmission blocking agents. The molecules were either isolated from insect hemolymph or recombinantly expressed in tobacco and designed to act either directly on the mosquito midgut stages or cover receptors on mosquito tissues like the midgut epithelium which the parasite would need for transit. We were able to show an antiplasmodial and transmission blocking effect of the anti-microbial molecule harmonine, a defense compound isolated from the hemolymph of the Asian ladybug Harmonia axyridis. Harmonine thus represents a potential lead structure for the development of novel antimalarials.
Nitrogen-regulated pathogenesis describes the expression of virulence attributes as direct response to the quantity and quality of an available nitrogen source. As consequence of nitrogen availability, the opportunistic human fungal pathogen Candida albicans changes its morphology and secretes aspartic proteases [SAPs], both well characterized virulence attributes. C. albicans, contrarily to its normally non-pathogenic relative Saccharomyces cerevisiae, is able to utilize proteins, which are considered as abundant and important nitrogen source within the human host. To assimilate complex proteinaceous matter, extracellular proteolysis is followed by uptake of the degradation products through dedicated peptide transporters (di-/tripeptide transporters [PTRs] and oligopeptide transporters [OPTs]). The expression of both traits is transcriptionally controlled by Stp1 - the global regulator of protein utilization - in C. albicans. The aim of the present study was to elucidate the regulation of virulence attributes of the pathogenic fungus C. albicans by nitrogen availability in more detail. Within a genome wide binding profile of Stp1, during growth with proteins, more than 600 Stp1 target genes were identified, thereby confirming its role in the usage of proteins, but also other nitrogenous compounds as nitrogen source. Moreover, the revealed targets suggest an involvement of Stp1 in the general adaption to nutrient availability as well as in the environmental stress response. With the focus on protein utilization and nitrogen-regulated pathogenesis, the regulation of the major secreted aspartic protease Sap2 - additionally one of the prime examples of allelic heterogeneity in C. albicans - was investigated in detail. Thereby, the heterogezygous SAP2 promoter helped to identify an unintended genomic alteration as the true cause of a growth defect of a C. albicans mutant. Additionally, the promoter region, which was responsible for the differential activation of the SAP2 alleles, was delimited. Furthermore, general Sap2 induction was demonstrated to be mediated by distinct cis-acting elements that are required for a high or a low activity of SAP2 expression. For the utilization of proteins as nitrogen source it is also crucial to take up the peptides that are produced by extracellular proteolysis. Therefore, the function and importance of specific peptide transporters was investigated in C. albicans mutants, unable to use peptides as nitrogen source (opt1Δ/Δ opt2Δ/Δ opt3Δ/Δ opt4Δ/Δ opt5Δ/Δ ptr2Δ/Δ ptr22Δ/Δ septuple null mutants). The overexpression of individual transporters in these mutants revealed differential substrate specificities and expanded the specificity of the OPTs to dipeptides, a completely new facet of these transporters. The peptide-uptake deficient mutants were further used to elucidate, whether indeed proteins and peptides are an important in vivo nitrogen source for C. albicans. It was found that during competitive colonization of the mouse intestine these mutants exhibited wild-type fitness, indicating that neither proteins nor peptides are primary nitrogen sources required to efficiently support growth of C. albicans in the mouse gut. Adequate availability of the preferred nitrogen source ammonium represses the utilization of proteins and other alternative nitrogen sources, but also the expression of virulence attributes, like Sap secretion and nitrogen-starvation induced filamentation. In order to discriminate, whether ammonium availability is externally sensed or determined inside the cell by C. albicans, the response to exterior ammonium concentrations of ammonium-uptake deficient mutants (mep1Δ/Δ mep2Δ/Δ null mutants) was investigated. This study showed that presence of an otherwise suppressing ammonium concentration did not inhibit Sap2 proteases secretion and arginine-induced filamentation in these mutants. Conclusively, ammonium availability is primarily determined inside the cell in order to control the expression of virulence traits. In sum, the present work contributes to the current understanding of how C. albicans regulates expression of virulence-associated traits in response to the presence of available nitrogen sources - especially proteins and peptides - in order to adapt its lifestyle within a human host.
Background
Malignant pleural effusion (MPE) is associated with advanced stages of lung cancer and is mainly dependent on invasion of the pleura and expression of vascular endothelial growth factor (VEGF) by cancer cells. As MPE indicates an incurable disease with limited palliative treatment options and poor outcome, there is an urgent need for new and efficient treatment options.
Methods
In this study, we used subcutaneously generated PC14PE6 lung adenocarcinoma xenografts in athymic mice that developed subcutaneous malignant effusions (ME) which mimic pleural effusions of the orthotopic model. Using this approach monitoring of therapeutic intervention was facilitated by direct observation of subcutaneous ME formation without the need of sacrificing mice or special imaging equipment as in case of MPE. Further, we tested oncolytic virotherapy using Vaccinia virus as a novel treatment modality against ME in this subcutaneous PC14PE6 xenograft model of advanced lung adenocarcinoma.
Results
We demonstrated significant therapeutic efficacy of Vaccinia virus treatment of both advanced lung adenocarcinoma and tumor-associated ME. We attribute the efficacy to the virus-mediated reduction of tumor cell-derived VEGF levels in tumors, decreased invasion of tumor cells into the peritumoral tissue, and to viral infection of the blood vessel-invading tumor cells. Moreover, we showed that the use of oncolytic Vaccinia virus encoding for a single-chain antibody (scAb) against VEGF (GLAF-1) significantly enhanced mono-therapy of oncolytic treatment.
Conclusions
Here, we demonstrate for the first time that oncolytic virotherapy using tumor-specific Vaccinia virus represents a novel and promising treatment modality for therapy of ME associated with advanced lung cancer.
Escherichia coli Nissle 1917 (EcN) gehört zu den am besten untersuchten und charakterisierten probiotischen Bakterienstämmen. Seit Beginn des letzten Jahrhunderts wird er als Medikament eingesetzt, um verschiedene Darmerkrankungen wie z.B. Diarrhöe, entzündliche Darmerkrankungen und Verstopfung zu behandeln. Die Flagelle des EcN vermittelt Beweglichkeit und kann die Produktion von humanem β-Defensin 2 (hBD2) durch Epithelzellen induzieren. Somit ist dieses Organell direkt in die probiotische Funktion des EcN involviert. Es konnte gezeigt werden, dass die Flagellen anderer Bakterien, wie z.B. dem probiotischen Stamm Bacillus cereus CH oder den pathogenen Stämmen Pseudomonas aeruginosa und Clostridium difficile, die Adhäsion an intestinalen Mucus, welcher von Epithelzellen sekretiert wird, vermitteln. Allerdings blieb unklar, welcher Teil der Flagelle an welche Mucuskomponente bindet. Die Fähigkeit effizient an Wirtgewebe zu adhärieren wird als wichtiges Attribut eines probiotischen Stammes angesehen. Ex vivo Adhäsionsstudien mit Kryoschnitten humaner Darmbiopsien haben gezeigt, dass die Flagelle des EcN in die effiziente Adhäsion an humanes Darmgewebe involviert sein muss. Aus diesem Grund wurde in dieser Arbeit die Funktion der Flagelle des EcN als Adhäsin untersucht. Zunächst wurde die hyperflagellierte Variante EcN ATHF isoliert und durch verschiedene Experimente, z.B. Schwärmagartests und Elektronenmikroskopie, charakterisiert. Weitere ex vivo Adhäsionsstudien mit EcN ATHF zeigten eine höhere Adhäsionseffizienz dieser hyperflagellierten Variante und bestätigten damit die Rolle der Flagelle bei der effizienten Adhäsion von EcN an die Kryoschnitte der humanen Darmbiopsien. Interessanterweise fungierte die Flagelle in in vitro Studien mit den humanen Epithelzellen Caco-2 und T24 nicht als Adhäsin. Diese Unterschiede zwischen den in vitro und ex vivo Studien führten zu der Annahme, dass die Flagelle des EcN in vivo die Adhäsion an Mucus vermittelt, welcher von den Caco-2- und T24-Zellen nicht produziert wird, aber in den Kryoschnitten der Darmbiopsien nachgewiesen wurde. Diese Vermutung wurde durch in vitro Adhäsionsstudien mit der Mucin-produzierenden Epithelzelllinie LS174-T bestätigt, da die Flagellen für eine effektive Adhäsion an diese Zellen essentiell waren. Zudem reduzierte die Präinkubation flagellierter EcN-Stämme mit Mucin2 ihre Adhäsionseffizienz an Kryoschnitte humaner Darmbiopsien. Um die direkte Interaktion zwischen Flagellen des EcN Wildtyps und Mucus zu zeigen, wurde ein ELISA etabliert. Es konnte eine direkte konzentrationsabhängige Interaktion zwischen isolierten Flagellen des EcN Wildtyps und Mucin2, bzw. humanem Mucus (Kolon) beobachtet werden. Interessanterweise konnte keine Interaktion zwischen isolierten Flagellen des EcN Wildtyps und murinem Mucus (Duodenum, Ileum, Caecum, Colon) festgestellt werden. Dies weist darauf hin, dass die Mucuszusammensetzung zwischen verschiedenen Spezies variiert. Verschiedene Kohlenhydrate, welche bekannte Mucusbestandteile sind, wurden auf ihre Interaktion mit der Flagelle von EcN getestet und Gluconat wurde als ein Rezeptor identifiziert. Die Präinkubation isolierter Flagellen mit Gluconat reduzierte ihre Interaktion mit Mucin2, bzw. humanem Mucus signifikant. Zudem wurde die oberflächenexponierte Domäne D3 des Flagellins, der Hauptuntereinheit der Flagelle, als möglicher Interaktionspartner von Mucin2, bzw. humanem Mucus ausgeschlossen. Flagellen, die aus einer Domäne D3 Deletionsmutante isoliert wurden, zeigten sogar eine effizientere Bindung an Mucin2, bzw. humanen Mucus. Weiterhin konnte gezeigt werden, dass Änderungen des pH-Wertes signifikante Effekte auf die Interaktion zwischen Mucus und isolierten Flagellen hatten, vermutlich aufgrund von Konformationsänderungen. Zusammenfassend wurde in dieser Arbeit die Flagelle als neues und scheinbar wichtigstes Adhäsin in vivo für den probiotischen Stamm EcN identifiziert. Hierfür wurden sowohl eine hyperflagellierte Variante, eine ΔfliC Mutante, sowie der dazugehörige komplementierte Stamm verwendet. EcN ist zudem der erste probiotische Stamm für den eine direkte Bindung der Flagellen an humanen Mucus nachgewiesen werden konnte. Die Mucuskomponente Gluconat konnte dabei als wichtiger Rezeptor identifiziert werden. Da einige pathogene Bakterien ihre Flagelle zur Adhäsion an Wirtsgewebe nutzen, könnte dieses Organell EcN dazu befähigen, mit Pathogenen um die erfolgreiche Kolonisierung des Darms zu konkurrieren, was als wichtige Eigenschaft eines Probiotikums betrachtet wird.
Malaria and HIV are among the most important global health problems of our time and together are responsible for approximately 3 million deaths annually. These two diseases overlap in many regions of the world including sub-Saharan Africa, Southeast Asia and South America, leading to a higher risk of co-infection. In this study, we generated and characterized hybrid molecules to target P. falciparum and HIV simultaneously for a potential HIV/malaria combination therapy. Hybrid molecules were synthesized by covalent fusion between azidothymidine (AZT) and dihydroartemisinin (DHA), tetraoxane or chloroquine (CQ); and a small library was generated and tested for antiviral and antimalarial activity. Our data suggest that dihyate is the most potent molecule in vitro, with antiplasmodial activity comparable to that of DHA (IC50 = 26 nM, SI > 3000), a moderate activity against HIV (IC50 = 2.9 µM; SI > 35) and safe to HeLa cells at concentrations used in the assay (CC50 > 100 µM). Pharmacokinetic studies further revealed that dihyate is metabolically unstable and is cleaved following an O-dealkylation once in contact with cytochrome P450 enzymes. The later further explains the uneffectiveness of dihyate against the CQ-sensitive P. berghei N strain in mice when administered by oral route at 20 mg/kg. Here, we report on a first approach to develop antimalarial/anti-HIV hybrid molecules and future optimization efforts will aim at producing second generation hybrid molecules to improve activity against HIV as well as compound bioavailability. With the emergence of resistant parasites against all the counterpart drugs of artemisinin derivatives used in artemisinin based combination therapies (ACTs), the introduction of antibiotics in the treatment of malaria has renewed interest on the identification of antibiotics with potent antimalarial properties. In this study we also investigated the antiplasmodial potential of thiostrepton and derivatives, synthesized using combinations of tail truncation, oxidation, and addition of lipophilic thiols to the terminal dehydroamino acid. We showed that derivatives SS231 and SS234 exhibit a better antiplasmodial activity (IC50 = 1 µM SI > 59 and SI > 77 respectively) than thiostrepton (IC50 = 8.95 µM, SI = 1.7). The antiplasmodial activity of these derivatives was observed at concentrations which are not hemolytic and non-toxic to human cell lines. Thiostrepton and derivatives appeared to exhibit transmission blocking properties when administered at their IC50 or IC90 concentrations and our data also showed that they attenuate proteasome activity of Plasmodium, which resulted in an accumulation of ubiquitinated proteins after incubation with their IC80 concentrations. Our results indicate that the parasite’s proteasome could be an attractive target for therapeutic intervention. In this regard, thiostrepton derivatives are promising candidates by dually acting on two independent targets, the proteasome and the apicoplast, with the capacity to eliminate both intraerythrocytic asexual and transmission stages of the parasite. To further support our findings, we evaluated the activity of a new class of antimalarial and proteasome inhibitors namely peptidyl sulfonyl fluorides on gametocyte maturation and analogues AJ34 and AJ38 were able to completely suppress gametocytogenesis at IC50 concentrations (0.23 µM and 0.17 µM respectively) suggesting a strong transmission blocking potential. The proteasome, a major proteolytic complex, responsible for the degradation and re-cycling of non-functional proteins has been studied only indirectly in P. falciparum. In addition, an apparent proteasome-like protein with similarity to bacterial ClpQ/hslV threonine-peptidases was predicted in the parasite. Antibodies were generated against the proteasome subunits alpha type 5 (α5-SU), beta type 5 (β5-SU) and pfhslV in mice and we showed that the proteasome is expressed in both sexual and asexual blood stages of P. falciparum, where they localize in the nucleus and in the cytoplasm. However, expression of PfhslV was only observed in trophozoites and shizonts. The trafficking of the studied proteasome subunits was further investigated by generating parasites expressing GFP tagged proteins. The expression of α5-SU-GFP in transgenic parasite appeared to localize abundantly in the cytoplasm of all blood stages, and no additional information was obtained from this parasite line. In conclusion, our data highlight two new tools towards combination therapy. Hybrid molecules represent promising tools for the cure of co-infected individuals, while very potent antibiotics with a wide scope of activities could be useful in ACTs by eliminating resistant parasites and limiting transmission of both, resistances and disease.
Dendritic cell-based vaccination is a well established technique for preventive and therapeutic instruction of the immune system where conservative vaccine formulations fail to cure or prevent diseases, respectively. Efficiency of this technique already was demonstrated in infectious diseases as well as for cancer in animal or human studies. Well controlled manipulation and antigen-loading of immature DC is most beneficial to this technique. But, time-consuming and cost-extensive procedures for preparation of DC precursors, expansion and stimulation of DC and inpatient administration are big disadvantages regarding vaccine development for pandemic infectious diseases that occur mainly in underdeveloped countries. Therefore vaccines are needed that are pathogen-tailored and able to induce equal immune responses as their DC-based vaccine models. For vaccination against Leishmania parasites such a DC-based vaccine is feasible and its efficacy to induce protective Th1-based immune responses was already demonstrated in several animal studies. But, one of our own studies indicated supportive activity of host cells exceeding the allocation of T cells to become activated by transferred DC. IL-12, an important cytokine for the induction of Th1-related immune responses, has to be produced by host cells. Therefore, the aim of this study was to investigate the mechanism of BMDC-based vaccination with regard to simplification of the vaccine formulation. Key questions that have been addressed are: Which cells process the information that is transferred by the injected DC and what are the key components of this information? Further more, it was looked at whether altered vaccine formulations are able to induce protective immunity and whether they share equal molecular mechanisms. The current paradigm of BMDC-based vaccination proposes direct interaction of transferred BMDC with host T cells. These BMDC have to be antigen-loaded for stimulation via antigen-peptide-MHC molecule-complexes and they have to be activated for proper co-stimulation of T cells. Here, this study demonstrates that neither activation for co-stimulation nor direct interaction with adequate MHC molecules is needed for the induction of protective immunity against infection with Leishmania-parasites. Disrupted antigen-loaded BMDC are able to induce protective immunity in BALB/c mice without pre-stimulation via CpG ODN. Beyond, if BMDC were used with a different MHC-background than recipient mice then the vaccine still would be efficient in terms of reduction of footpad swelling and parasite load in draining lymph nodes. Even more, DC-specific features are no key component that leads to protective immunity as vaccination with disrupted antigen-loaded MΦ shows equal properties than before mentioned vaccine formulations. Further more, it was found that host DC play a major role in transforming the incoming signal, received from transferred antigen-loaded DC, into Th1-related stimuli and Leishmania-antigen-specific T cell activation. Suspensions of disrupted antigen-loaded DC resemble a combination of laid off soluble molecules together with exosome-like vesicles that formed after disruption of membranes. Here it was shown that separation of the membranous and soluble fractions and subsequent transfer into BALB/c mice will lead to protection of these mice against infection with L. major promastigotes only if the membranous fraction is used as vaccine. More, this vaccine formulation takes advantage of easy storage at -80°C with no need of fresh production. This clearly demonstrates that the immunity-inducing principle of disrupted DC-based vaccination lies within the membrane enclosed fraction. On a molecular level, disrupted antigen-loaded DC induce Th1-related cytokines during vaccination and as response on pathogen encounter. In vivo assays revealed IL-12 production and antigen-specific T cell proliferation among splenocytes that were stimulated with disrupted antigen-loaded DC. Splenocytes of accordingly vaccinated mice produce tremendous amounts of IFNγ after stimulation with Leishmania parasites. In summary, disrupted antigen-loaded BMDC fulfil all characteristics of DC-based vaccination against Leishmania major. But, while purification of membranes of antigen-loaded DC and subsequent transfer to BALB/c mice leads to control of the disease in the animal model, only slight levels of Th1-related cytokines are seen in the in vivo assays. Whether this points towards a loss of vaccine activity on unseen levels or unknown sites where Th1-related immunity is induced by both, complete solution and purified membranes, still has to be determined.
Cutaneous leishmaniasis is endemic in tropical and subtropical regions of the world. Effective vaccination strategies are urgently needed because of the emergence of drug-resistant parasites and severe side effects of chemotherapy. The research group of Heidrun Moll previously established a DC-based vaccination strategy to induce complete and long-lasting immunity to experimental leishmaniasis using LmAg-loaded and CpG ODN-activated DC as a vaccine carrier. Prevention of tissue damages at the site of L. major inoculation can be achieved if the BALB/c mice were systemically given LmAg-loaded BMDC that had been exposed to CpG ODN. The interest in further exploring the role of IL-4 aroused as previous studies allowed establishing that IL-4 was involved in the redirection of the immune response towards a type 1 profile. Thus, wt BALB/c mice or DC-specific CD11ccreIL-4Rα-/lox BALB/c mice were given either wt or IL-4Rα-deficient LmAg-loaded BMDC exposed or not to CpG ODN prior to inoculation of 2 x 105 stationary phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4Rα-mediated signaling in the vaccinating DC is required to prevent tissue damages at the site of L. major inoculation, as properly conditioned wt DC but not IL-4Rα-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining LN in CD11ccreIL-4Rα-/lox mice immunized with CpG ODN-exposed LmAg-loaded IL-4Rα-deficient DC, indicating the influence of IL-4R-mediated signaling in host DC to control parasite replication. In addition, no footpad damage was observed in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. Discussing these findings allow the assumption that triggering the IL4/IL4Rα signaling pathway could be a precondition when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms.
The probiotic Escherichia coli strain Nissle 1917 (EcN) is one of the few probiotics licensed as a medication in several countries. Best documented is its effectiveness in keeping patients suffering from ulcerative colitis (UC) in remission. This might be due to its ability to induce the production of human beta defensin 2 (HBD2) in a flagellin-dependent way in intestinal epithelial cells. In contrast to ulcerative colitis, for Crohn´s disease (CD) convincing evidence is lacking that EcN might be clinically effective, most likely due to the genetically based inability of sufficient defensin production in CD patients. As a first step in the development of an alternative approach for the treatment of CD patients, EcN strains were constructed which were able to produce human alpha-defensin 5 (HD5) or beta-defensin 2 (HBD2). For that purpose codon-optimized defensin genes encoding either the proform with the signal sequence or the mature form of human alpha defensin 5 (HD5) or the gene encoding HBD2 with or without the signal sequence were cloned in an expression vector plasmid under the control of the T7 promoter. Synthesis of the encoded defensins was shown by Western blots after induction of expression and lysis of the recombinant EcN strains. Recombinant mature HBD2 with an N-terminal His-tag could be purified by Ni-column chromatography and showed antimicrobial activity against E. coli, Salmonella enterica serovar Typhimurium and Listeria monocytogenes. In a second approach, that part of the HBD2-gene which encodes mature HBD2 was fused with yebF gene. The resulting fusion protein YebFMHBD2 was secreted from the encoding EcN mutant strain after induction of expression. Presence of YebFMHBD2 in the medium was not the result of leakage from the bacterial cells, as demonstrated in the spent culture supernatant by Western blots specific for ß-galactosidase and maltose-binding protein. The dialyzed and concentrated culture supernatant inhibited the growth of E. coli, Salmonella enterica serovar Typhimurium and Listeria monocytogenes in radial diffusion assays as well as in liquid coculture. This demonstrates EcN to be a suitable probiotic E. coli strain for the production of certain defensins.
The sexual phase of Plasmodium falciparum begins with the differentiation of intraerythrocytic sexual stages, termed gametocytes, in the human host. Mature gametocytes circulate in the peripheral blood and are taken up by the mosquito during the blood meal. These stages are essential for the spread of the malaria disease and form gametes in the mosquito midgut within minutes. A highly conserved family of six secreted proteins has been identified in Plasmodium falciparum. They comprise multiple adhesive domains and are termed PfCCp1 through PfCCp5, and PfFNPA. It was revealed in this work that PfCCp multi-domain adhesion proteins form protein complexes in gametocytes and on the surface of newly emerged macrogametes by adhesion domain-mediated binding. Co-Immunoprecipitation assays with activated gametocyte lysates show interactions between PfCCp proteins and indicate surface association via Pfs230 and Pfs25. Pfs230 is connected with the plasma membrane of the parasite by its interaction partner Pfs48/45. This protein is linked to the plasma membrane by a GPI anchor and presumably retains the multi-protein complex on the surface of newly emerged macrogametes in the mosquito midgut. A WD40 domain containing protein was identified to be part of this protein complex. It might serve as platform for the assembly of the multi protein complex or mediate the interplay among proteins, as suggested from known functions of the WD40 domain repeats. During egress from the host erythrocyte, the emerging gametes become vulnerable to factors of the human complement, which is taken up with the blood meal. In this thesis it was found that the complement system is active for about one hour post feeding. Macrogametes defend against complement-mediated lysis by co-opting the human complement regulators Factor H and FHL-1 from the blood-meal. These serum proteins bind via its SCR domains 5-7 to the surface of macrogametes. Once bound, they trigger complement inactivation of the alternative pathway, which prevents induction of complement lysis on the surface of the malaria parasite. Antibodies against Factor H are able to impair the sexual development in vitro and are able to block transmission to the mosquito. Interaction studies on endogenous proteins and immobilized recombinant proteins revealed the PfGAP50 protein as binding partner of Factor H and FHL-1. This protein was hitherto described as a glideosome-associated protein in invasive parasite stages, but has not yet been characterized in gametes. First localization studies indicate a relocation of PfGAP50 from the inner membrane complex to the surface of macrogametes. Malaria still persists as one of the deadliest infectious diseases worldwide. Investigations on the essential transmissive stages, gametocytes and gametes of Plasmodium falciparum, stood in the background of research for a long time. This work deciphered details on protein interactions on the surface of the malaria parasite and provides first information about coactions between the parasite and the human complement in the mosquito midgut.
Global Regulatory Functions of the Staphylococcus aureus Endoribonuclease III in Gene Expression
(2012)
RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus. However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family of double-strand-specific endoribonucleases. To define genomic transcripts that are bound and processed by RNase III, we performed deep sequencing on cDNA libraries generated from RNAs that were co-immunoprecipitated with wild-type RNase III or two different cleavage-defective mutant variants in vivo. Several newly identified RNase III targets were validated by independent experimental methods. We identified various classes of structured RNAs as RNase III substrates and demonstrated that this enzyme is involved in the maturation of rRNAs and tRNAs, regulates the turnover of mRNAs and non-coding RNAs, and autoregulates its synthesis by cleaving within the coding region of its own mRNA. Moreover, we identified a positive effect of RNase III on protein synthesis based on novel mechanisms. RNase III–mediated cleavage in the 5′ untranslated region (5′UTR) enhanced the stability and translation of cspA mRNA, which encodes the major cold-shock protein. Furthermore, RNase III cleaved overlapping 5′UTRs of divergently transcribed genes to generate leaderless mRNAs, which constitutes a novel way to co-regulate neighboring genes. In agreement with recent findings, low abundance antisense RNAs covering 44% of the annotated genes were captured by co-immunoprecipitation with RNase III mutant proteins. Thus, in addition to gene regulation, RNase III is associated with RNA quality control of pervasive transcription. Overall, this study illustrates the complexity of post-transcriptional regulation mediated by RNase III.
The harlequin ladybird beetle Harmonia axyridis has been introduced in many countries as a biological control agent, but has become an invasive species threatening the biodiversity of native ladybirds. Its invasive success has been attributed to its vigorous resistance against diverse pathogens. This study demonstrates that harmonine ((17R,9Z)-1,17-diaminooctadec-9-ene), which is present in H. axyridis haemolymph, displays broad-spectrum antimicrobial activity that includes human pathogens. Antibacterial activity is most pronounced against fast-growing mycobacteria and Mycobacterium tuberculosis, and the growth of both chloroquine-sensitive and -resistant Plasmodium falciparum strains is inhibited. Harmonine displays gametocytocidal activity, and inhibits the exflagellation of microgametocytes and zygote formation. In an Anopheles stephensi mosquito feeding model, harmonine displays transmission-blocking activity.
Agrobacterium species are capable of interkingdom gene transfer between bacteria and plants. The genome of Agrobacterium tumefaciens consists of a circular and a linear chromosome, the At-plasmid and the Ti-plasmid, which harbors bacterial virulence genes required for tumor formation in plants. Little is known about promoter sequences and the small RNA (sRNA) repertoire of this and other α-proteobacteria. We used a differential RNA sequencing (dRNA-seq) approach to map transcriptional start sites of 388 annotated genes and operons. In addition, a total number of 228 sRNAs was revealed from all four Agrobacterium replicons. Twenty-two of these were confirmed by independent RNA gel blot analysis and several sRNAs were differentially expressed in response to growth media, growth phase, temperature or pH. One sRNA from the Ti-plasmid was massively induced under virulence conditions. The presence of 76 cis-antisense sRNAs, two of them on the reverse strand of virulence genes, suggests considerable antisense transcription in Agrobacterium. The information gained from this study provides a valuable reservoir for an in-depth understanding of sRNA-mediated regulation of the complex physiology and infection process of Agrobacterium.
Numerous small non-coding RNAs (sRNAs) in bacteria modulate rates of translation initiation and degradation of target mRNAs, which they recognize through base-pairing facilitated by the RNA chaperone Hfq. Recent evidence indicates that the ternary complex of Hfq, sRNA and mRNA guides endoribonuclease RNase E to initiate turnover of both the RNAs. We show that a sRNA not only guides RNase E to a defined site in a target RNA, but also allosterically activates the enzyme by presenting a monophosphate group at the 5′-end of the cognate-pairing “seed.” Moreover, in the absence of the target the 5′-monophosphate makes the sRNA seed region vulnerable to an attack by RNase E against which Hfq confers no protection. These results suggest that the chemical signature and pairing status of the sRNA seed region may help to both ‘proofread’ recognition and activate mRNA cleavage, as part of a dynamic process involving cooperation of RNA, Hfq and RNase E.
Incidence rates of infections caused by environmental opportunistic fungi have risen over recent decades. Aspergillus species have emerged as serious threat for the immunecompromised, and detailed knowledge about virulence-determining traits is crucial for drug target identification. As a prime saprobe, A. fumigatus has evolved to efficiently adapt to various stresses and to sustain nutritional supply by osmotrophy, which is characterized by extracellular substrate digestion followed by efficient uptake of breakdown products that are then fed into the fungal primary metabolism. These intrinsic metabolic features are believed to be related with its virulence ability. The plethora of genes that encode underlying effectors has hampered their in-depth analysis with respect to pathogenesis. Recent developments in Aspergillus molecular biology allow conditional gene expression or comprehensive targeting of gene families to cope with redundancy. Furthermore, identification of essential genes that are intrinsically connected to virulence opens accurate perspectives for novel targets in antifungal therapy.
Compared to transcriptional activation, other mechanisms of gene regulation have not been widely exploited for the control of transgenes. One barrier to the general use and application of alternative splicing is that splicing-regulated transgenes have not been shown to be reliably and simply designed. Here, we demonstrate that a cassette bearing a suicide exon can be inserted into a variety of open reading frames (ORFs), generating transgenes whose expression is activated by exon skipping in response to a specific protein inducer. The surprisingly minimal sequence requirements for the maintenance of splicing fidelity and regulation indicate that this splicing cassette can be used to regulate any ORF containing one of the amino acids Glu, Gln or Lys. Furthermore, a single copy of the splicing cassette was optimized by rational design to confer robust gene activation with no background expression in plants. Thus, conditional splicing has the potential to be generally useful for transgene regulation.
Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 angstrom resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.
The proteasome of malaria parasites: A multi-stage drug target for chemotherapeutic intervention?
(2012)
The ubiquitin/proteasome system serves as a regulated protein degradation pathway in eukaryotes, and is involved in many cellular processes featuring high protein turnover rates, such as cell cycle control, stress response and signal transduction. In malaria parasites, protein quality control is potentially important because of the high replication rate and the rapid transformations of the parasite during life cycle progression. The proteasome is the core of the degradation pathway, and is a major proteolytic complex responsible for the degradation and recycling of non-functional ubiquitinated proteins. Annotation of the genome for Plasmodium falciparum, the causative agent of malaria tropica, revealed proteins with similarity to human 26S proteasome subunits. In addition, a bacterial ClpQ/hslV threonine peptidase-like protein was identified. In recent years several independent studies indicated an essential function of the parasite proteasome for the liver, blood and transmission stages. In this review, we compile evidence for protein recycling in Plasmodium parasites and discuss the role of the 26S proteasome as a prospective multi-stage target for antimalarial drug discovery programs.
A remarkable feature of many small non-coding RNAs (sRNAs) of Escherichia coli and Salmonella is their accumulation in the stationary phase of bacterial growth. Several stress response regulators and sigma factors have been reported to direct the transcription of stationary phase-specific sRNAs, but a widely conserved sRNA gene that is controlled by the major stationary phase and stress sigma factor, Sigma(S) (RpoS), has remained elusive. We have studied in Salmonella the conserved SdsR sRNA, previously known as RyeB, one of the most abundant stationary phase-specific sRNAs in E. coli. Alignments of the sdsR promoter region and genetic analysis strongly suggest that this sRNA gene is selectively transcribed by Sigma(S). We show that SdsR down-regulates the synthesis of the major Salmonella porin OmpD by Hfq-dependent base pairing; SdsR thus represents the fourth sRNA to regulate this major outer membrane porin. Similar to the InvR, MicC and RybB sRNAs, SdsR recognizes the ompD mRNA in the coding sequence, suggesting that this mRNA may be primarily targeted downstream of the start codon. The SdsR-binding site in ompD was localized by 3'-RACE, an experimental approach that promises to be of use in predicting other sRNA-target interactions in bacteria.
Background: CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake.
Principal Findings: In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria.
Conclusions: Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment.
We report on the characterization and target analysis of the small (s) RNA\(_{162}\) in the methanoarchaeon Methanosarcina mazei. Using a combination of genetic approaches, transcriptome analysis and computational predictions, the bicistronic MM2441-MM2440 mRNA encoding the transcription factor MM2441 and a protein of unknown function was identified as a potential target of this sRNA, which due to processing accumulates as three stabile 5' fragments in late exponential growth. Mobility shift assays using various mutants verified that the non-structured single-stranded linker region of sRNA\(_{162}\) (SLR) base-pairs with the MM2440-MM2441 mRNA internally, thereby masking the predicted ribosome binding site of MM2441. This most likely leads to translational repression of the second cistron resulting in dis-coordinated operon expression. Analysis of mutant RNAs in vivo confirmed that the SLR of sRNA\(_{162}\) is crucial for target interactions. Furthermore, our results indicate that sRNA\(_{162}\)-controlled MM2441 is involved in regulating the metabolic switch between the carbon sources methanol and methylamine. Moreover, biochemical studies demonstrated that the 50 end of sRNA\(_{162}\) targets the 5'-untranslated region of the cis-encoded MM2442 mRNA. Overall, this first study of archaeal sRNA/mRNA-target interactions unraveled that sRNA\(_{162}\) acts as an antisense (as) RNA on cis- and trans-encoded mRNAs via two distinct domains, indicating that cis-encoded asRNAs can have larger target regulons than previously anticipated.
Prevention of tissue damages at the site of Leishmania major inoculation can be achieved if the BALB/c mice are systemically given L. major antigen (LmAg)-loaded bone marrow-derived dendritic cells (DC) that had been exposed to CpG-containing oligodeoxynucleotides (CpG ODN). As previous studies allowed establishing that interleukin-4 (IL-4) is involved in the redirection of the immune response towards a type 1 profile, we were interested in further exploring the role of IL-4. Thus, wild-type (wt) BALB/c mice or DC-specific IL-4 receptor \(\alpha\) (IL-4R \(\alpha\))-deficient (CD11c\(^{cre}\)IL-4R \(\alpha^{-/lox}\) BALB/c mice were given either wt or IL-4R \(\alpha\)-deficient LmAg-loaded bone marrow-derived DC exposed or not to CpG ODN prior to inoculation of 2x10\(^5\) stationary-phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4R alpha-mediated signaling in the vaccinating DC is required to prevent tissue damage at the site of L. major inoculation, as properly conditioned wt DC but not IL-4R alpha-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining lymph nodes of CD11c\(^{cre}\)IL-4R \(\alpha^{-/lox}\) mice immunized with CpG ODN-exposed LmAg-loaded IL-4R \(\alpha\)-deficient DC, indicating the influence of IL-4R \(\alpha\)-mediated signaling in host DC to control parasite replication. In addition, no footpad damage occurred in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. We discuss these findings and suggest that the IL4/IL4R \(\alpha\) signaling pathway could be a key pathway to trigger when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms.
Background: Invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium (S. Typhimurium) requires expression of the extracellular virulence gene expression programme (STEX), activation of which is dependent on the signalling molecule guanosine tetraphosphate (ppGpp). Recently, next-generation transcriptomics (RNA-seq) has revealed the unexpected complexity of bacterial transcriptomes and in this report we use differential RNA sequencing (dRNA-seq) to define the high-resolution transcriptomic architecture of wildtype S. Typhimurium and a ppGpp null strain under growth conditions which model STEX. In doing so we show that ppGpp plays a much wider role in regulating the S. Typhimurium STEX primary transcriptome than previously recognised.
Results: Here we report the precise mapping of transcriptional start sites (TSSs) for 78% of the S. Typhimurium open reading frames (ORFs). The TSS mapping enabled a genome-wide promoter analysis resulting in the prediction of 169 alternative sigma factor binding sites, and the prediction of the structure of 625 operons. We also report the discovery of 55 new candidate small RNAs (sRNAs) and 302 candidate antisense RNAs (asRNAs). We discovered 32 ppGpp-dependent alternative TSSs and determined the extent and level of ppGpp-dependent coding and non-coding transcription. We found that 34% and 20% of coding and non-coding RNA transcription respectively was ppGpp-dependent under these growth conditions, adding a further dimension to the role of this remarkable small regulatory molecule in enabling rapid adaptation to the infective environment.
Conclusions: The transcriptional architecture of S. Typhimurium and finer definition of the key role ppGpp plays in regulating Salmonella coding and non-coding transcription should promote the understanding of gene regulation in this important food borne pathogen and act as a resource for future research.
Background: Recent data suggest that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence, novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy.
Methods: Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance, irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover, we identified and isolated CD44\(^+\)CD24\(^+\)ESA\(^+\) cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models.
Results: We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore, GLV-1h68 also showed preferential replication in CD44\(^+\)CD24\(^+\)ESA\(^+\) cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44\(^+\)CD24\(^-\)ESA\(^+\) cells.
Conclusions: Taken together, our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus, GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors, especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors.
Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS.
Many microRNAs (miRNAs) are co-regulated during the same physiological process but the underlying cellular logic is often little understood. The conserved, immunomodulatory miRNAs miR-146 and miR-155, for instance, are co-induced in many cell types in response to microbial lipopolysaccharide (LPS) to feedback-repress LPS signalling through Toll-like receptor TLR4. Here, we report that these seemingly co-induced regulatory RNAs dramatically differ in their induction behaviour under various stimuli strengths and act non-redundantly through functional specialization; although miR-146 expression saturates at sub-inflammatory doses of LPS that do not trigger the messengers of inflammation markers, miR-155 remains tightly associated with the pro-inflammatory transcriptional programmes. Consequently, we found that both miRNAs control distinct mRNA target profiles; although miR-146 targets the messengers of LPS signal transduction components and thus downregulates cellular LPS sensitivity, miR-155 targets the mRNAs of genes pervasively involved in pro-inflammatory transcriptional programmes. Thus, miR-155 acts as a broad limiter of pro-inflammatory gene expression once the miR-146 dependent barrier to LPS triggered inflammation has been breached. Importantly, we also report alternative miR-155 activation by the sensing of bacterial peptidoglycan through cytoplasmic NOD-like receptor, NOD2. We predict that dosedependent responses to environmental stimuli may involve functional specialization of seemingly coinduced miRNAs in other cellular circuitries as well.
Non-coding RNAs constitute a major class of regulators involved in bacterial gene expression. A group of riboregulators of heterogeneous size and shape referred to as small regulatory RNAs (sRNAs) control trans- or cis-encoded genes through direct base-pairing with their mRNAs. Although mostly inhibiting their target mRNAs, several sRNAs also induce gene expression. An important co-factor for sRNA activity is the RNA chaperone, Hfq, which is able to rearrange intramolecular secondary structures and to promote annealing of complementary RNA sequences. In addition, Hfq protects unpaired RNA from degradation by ribonucleases and thus increases sRNA stability. Co-immunoprecipitation of RNA with the Hfq protein, and further experimental as well as bioinformatical studies performed over the last decade suggested the presence of more than 150 different sRNAs in various Enterobacteria including Escherichia coli and Salmonellae. So-called core sRNAs are considered to fulfill central cellular activities as deduced from their high degree of conservation among different species. Approximately 25 core sRNAs have been implicated in gene regulation under a variety of environmental responses. However, for the majority of sRNAs, both the riboregulators’ individual biological roles as well as modes of action remain to be elucidated. The current study aimed to define the cellular functions of the two highly conserved, Hfq-dependent sRNAs, SdsR and RydC, in the model pathogen Salmonella Typhimurium. SdsR had been known as one of the most abundant sRNAs during stationary growth phase in E. coli. Examination of the conservation patterns in the sdsR promoter region in combination with classic genetic analyses revealed SdsR as the first sRNA under direct transcriptional control of the alternative σ factor σS. In Salmonella, over-expression of SdsR down-regulates the synthesis of the major porin OmpD, and the interaction site in the ompD mRNA coding sequence was mapped by a 3'RACE-based approach. At the post-transcriptional level, expression of ompD is controlled by three additional sRNAs, but SdsR plays a specific role in porin regulation during the stringent response. Similarly, RydC, the second sRNA adressed in this study, was initially discovered in E. coli but appeared to be conserved in many related γ-proteobacteria. An interesting aspect of this Hfq-dependent sRNAs is its secondary structure involving a pseudo-knot configuration, while the 5’ end remains single stranded. A transcriptomic approach combining RydC pulse-expression and scoring of global mRNA changes on microarrays was employed to identify the targets of this sRNA. RydC specifically activated expression of the longer of two versions of the cfa mRNA encoding for the phospholipid-modifying enzyme cyclopropane fatty acid synthase. Employing its conserved single-stranded 5' end, RydC acts as a positive regulator and masks a recognition site of the endoribonuclease, RNase E, in the cfa leader.
Single-cell time-lapse analysis of depletion of the universally conserved essential protein YgjD
(2011)
Background:
The essential Escherichia coli gene ygjD belongs to a universally conserved group of genes whose function has been the focus of a number of recent studies. Here, we put ygjD under control of an inducible promoter, and used time-lapse microscopy and single cell analysis to investigate the phenotypic consequences of the depletion of YgjD protein from growing cells.
Results:
We show that loss of YgjD leads to a marked decrease in cell size and termination of cell division. The transition towards smaller size occurs in a controlled manner: cell elongation and cell division remain coupled, but cell size at division decreases. We also find evidence that depletion of YgjD leads to the synthesis of the intracellular signaling molecule (p) ppGpp, inducing a cellular reaction resembling the stringent response. Concomitant deletion of the relA and spoT genes - leading to a strain that is uncapable of synthesizing (p) ppGpp abrogates the decrease in cell size, but does not prevent termination of cell division upon YgjD depletion.
Conclusions:
Depletion of YgjD protein from growing cells leads to a decrease in cell size that is contingent on (p) ppGpp, and to a termination of cell division. The combination of single-cell time-lapse microscopy and statistical analysis can give detailed insights into the phenotypic consequences of the loss of essential genes, and can thus serve as a new tool to study the function of essential genes.
Background:
A substantial amount of data has been accumulated supporting the important role of genomic islands (GEIs) - including pathogenicity islands (PAIs) - in bacterial genome plasticity and the evolution of bacterial pathogens. Their instability and the high level sequence similarity of different (partial) islands suggest an exchange of PAIs between strains of the same or even different bacterial species by horizontal gene transfer (HGT). Transfer events of archetypal large genomic islands of enterobacteria which often lack genes required for mobilisation or transfer have been rarely investigated so far.
Results:
To study mobilisation of such large genomic regions in prototypic uropathogenic E. coli (UPEC) strain 536, PAI II(536) was supplemented with the mob(RP4) region, an origin of replication (oriV(R6K)), an origin of transfer (oriT(RP4)) and a chloramphenicol resistance selection marker. In the presence of helper plasmid RP4, conjugative transfer of the 107-kb PAI II(536) construct occured from strain 536 into an E. coli K-12 recipient. In transconjugants, PAI II(536) existed either as a cytoplasmic circular intermediate (CI) or integrated site-specifically into the recipient's chromosome at the leuX tRNA gene. This locus is the chromosomal integration site of PAI II(536) in UPEC strain 536. From the E. coli K-12 recipient, the chromosomal PAI II(536) construct as well as the CIs could be successfully remobilised and inserted into leuX in a PAI II(536) deletion mutant of E. coli 536.
Conclusions:
Our results corroborate that mobilisation and conjugal transfer may contribute to evolution of bacterial pathogens through horizontal transfer of large chromosomal regions such as PAIs. Stabilisation of these mobile genetic elements in the bacterial chromosome result from selective loss of mobilisation and transfer functions of genomic islands.
Background:
We have shown that insertion of the three vaccinia virus (VACV) promoter-driven foreign gene expression cassettes encoding Renilla luciferase-Aequorea GFP fusion protein, beta-galactosidase, and beta-glucuronidase into the F14.5L, J2R, and A56R loci of the VACV LIVP genome, respectively, results in a highly attenuated mutant strain GLV 1h68. This strain shows tumor specific replication and is capable of eradicating tumors with little or no virulence in mice. This study aimed to distinguish the contribution of added VACV promoter-driven transcriptional units as inserts from the effects of insertional inactivation of three viral genes, and to determine the correlation between replication efficiency of oncolytic vaccinia virus in cell cultures and the virulence and antitumor efficacy in mice
Methods:
A series of recombinant VACV strains was generated by replacing one, two, or all three of the expression cassettes in GLV 1h68 with short non coding DNA sequences. The replication efficiency and tumor cell killing capacity of these newly generated VACV strains were compared with those of the parent virus GLV-1h68 in cell cultures. The virus replication efficiency in tumors and antitumor efficacy as well as the virulence were evaluated in nu/nu (nude) mice bearing human breast tumor xenografts.
Results:
we found that virus replication efficiency increased with removal of each of the expression cassettes. The increase in virus replication efficiency was proportionate to the strength of removed VACV promoters linked to foreign genes. The replication efficiency of the new VACV strains paralleled their cytotoxicity in cell cultures. The increased replication efficiency in tumor xenografts resulted in enhanced antitumor efficacy in nude mice. Similarly, the enhanced virus replication efficiency was indicative of increased virulence in nude mice.
Conclusions:
These data demonstrated that insertion of VACV promoter-driven transcriptional units into the viral genome for the purpose of insertional mutagenesis did modulate the efficiency of virus replication together with antitumor efficacy as well as virulence. Replication efficiency of oncolytic VACV in cell cultures can predict the virulence and therapeutic efficacy in nude mice. These findings may be essential for rational design of safe and potent VACV strains for vaccination and virotherapy of cancer in humans and animals.
Background:
Recent studies have shown that human ferritin can be used as a reporter of gene expression for magnetic resonance imaging (MRI). Bacteria also encode three classes of ferritin-type molecules with iron accumulation properties.
Methods and Findings:
Here, we investigated whether these bacterial ferritins can also be used as MRI reporter genes and which of the bacterial ferritins is the most suitable reporter. Bacterial ferritins were overexpressed in probiotic E. coli Nissle 1917. Cultures of these bacteria were analyzed and those generating highest MRI contrast were further investigated in tumor bearing mice. Among members of three classes of bacterial ferritin tested, bacterioferritin showed the most promise as a reporter gene. Although all three proteins accumulated similar amounts of iron when overexpressed individually, bacterioferritin showed the highest contrast change. By site-directed mutagenesis we also show that the heme iron, a unique part of the bacterioferritin molecule, is not critical for MRI contrast change. Tumor-specific induction of bacterioferritin-expression in colonized tumors resulted in contrast changes within the bacteria-colonized tumors.
Conclusions:
Our data suggest that colonization and gene expression by live vectors expressing bacterioferritin can be monitored by MRI due to contrast changes.
Background:
During the last years, (19)F-MRI and perfluorocarbon nanoemulsion (PFC) emerged as a powerful contrast agent methodology to track cells and to visualize inflammation. We applied this new modality to visualize deep tissue abscesses during acute and chronic phase of inflammation caused by Staphylococcus aureus infection.
Methodology and Principal Findings:
In this study, a murine thigh infection model was used to induce abscess formation and PFC or CLIO (cross linked ironoxides) was administered during acute or chronic phase of inflammation. 24 h after inoculation, the contrast agent accumulation was imaged at the site of infection by MRI. Measurements revealed a strong accumulation of PFC at the abscess rim at acute and chronic phase of infection. The pattern was similar to CLIO accumulation at chronic phase and formed a hollow sphere around the edema area. Histology revealed strong influx of neutrophils at the site of infection and to a smaller extend macrophages during acute phase and strong influx of macrophages at chronic phase of inflammation.
Conclusion and Significance:
We introduce (19)F-MRI in combination with PFC nanoemulsions as a new platform to visualize abscess formation in a murine thigh infection model of S. aureus. The possibility to track immune cells in vivo by this modality offers new opportunities to investigate host immune response, the efficacy of antibacterial therapies and the influence of virulence factors for pathogenesis.
Background
MicroRNAs, post-transcriptional regulators of eukaryotic gene expression, are implicated in host defense against pathogens. Viruses and bacteria have evolved strategies that suppress microRNA functions, resulting in a sustainable infection. In this work we report that Helicobacter pylori, a human stomach-colonizing bacterium responsible for severe gastric inflammatory diseases and gastric cancers, downregulates an embryonic stem cell microRNA cluster in proliferating gastric epithelial cells to achieve cell cycle arrest.
Results
Using a deep sequencing approach in the AGS cell line, a widely used cell culture model to recapitulate early events of H. pylori infection of gastric mucosa, we reveal that hsa-miR-372 is the most abundant microRNA expressed in this cell line, where, together with hsa-miR-373, it promotes cell proliferation by silencing large tumor suppressor homolog 2 (LATS2) gene expression. Shortly after H. pylori infection, miR-372 and miR-373 synthesis is highly inhibited, leading to the post-transcriptional release of LATS2 expression and thus, to a cell cycle arrest at the G1/S transition. This downregulation of a specific cell-cycle-regulating microRNA is dependent on the translocation of the bacterial effector CagA into the host cells, a mechanism highly associated with the development of severe atrophic gastritis and intestinal-type gastric carcinoma.
Conclusions
These data constitute a novel example of host-pathogen interplay involving microRNAs, and unveil the couple LATS2/miR-372 and miR-373 as an unexpected mechanism in infection-induced cell cycle arrest in proliferating gastric cells, which may be relevant in inhibition of gastric epithelium renewal, a major host defense mechanism against bacterial infections.
Der Hefepilz Candida albicans gehört zu den fakultativ pathogenen Infektionserregern und ist Teil der natürlichen Mikroflora der Schleimhäute des Verdauungs- und Urogenitaltraktes der meisten gesunden Menschen. Ist das Gleichgewicht der Flora gestört, kann es zu oberflächlichen Mykosen kommen, wie z.B. der oropharyngealen Candidiasis (Mundsoor), die in der Regel durch die Gabe eines Antimykotikums in wenigen Tagen zu behandeln sind. In seltenen Fällen kann es auch zu schwerwiegenden Infektionsverläufen bis hin zu lebensbedrohlichen systemischen Mykosen kommen. Hauptsächlich immunsupprimierte Patienten, wie z.B. AIDS-Patienten oder Personen, die kürzlich einer Organ- oder Knochenmarkstransplantation unterzogen wurden, leiden häufig an oberflächlichen C. albicans-Infektionen. Insbesondere bei wiederkehrenden Infektionen ist der Pilz in der Lage, gegen das häufig verabreichte Medikament Fluconazol eine Resistenz zu entwickeln. Ein wichtiger Mechanismus dieser Resistenzentwicklung ist die Überexpression von Effluxpumpen, die das Medikament aus der Zelle heraustransportieren. Zwei Arten von Effluxpumpen, die eine Rolle in der Resistenzentwicklung in C. albicans spielen, konnten bisher identifiziert werden, die ABC (ATP binding cassette)-Transporter Cdr1 und Cdr2 sowie der MFS (major facilitator superfamily)-Transporter Mdr1. Der Zinc-Cluster Transkriptionsfaktor Mrr1 spielt eine wichtige Rolle in der Regulation der MDR1-E¬ffluxpumpe. Er kontrolliert die MDR1-Expression in Anwesenheit induzierender Substanzen und sogenannte "gain-of-function" Mutationen in MRR1 konnten als die Ursache der konstitutiven MDR1-Hochregulierung und der "Multidrug-Resistance" in C. albicans identifiziert werden. In dieser Arbeit konnte ein Ortholog zu MRR1 aus C. albicans in Candida dubliniensis, einer zu C. albicans nahe verwandten Hefe, identifiziert werden. Es wurde gezeigt, dass in den untersuchten klinischen und in vitro generierten Fluconazol-resistenten C. dubliniensis-Stämmen ebenfalls gain-of-funcion Mutationen in MRR1 die MDR1-Überexpression und eine Resistenz bewirken. Die Ergebnisse demonstrieren, dass der Transkriptionsfaktor Mrr1 eine wichtige Rolle in der Entwicklung der Resistenz in diesen humanpathogenen Pilzen spielt. Bisher ist nicht bekannt, wie der Zinc-Cluster Transkriptionsfaktor MRR1 durch induzierende Substanzen oder gain-of-function Mutationen aktiviert wird. Um zu verstehen, wie die Mrr1- Aktivität reguliert wird, wurden in dieser Arbeit durch Deletionsstudien funktionelle Domänen des Transkriptionsfaktors identifiziert. Um einen besseren Einblick in die Regulation der MDR1-vermittelten Resistenz in C. albicans zu bekommen, wurde in dieser Arbeit die gegenseitige Abhängigkeit von Mrr1 und Cap1 bzw. Upc2 in Bezug auf die MDR1-Expression untersucht. Es wurden ChIP-on-chip Analysen und Transkriptionsprofile mit aktiviertem Mrr1 durchgeführt, um direkte Targets von Mrr1 zu identifizieren. Mit der vorliegenden Arbeit wurde ein wichtiger Beitrag zum Verständnis der Entwicklung der Multidrug-Resistenz in C. albicans geleistet. E¬ffluxpumpen und deren Regulatoren stellen in der Bekämpfung von C. albicans-Infektionen ein interessantes Angriffsziel für die Entwicklung neuer Medikamente und die Weiterentwicklung bereits vorhandender Antimykotika dar.
The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis, by the overexpression of genes encoding multidrug efflux pumps or ergosterol biosynthesis enzymes. Zinc cluster transcription factors play a central role in the transcriptional regulation of drug resistance. Mrr1 regulates the expression of the major facilitator MDR1, Tac1 controls the expression of the ABC transporters CDR1 and CDR2, and Upc2 regulates ergosterol biosynthesis (ERG) genes. Gain-of-function mutations in these transcription factors result in constitutive overexpression of their target genes and are responsible for fluconazole resistance in many clinical C. albicans isolates. The transcription factor Ndt80 contributes to the drug-induced upregulation of CDR1 and ERG genes and also binds to the MDR1 and CDR2 promoters, suggesting that it is an important component of all major transcriptional mechanisms of fluconazole resistance. However, we found that Ndt80 is not required for the induction of MDR1 and CDR2 expression by inducing chemicals. CDR2 was even partially derepressed in ndt80D mutants, indicating that Ndt80 is a repressor of CDR2 expression. Hyperactive forms of Mrr1, Tac1, and Upc2 promoted overexpression of MDR1, CDR1/CDR2, and ERG11, respectively, with the same efficiency in the presence and absence of Ndt80. Mrr1- and Tac1-mediated fluconazole resistance was even slightly enhanced in ndt80D mutants compared to wild-type cells. These results demonstrate that Ndt80 is dispensable for the constitutive overexpression of Mrr1, Tac1, and Upc2 target genes and the increased fluconazole resistance of strains that have acquired activating mutations in these transcription factors.
Die asexuellen Sporen von Aspergillus fumigatus sind ubiquitär verbreitete Luftkeime. Als Saprophyt ist dieser opportunistisch humanpathogene Pilz darauf spezialisiert, polymere Substanzen aus dem umgebenden Milieu zu zersetzen, um daraus die von ihm benötigten Nährstoffe zu generieren und aufzunehmen. Die Fähigkeit, verschiedene Stickstoff- und Kohlenstoffquellen zu verwerten, trägt dabei zu seiner Virulenz bei und hierbei scheint die extrazelluläre Proteolyse eine wichtige Rolle zu spielen. Sekretierte Proteasen, die das umgebende Gewebe während einer Infektion mit A. fumigatus erschließen, könnten somit zu dessen Pathogenität beitragen. Dementsprechend sollte im Rahmen dieser Arbeit die Bedeutung einer Regulation der extrazellulären proteolytischen Aktivität von A. fumigatus für dessen Virulenz untersucht werden. Dies geschah durch Untersuchungen eines konservierten Transkriptionsfaktors, PrtT. Dabei stellte sich heraus, dass PrtT die Expression der drei Hauptproteasen von A. fumigatus, Alp, Mep und Pep stark beeinflusst, in einem murinen Tiermodell der pulmonaren Aspergillose scheint dieser Regulator jedoch keine Rolle für die Pathogenität von A. fumigatus zu spielen. Um einen weiteren Aspekt des pilzlichen Aminosäurestoffwechsels zu beleuchten, wurde die Biosynthese der aromatischen Aminosäuren als mögliche Virulenzdeterminate untersucht. Für den Menschen sind diese Aminosäuren essentiell, weshalb dieser Syntheseweg ein mögliches Ziel für antimykotische Substanzen darstellen könnte. Es konnten mehrere für A. fumigatus essentielle Komponenten des Shikimatweges identifiziert werden, des Weiteren wurden Deletionsmutanten in den Genen aroC und trpA, die für die Chorismatmutase bzw. Anthranilatsynthase der Biosynthese von Phenylalanin und Tyrosin bzw. Tryptophan kodieren, erzeugt und phänotypisch charakterisiert. Deren Untersuchung in einem alternativen Tiermodell der Aspergillose zeigte eine deutlich attenuierte Virulenz. Diese Ergebnisse verdeutlichen, wie wichtig die Biosynthese der aromatischen Aminosäuren für das Wachstum von A. fumigatus ist, und dass ein Eingriff in diesen Syntheseweg eine lohnende Strategie zur Entwicklung neuer Antimykotika sein könnte. Die hier präsentierten Ergebnisse unterstreichen die für den Schimmelpilz A. fumigatus typische Redundanz bezüglich extrazellulärer proteolytischer Enzyme und dass diese nur bedingt hinsichtlich ihres Virulenzbeitrags untersucht werden können. Im Gegensatz hierzu lassen sich bestimmte Stoffwechselwege, die oftmals durch einzigartige Genprodukte katalysiert werden, unter Umständen besser als unspezifische aber vielversprechende Virulenzdeterminanten identifizieren.
Bacterial glucuronidase as general marker for oncolytic virotherapy or other biological therapies
(2011)
Background: Oncolytic viral tumor therapy is an emerging field in the fight against cancer with rising numbers of clinical trials and the first clinically approved product (Adenovirus for the treatment of Head and Neck Cancer in China) in this field. Yet, until recently no general (bio)marker or reporter gene was described that could be used to evaluate successful tumor colonization and/or transgene expression in other biological therapies. Methods: Here, a bacterial glucuronidase (GusA) encoded by biological therapeutics (e.g. oncolytic viruses) was used as reporter system. Results: Using fluorogenic probes that were specifically activated by glucuronidase we could show 1) preferential activation in tumors, 2) rena l excretion of the activated fluorescent compounds and 3) reproducible detection of GusA in the serum of oncolytic vaccinia virus treated, tumor bearing mice in several tumor models. Time course studies revealed that reliable differentiation between tumor bearing and healthy mice can be done as early as 9 days post injection of the virus. Regarding the sensitivity of the newly developed assay system, we could show that a single infected tumor cell could be reliably detected in this assay. Conclusion: GusA therefore has the potential to be used as a general marker in the preclinical and clinical evaluation of (novel) biological therapies as well as being useful for the detection of rare cells such as circulating tumor cells
While beneficial sponge-microbe associations have received much attention in recent years, less effort has been undertaken to investigate the interactions of sponges with potentially pathogenic microorganisms. Thus, the aim of this study was to examine two selected Caribbean disease conditions, termed “Sponge Orange Band” and “Sponge White Patch”, via ecological and molecular methods. Sponge Orange Band (SOB) disease affects the prominent Caribbean barrel sponge Xestospongia muta that is counted among the high-microbial-abundance (HMA) sponges, whereas Sponge White Patch (SWP) disease affects the abundant rope sponge Amphimedon compressa that belongs to the low-microbial-abundance (LMA) sponges. I have documented for both Caribbean sponge diseases a disease progression going along with massive tissue destruction as well as loss of the characteristic microbial signatures. Even though new bacteria were shown to colonize the bleached areas, the infection trials revealed in both cases no indication for the involvement of a microbial pathogen as an etiologic agent of disease leaving us still in the dark about the cause of Sponge Orange Band as well as Sponge White Patch disease.
Die Therapie von bakteriellen Infektionen beruht heutzutage zum Großteil auf dem Einsatz von Antibiotika. Die schnelle Entwicklung und rasche Verbreitung von resistenten Stämmen mancher Erreger gegen diese Antibiotika stellt ein enormes Problem für das Gesundheitswesen dar. Da momentan zur Antibiotikatherapie keine Alternativen bestehen, kommt der Erforschung neuer potenzieller Wirkstoffe eine sehr große Bedeutung zu. In einem Screening-Verfahren lagen die minimalen Hemmkonzentrationen einiger bisquartärer Bisnaphthalimide gegen Staphylococcus aureus und S. epidermidis im Bereich von 0,6 bis 2,5 µg/ml. Die Substanz mit den geringsten minimalen Hemmkonzentrationen war MT02. Daraufhin wurde das Wirkungsspektrum von MT02 gegen Bakterien detaillierter untersucht und gefunden, dass die Substanz vorwiegend gegen Gram-positive Erreger und nicht gegen Gram-negative Bakterien wirksam ist. Zytotoxizitätstests ergaben eine geringe bis nicht nachweisbare Toxizität gegen verschiedene Zelllinien im Bereich von 73 bis mehr als 150 µg/ml. Um die Wirkungsweise von MT02 genauer zu untersuchen wurden zunächst DNA-Microarray-Untersuchungen an S. aureus durchgeführt. Deren Ergebnisse ließen einen Einfluss der Substanz auf viele Gene des DNA-Metabolismus erkennen. Inkorporationsstudien mittels radioaktiver Ganzzellmarkierung bestätigten die Auswirkung von MT02 auf den DNA-Stoffwechsel. Durch kompetitive Inkubation wurde festgestellt, dass MT02 in der Lage ist Ethidiumbromid von DNA zu verdrängen bzw. dessen Bindung zu verhindern. Genauere Untersuchungen mittels Oberflächen-Plasmon-Resonanz ergaben, dass MT02 konzentrationsabhängig, reversibel und sequenzunspezifisch an DNA bindet. Die thermodynamischen Dissoziationskonstanten lagen im Mittel bei ca. 4 x 10-8 mol/l und beschrieben somit eine relativ starke Bindung von MT02 an DNA. Neben diesem primären Wirkungsmechanismus der DNA-Bindung gaben mehrere Befunde Hinweise auf einen sekundären Wirkmechanismus, der die Zellwand-Struktur bzw. Zellwand-Biosynthese beinhaltet. Eine MT02-resistente Mutante von S. aureus HG001 konnte durch vielfaches Passagieren in MT02-haltigem Medium generiert werden. Diese erzeugte bei Wachstum mit hohen Konzentrationen an MT02 einen roten Phänotyp. Die Natur dieses roten Farbstoffes konnte bislang nicht aufgeklärt werden, jedoch gibt es Hinweise, dass dieser auf Abbauprodukte von MT02 zurückzuführen ist. In einem weiteren Projekt wurde mittels Transkriptionsstudien die Auswirkung von verschiedenen bekannten Antibiotika sowie von neuen Wirkstoffen auf das Transkriptom von S. epidermidis untersucht. Die Ergebnisse dieser Studien können durch vergleichende Analysen als Grundlage für die Einordnung des Wirkmechanismus neuer Substanzen dienen.
Background: In principle, the elimination of malignancies by oncolytic virotherapy could proceed by different mechanisms - e.g. tumor cell specific oncolysis, destruction of the tumor vasculature or an anti-tumoral immunological response. In this study, we analyzed the contribution of these factors to elucidate the responsible mechanism for regression of human breast tumor xenografts upon colonization with an attenuated vaccinia virus (VACV). Methods: Breast tumor xenografts were analyzed 6 weeks post VACV infection (p.i.; regression phase) by immunohistochemistry and mouse-specific expression arrays. Viral-mediated oncolysis was determined by tumor growth analysis combined with microscopic studies of intratumoral virus distribution. The tumor vasculature was morphologically characterized by diameter and density measurements and vessel functionality was analyzed by lectin perfusion and extravasation studies. Immunological aspects of viral-mediated tumor regression were studied in either immune-deficient mouse strains (T-, B-, NK-cell-deficient) or upon cyclophosphamide-induced immunosuppression (MHCII+-cell depletion) in nude mice. Results: Late stage VACV-infected breast tumors showed extensive necrosis, which was highly specific to cancer cells. The tumor vasculature in infected tumor areas remained functional and the endothelial cells were not infected. However, viral colonization triggers hyperpermeability and dilatation of the tumor vessels, which resembled the activated endothelium in wounded tissue. Moreover, we demonstrated an increased expression of genes involved in leukocyte-endothelial cell interaction in VACV-infected tumors, which orchestrate perivascular inflammatory cell infiltration. The immunohistochemical analysis of infected tumors displayed intense infiltration of MHCII-positive cells and colocalization of tumor vessels with MHCII+/CD31+ vascular leukocytes. However, GI-101A tumor growth analysis upon VACV-infection in either immunosuppressed nude mice (MHCII+-cell depleted) or in immune-deficient mouse strains (T-, B-, NK-cell-deficient) revealed that neither MHCII-positive immune cells nor T-, B-, or NK cells contributed significantly to VACV-mediated tumor regression. In contrast, tumors of immunosuppressed mice showed enhanced viral spreading and tumor necrosis. Conclusions: Taken together, these results indicate that VACV-mediated oncolysis is the primary mechanism of tumor shrinkage in the late regression phase. Neither the destruction of the tumor vasculature nor the massive VACV-mediated intratumoral inflammation was a prerequisite for tumor regression. We propose that approaches to enhance viral replication and spread within the tumor microenvironment should improve therapeutical outcome.
A Candidate Approach Implicates the Secreted Salmonella Effector Protein SpvB in P-Body Disassembly
(2011)
P-bodies are dynamic aggregates of RNA and proteins involved in several post-transcriptional regulation processes. Pbodies have been shown to play important roles in regulating viral infection, whereas their interplay with bacterial pathogens, specifically intracellular bacteria that extensively manipulate host cell pathways, remains unknown. Here, we report that Salmonella infection induces P-body disassembly in a cell type-specific manner, and independently of previously characterized pathways such as inhibition of host cell RNA synthesis or microRNA-mediated gene silencing. We show that the Salmonella-induced P-body disassembly depends on the activation of the SPI-2 encoded type 3 secretion system, and that the secreted effector protein SpvB plays a major role in this process. P-body disruption is also induced by the related pathogen, Shigella flexneri, arguing that this might be a new mechanism by which intracellular bacterial pathogens subvert host cell function.
The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is an important model to elucidate the mechanisms involved in the interaction with the host. To gain insight into the transcriptome of the Xcv strain 85-10, we took a differential RNA sequencing (dRNA-seq) approach. Using a novel method to automatically generate comprehensive transcription start site (TSS) maps we report 1421 putative TSSs in the Xcv genome. Genes in Xcv exhibit a poorly conserved -10 promoter element and no consensus Shine-Dalgarno sequence. Moreover, 14% of all mRNAs are leaderless and 13% of them have unusually long 5'-UTRs. Northern blot analyses confirmed 16 intergenic small RNAs and seven cis-encoded antisense RNAs in Xcv. Expression of eight intergenic transcripts was controlled by HrpG and HrpX, key regulators of the Xcv type III secretion system. More detailed characterization identified sX12 as a small RNA that controls virulence of Xcv by affecting the interaction of the pathogen and its host plants. The transcriptional landscape of Xcv is unexpectedly complex, featuring abundant antisense transcripts, alternative TSSs and clade-specific small RNAs.
Background:
The probiotic Escherichia coli strain Nissle 1917 (EcN) has been shown to interfere in a human in vitro model with the invasion of several bacterial pathogens into epithelial cells, but the underlying molecular mechanisms are not known.
Methodology/Principal Findings:
In this study, we investigated the inhibitory effects of EcN on Salmonella Typhimurium invasion of porcine intestinal epithelial cells, focusing on EcN effects on the various stages of Salmonella infection including intracellular and extracellular Salmonella growth rates, virulence gene regulation, and adhesion. We show that EcN affects the initial Salmonella invasion steps by modulating Salmonella virulence gene regulation and Salmonella SiiE-mediated adhesion, but not extra-and intracellular Salmonella growth. However, the inhibitory activity of EcN against Salmonella invasion always correlated with EcN adhesion capacities. EcN mutants defective in the expression of F1C fimbriae and flagellae were less adherent and less inhibitory toward Salmonella invasion. Another E. coli strain expressing F1C fimbriae was also adherent to IPEC-J2 cells, and was similarly inhibitory against Salmonella invasion like EcN.
Conclusions:
We propose that EcN affects Salmonella adhesion through secretory components. This mechanism appears to be common to many E. coli strains, with strong adherence being a prerequisite for an effective reduction of SiiE-mediated Salmonella adhesion.
Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In this study, we analyzed for the first time the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 in two human hepatocellular carcinoma cell lines HuH7 and PLC/PRF/5 (PLC) in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 efficiently colonized, replicated in, and did lyse these cancer cells in culture. Experiments with HuH7 and PLC xenografts have revealed that a single intravenous injection (i.v.) of mice with GLV-1h68 resulted in a significant reduction of primary tumor sizes compared to uninjected controls. In addition, replication of GLV-1h68 in tumor cells led to strong inflammatory and oncolytic effects resulting in intense infiltration of MHC class II-positive cells like neutrophils, macrophages, B cells and dendritic cells and in up-regulation of 13 pro-inflammatory cytokines. Furthermore, GLV-1h68 infection of PLC tumors inhibited the formation of hemorrhagic structures which occur naturally in PLC tumors. Interestingly, we found a strongly reduced vascular density in infected PLC tumors only, but not in the non-hemorrhagic HuH7 tumor model. These data demonstrate that the GLV-1h68 vaccinia virus may have an enormous potential for treatment of human hepatocellular carcinoma in man.
Scientific research is a process concerned with the creation, collective accumulation, contextualization, updating and maintenance of knowledge. Wikis provide an environment that allows to collectively accumulate, contextualize, update and maintain knowledge in a coherent and transparent fashion. Here, we examine the potential of wikis as platforms for scholarly publishing. In the hope to stimulate further discussion, the article itself was drafted on Species-ID – a wiki that hosts a prototype for wiki-based scholarly publishing – where it can be updated, expanded or otherwise improved.
Staphylococcus aureus ist einer der häufigsten Erreger von nosokomialen Infektionen. Diese grampositiven Bakterien verursachen neben harmlosen oberflächlichen Hautinfektionen auch lebensbedrohliche Systeminfektionen. Ein großes Problem in der Therapie von S. aureus-Infektionen stellen die zunehmenden Multiresistenzen dar. Die Entwicklung neuer Antibiotika wird zukünftig wahrscheinlich nicht ausreichen, da immer wieder neue Resistenzen der Bakterien zu erwarten sind. Es besteht daher dringender Bedarf an der Entwicklung alternativer Therapieformen im Kampf gegen multiresistente Problemkeime wie S. aureus. Eine Möglichkeit besteht in der Immuntherapie, zum Beispiel durch Gewinnung von monoklonalen Antikörpern gegen geeignete Targetstrukturen von S. aureus. Ziel dieser Arbeit war es, zunächst zwei Proteine IsaA und IsaB herzustellen, um diese Proteine für Immunisierungsstudien zu nutzen. Zunächst wurde das gereinigte IsaA-Protein verwendet, um ein Kaninchen zu immunisieren. Mit den daraus gewonnenen Antikörpern wurden dann erste Tierversuche begonnen, um die Bedingungen für den therapeutischen Einatz von gegen IsaA-gerichteten Antikörpern zu ermitteln und die Wirksamkeit einer Antikörper-Behandlung zu evaluieren. Für die Herstellung der gewünschten Proteine wurden die Gensequenzen zunächst aus verschiedenen S. aureus-Stämmen mittels PCR amplifiziert und in den kommerziellen Expressionsvektor pQE30 kloniert. Die amplifizierte Gensequenz stammt aus den klinischen Stämmen 418 (IsaA) bzw. 134 (IsaB). Nach der Klonierung wurden geeignete Expressions- und Reinigungsstrategien entwickelt. Dabei wurden folgende Bedingungen als optimal für Wachstum und Überexpression herausgearbeitet: IsaA: Induktion der Überexpression mit 100 µM IPTG, 3 h Wachstum bei 37°C. IsaB: Induktion der Überexpression mit 100 µM IPTG, 4 h Wachstum bei 37°C. Es stellte sich auch heraus, dass IsaA zunächst in nur unzureichender Quantität vorhanden bzw. exprimiert worden war. Die Vermutung, dass IsaA überwiegend im Pellet in sogenannten Einschlusskörpern (inclusion bodies) eingeschlossen war, erklärte dieses Phänomen. Das Protein konnte erfolgreich aus dem Pellet isoliert werden. Die Produktion und Aufreinigung beider Proteine IsaA und IsaB unter optimierten Bedingungen ergab, dass beide Proteine nun in ausreichender Menge und Konzentration für die folgende Immunisierung und die weiteren Arbeiten vorlagen. Aus Kaninchen, die mit IsaA immunisiert wurden, konnten polyklonale Antikörper gewonnen werden, die die Grundlage für einen ersten Tierversuch mit 24 Ratten bildeten. Hierbei zeigte sich, dass die Tiere, die mit 1.000.000.000 Bakterien infiziert worden waren deutlich stärkere Infektionszeichen aufwiesen als diejenigen, die mit 100.000.000 Bakterien infiziert worden waren. Weiterhin wurde deutlich, dass die Tiere, die Serum (mit Antikörper gegen IsaA) erhalten hatten, gegenüber den Vergleichstieren mit Placebo einen deutlichen Vorteil hinsichtlich Infektionszeichen und Immunantwort hatten. Somit belegen die tierexperimentiellen Ergebnisse in dieser Arbeit erstmalig den therapeutischen Nutzen von Antikörpern gegen IsaA. IsaA ist demnach ein geeignetes Target für eine Immuntherapie gegen S. aureus.
Despite the internet's dynamic and collaborative nature, scientists continue to produce grant proposals, lab notebooks, data files, conclusions etc. that stay in static formats or are not published online and therefore not always easily accessible to the interested public. Because of limited adoption of tools that seamlessly integrate all aspects of a research project (conception, data generation, data evaluation, peerreviewing and publishing of conclusions), much effort is later spent on reproducing or reformatting individual entities before they can be repurposed independently or as parts of articles.
We propose that workflows - performed both individually and collaboratively - could potentially become more efficient if all steps of the research cycle were coherently represented online and the underlying data were formatted, annotated and licensed for reuse. Such a system would accelerate the process of taking projects from conception to publication stages and allow for continuous updating of the data sets and their interpretation as well as their integration into other independent projects.
A major advantage of such work ows is the increased transparency, both with respect to the scientific process as to the contribution of each participant. The latter point is important from a perspective of motivation, as it enables the allocation of reputation, which creates incentives for scientists to contribute to projects. Such work ow platforms offering possibilities to fine-tune the accessibility of their content could gradually pave the path from the current static mode of research presentation into a more coherent practice of open science.
Bacteria lose or gain genetic material and through selection, new variants become fixed in the population. Here we provide the first, genome-wide example of a single bacterial strain’s evolution in different deliberately colonized patients and the surprising insight that hosts appear to personalize their microflora. By first obtaining the complete genome sequence of the prototype asymptomatic bacteriuria strain E. coli 83972 and then resequencing its descendants after therapeutic bladder colonization of different patients, we identified 34 mutations, which affected metabolic and virulence-related genes. Further transcriptome and proteome analysis proved that these genome changes altered bacterial gene expression resulting in unique adaptation patterns in each patient. Our results provide evidence that, in addition to stochastic events, adaptive bacterial evolution is driven by individual host environments. Ongoing loss of gene function supports the hypothesis that evolution towards commensalism rather than virulence is favored during asymptomatic bladder colonization.
The tropical disease malaria, which results in more than one million deaths annually, is caused by protozoan parasites of the genus Plasmodium and transmitted by blood-feeding Anopheline mosquitoes. Parasite transition from the human host to the mosquito vector is mediated by gametocytes, sexual stages that are formed in human erythrocytes, which therefore play a crucial part in the spread of the tropical disease. The uptake by the blood-feeding mosquito triggers important molecular and cellular changes in the gametocytes, thus mediating the rapid adjustment of the parasite from the warm-blooded host to the insect host and subsequently initiating reproduction. The contact with midgut factors triggers gametocyte activation and results in their egress from the enveloping erythrocyte, which then leads to gamete formation and fertilization. This review summarizes recent findings on the role of gametocytes during transmission to themosquito and particularly focuses on the molecular mechanisms underlying gametocyte activation and emergence from the host erythrocyte during gametogenesis.
Malaria still persists as one of the deadliest infectious disease in addition to AIDS and tuberculosis. lt is a leading cause of high mortality and morbidity rates in the developing world despite of groundbreaking research on global eradication of the disease initiated by WHO, about half a century ago. Lack of a commercially available vaccine and rapid spread of drug resistance have hampered the attempts of extinguishing malaria, which still leads to an annual death toll of about one million people. Resistance to anti-malarial compounds thus renders search for new target proteins imperative. The kinome of the human malaria parasite Plasmodium falciparum comprises representatives of most eukaryotic protein kinase groups, including kinases which regulate proliferation and differentiation processes. Several reports till date have suggested involvement of parasite kinases in the human host and as well as in the mosquito vector. Kinases essential for life cycle stages of the parasite represent promising targets for anti-malarial compounds thus, provoking characterization of additional malarial kinases. Despite extensive research on most plasmodial enzymes, very little information is available regarding the four identified members of the cyclin dependent kinase like kinase (CLK) family. Thus, the present thesis dealt with the functional characterization of four members of the PfCLK kinase family of the parasite denoted as PfCLK-1/Lammer, PfCLK-2, PfCLK-3 and PfCLK-4 with a special focus on the first two kinases. Additionally, one Ca2+/Calmodulin dependent putative kinase-related protein, PfPKRP, presumed to be involved in sexual stage development of the parasite, was investigated for its expression in the life cycle of the parasite. In other eukaryotes, CLK kinases regulate mRNA splicing through phosphorylation of Serine/Arginine-rich proteins. Transcription analysis revealed abundance of PfCLK kinase genes throughout the asexual blood stages and in gametocytes. By reverse genetics approach it was demonstrated that all four kinases are essential for completion of the asexual replication cycle of P. falciparum. PfCLK 1/Lammer possesses two nuclear localization signals and PfCLK-2 possesses one of these signals upstream of the C-terminal catalytic domains. Protein level expression and sub-cellular localization of the two kinases was determined by generation of antiserum directed against the kinase domains of the respective kinase. Indirect immunofluorescence, Western blot and electron microscopy data confirm that the kinases are primarily localized in the parasite nucleus, and in vitro assays show that both enzymes are associated with phosphorylation activity. Finally, mass spectrometric analysis of co immunoprecipitated proteins shows interactions of the two PfCLK kinases with proteins, which have putative nuclease, phosphatase or helicase functions. PfPKRP on the other hand is predominantly expressed during gametocyte differentiation as identified from transcriptional analysis. Antiserum directed against the catalytic domain of PfPKRP detected the protein expression profile in both asexual and gametocyte parasite lysates. Via immunofluorescence assay, the kinase was localized in the parasite cytoplasm in a punctuated manner, mostly in the gametocyte stages. Reverse genetics resulted in the generation of PfPKRP gene-disruptant parasites, thus demonstrating that unlike CLK kinases, PfPKRP is dispensable for asexual parasite survival and hence might have crucial role in sexual development of the parasite. On one hand, characterization of PfCLK kinases exemplified the kinases involved in parasite replication cycle. Successful gene-disruption and protein expression of PfPKRP kinase on the other hand, demonstrated a role of the kinase in sexual stage development of the parasite. Both kinase families therefore, represent potential candidates for anti-plasmodial compounds.
The saprophytic filamentous fungus Aspergillus fumigatus has been gaining importance as an opportunistic human pathogen over the past decades. Advances in modern medicine have created a growing group of patients susceptible to infection with A. fumigatus, often contracting potentially deadly invasive aspergillosis. The virulence of this pathogen appears to be a multifactorial trait, a combination of physiological characteristics that enables the fungus to infect immunocompromised humans. This work concentrates on the nitrogen metabolism of A. fumigatus, which is essential for meeting the nutritional needs inside the human host. Using DNA microarrays, the transcriptional response during growth on three different secondary nitrogen sources was examined, which revealed the metabolic versatility of A. fumigatus, especially when challenged with proteins as the sole source of nitrogen. In-depth transcriptional profiling of the eight-member oligopeptide transporter (OPT) gene family underlined the importance of oligopeptide transport for growth on complex nitrogen sources like BSA or collagen. Heterologous expression of the opt genes in Saccharomyces cerevisiae showed their functionality as oligopeptide transporters, and characterized their substrate specificity. Using a Cre/loxP based genetic tool, a complete deletion of all opt genes in A. fumigatus was achieved. The resultant strain exhibited diminished growth on medium where the oligopeptide GPGG was the sole nitrogen source, but did not show any other in vitro phenotype. The opt deletion strain was not attenuated in virulence in a murine model of pulmonary aspergillosis, suggesting that the OPT gene family is not necessary for successful infection. The connection of oligopeptide transport and extracellular proteolytic activity was investigated by deleting the genes encoding Dpp4 and Dpp5, two dipeptidyl peptidases, or PrtT, the transcriptional regulator of major secreted proteases, in the complete opt deletion background. In contrast to the deletion of dpp4 and dpp5, which did not result in any additional phenotype, the absence of prtT led to a drastic growth defect on porcine lung agar. This suggests a synergistic action of extracellular proteolytic digest of proteins and transport of oligopeptide degradation products into the cell. Finally, this work established the bacterial β-Rec/six site-specific recombination system as a novel genetic tool for targeted gene deletion in A. fumigatus.
In dieser Arbeit werden die Ergebnisse der molekular-epidemiologischen Analyse von Virulenzgenen im Genom von insgesamt 222 Escherichia coli (E. coli)-Isolaten dargestellt, die von Mastitis-Fällen bei Rindern isoliert wurden. Mit Hilfe der Multiplex-Polymerase-Kettenreaktion wurde die Verbreitung von 42 potentiellen Virulenzfaktor-Genen extraintestinal pathogener E. coli (ExPEC) analysiert. Neben der quantitativen Bestimmung des Vorkommens jedes Einzelgens wurde in dieser Arbeit eine differenzierte Auswertung von Genkombinationen bei E. coli Mastitis-Isolaten vorgenommen. Diese ermittelten genetischen Muster werden zur 1. Prävalenz der in der Gesamtheit der Isolate, 2. Prävalenz in den phylogenetischen ECOR-Gruppen, 3. akut klinischen und chronischen Mastitis-Episoden und 4. dem Vorkommen spezifisch tierpathogener Adhäsine korreliert. Die Mastitis-Isolate konnten aufgrund der Virulenzmarkerverteilung und Phylogenie keinem bestimmten charakteristischen Pathotyp zugeordnet werden. Die überwiegende Mehrzahl der Mastitis-Isolate zeigte aufgrund einer geringen Prävalenz Virulenz-assoziierter Gene sowie der Zugehörigkeit zu den phylogenetischen Entwicklungslinien A und B1 ein geringes Virulenzpotential extraintestinal pathogener E. coli. Die Mehrzahl der Stämme enthielt eine singuläre Virulenzdeterminante (83 Stämme; 37,4 %), eine Zweierkombination (69 Stämme; 31,1 %) oder eine Dreierkombination von Virulenzgenen (34 Stämme; 15,3 %). Vier Gene für Virulenzfaktoren in Kombination zeigten sich lediglich in sieben Stämmen (3,1 %). Insbesondere die Anwesenheit von 5 bis 18 differenten Virulenzgenen pro Genom traten nur mit einer geringen Frequenz in zusammen 16 Isolaten (7,2 %) auf. Das absolut häufigste Virulenz-assoziierte Gen, das nachgewiesen wurde, war fimH, das für die mannosespezifische Adhäsinuntereinheit der Typ1-Fimbrien kodiert. Insgesamt gaben 88,7 % aller 222 untersuchten Stämme ein positives Signal in der Multiplex-PCR, und zwar 89,9 % der 199 klinischen Isolate sowie 85,7 % der Isolate chronischer Mastitiden. In etwa der Hälfte aller untersuchten Stämme trat auch das Gen traT auf, das Serumresistenz vermittelt (43,7 %). Die Genkombination fimH-traT wurde in wechselnden Konstellationen in insgesamt 83 Stämmen (37,3 %) gefunden. Sie ist damit die häufigste Virulenzgenkombination in den untersuchten E. coli-Genomen mit multiplen Virulenzdeterminanten. Da bei Rinder-Mastitis besonders in den schweren Fällen systemische Verläufe fördernde Faktoren wie Serumresistenz eine bedeutende Rolle spielen, könnte hier eine Selektion auf genetische Kopplung von traT mit fimH vorliegen. Deutlich geringere Prävalenzen wiesen die Virulenzgene für α-Hämolysin (hlyA, 10,8 %), den Yersiniabactinrezeptor (fyuA, 12,2 %) sowie das ebenfalls an der Serumresistenz beteiligte Gen iss (8,5 %) auf. Nur 13 (5,8 %) der 222 E. coli-Isolate besaßen keines der untersuchten Virulenzgene. Das Fehlen bekannter Virulenzgene in diesen Stämmen deutet darauf hin, dass weitere unberücksichtigte Faktoren eine Rolle bei der Virulenz von Mastitisisolaten spielen könnten oder der Status des Wirtsorganismus in diesen Fällen ausschlaggebend für eine erfolgreiche Infektion des Euters sein könnte. Offensichtlich sind die meisten der untersuchten E. coli- Virulenzfaktoren für die Pathogenese der Rindermastitis von untergeordneter Bedeutung. Von den 222 Isolaten zählten insgesamt 137 Stämme zur phylogenetischen Linie (ECOR-Gruppe) A, 62 zur ECOR-Gruppe B1, 20 zur ECOR-Gruppe B2 und 14 zur Gruppe D. Die Stämme, die zu den phylogenetischen Entwicklungslinien A, B1 und D gehören, unterschieden sich hinsichtlich der Prävalenz der Virulenzfaktormuster nicht vom Gesamtbild. Lediglich Isolate der ECOR-Gruppe B2 wiesen eine für sie typische Häufung von Virulenzgenclustern auf. Das relativ geringe Vorkommen bzw. weitgehende Fehlen (5 von 8) von Adhäsingenen spezifisch tierpathogener E. coli lässt darauf schließen, dass bislang beschriebenen Rinder-pathogenen E. coli keine Bedeutung als Verursacher einer Rindermastitis zukommt. Für die Analyse der chronischen Verlaufsform der Mastitis standen nur 21 Isolate zur Verfügung, die keinen hinreichend gesicherten Vergleich zu den Fällen mit akuter klinischer Mastitis (199 Stämme insgesamt) erlauben. Die auffällige Zunahme des Hämolysingens hlyA (23,8 %) gegenüber 9,5 % in den klinischen Isolaten (und 10,8 % in allen Stämmen) müsste in künftigen Untersuchungen invasiven Verhaltens der ExPEC beobachtet werden. Die Ergebnisse dieser Arbeit legen nahe, dass eine bovine Mastitis durch verschiedene E. coli-Varianten hervorgerufen werden kann und ein großes Potential extraintestinaler Virulenzfaktoren dazu nicht erforderlich ist. Entscheidend ist eine durch das fimH-Gen vermittelte Adhäsion, in der Hälfte der untersuchten Fälle unterstützt durch das Serumresistenz vermittelnde Gen traT.
Polyketide (PK) und nichtribosomale Peptide (NRP) sind zwei grosse Klassen von Naturstoffen, die eine grosse Vielfalt hinsichtlich ihrer Struktur und Funktion aufweisen. Sie werden von einer Reihe von Bakterien, Pilzen und Pflanzen als Sekundärmetabolite produziert und besitzen eine Vielzahl pharmakologisch wichtiger Aktivitäten, wie z.B. antimikrobielle, antimykotische, antitumorale oder antiparasitische Wirkungen. Ein Grossteil der bakteriellen Produzenten findet sich im Phylum Firmicutes, innerhalb der Gattungen Bacillus, Streptomyces und Mycobacterium. In E. coli sind Polyketide und nichtribosomale Proteine von eher geringer Bedeutung, mit Ausnahme der Siderophore Enterobactin und Yersiniabactin. Unerwartet war daher die Identifizierung eines neuen PKS/ NRPS-Gencluster in verschiedenen E. coli-Stämmen. Das 2006 durch NOUGAYRÈDE et al. zuerst beschriebene Colibactin-Gencluster kodiert für ein hybrides System aus modularen Polyketidsynthasen und nichtribosomalen Peptidsynthetasen sowie für zusätzliche editierende Enzyme und einen möglichen transkriptionellen Regulator (ClbR). Das Produkt der PKS/NRPS-Synthasen, Colibactin, übt in vitro einen zytopathischen Effekt (CPE) auf Säugerzelllinien aus. Die zytopathische Aktivität Colibactins zeichnet sich u.a. durch die Induktion von Doppelstrangbrüchen in der DNA der eukaryotischen Zellen aus. Darüber hinaus kommt es zu einer Unterbrechung des Zellzyklus in der G2-Phase nach einer transienten in vitro Infektion mit Colibactin-positiven Bakterienstämmen. Im Rahmen der vorliegenden Arbeit war besonders die weitere Aufklärung der Struktur des Colibactinclusters sowie die regulatorischen Mechanismen, die die Exression des hybriden nichtribosomalen Peptid-Polyketids von Interesse. Eine Transkriptionsanalyse führte zur Identifizierung der Transkriptionsstartpunkte der meisten relevanten Gene des Colibactinclusters. Basierend auf diesen neugewonnenen Informationen war eine Sequenzanalyse der upstream-Bereiche der Gene möglich, in deren Ergebnis neben den Elementen eines Sigma70-abhängigen Promotors, putative Bindestellen für mehrere Transkriptionsfaktoren identifiziert wurden. Untersuchungen zur Regulation der Colibactinsynthese zeigten, dass die Expression der Colibactin-Gene sowohl unter Kontrolle des Transkriptionsfaktors H-NS als auch des Colibactin-spezifischen Regulators ClbR stehen. Neben der Aufklärung der Struktur und Regulation der Colibactin-Gene bestand das Ziel dieser Arbeit in der Optimierung der Synthese des nichtribosomalen Peptid-Polyketids. Hierfür durchgeführte Expressionstudien zeigten einen Einfluss von Fettsäuren und Indol sowie von der Sauerstoffverfügbarkeit auf die Promotoraktivität einzelner Gene des Colibactin-Genclusters. Darüberhinaus konnte das pks-Genclusters erfolgreich in Pseudomonas putida KT2440 transferiert werden sowie der Nachweis der Funktionsfähigkeit Colibactins in diesem Wirtsorganismus nachgewiesen werden. Wenngleich die Stabilität des für diesen Zweck konstruierten Shuttle-Vektors nicht von Dauer ist, konnte gezeigt werden dass Pseudomonas putida prinzipiell als Wirtssystem für die Realisierbarkeit der heterologen Expression von Colibactin, geeignet ist. Zusätzlich zur Strukturanalyse des pks-Clusters und den Studien zur Expression der Colibactin-Gene befasste sich die hier vorliegende Arbeit mit der Fragestellung nach der biologischen Funktion Colibactins. Phänotypische Untersuchungen zeigen sowohl eine Beeinflussung der Eisenaufnahme als auch der Biofilmbildung durch das nichtribosomale Peptid-Polyketid. Dies sind die ersten Hinweise die zur Aufklärung der Funktion Colibactins beitragen könnten.
Avian pathogenic Escherichia coli (APEC) represent a subset of the so-called extraintestinal pathogenic Escherichia coli (ExPEC) pathotype that can cause various extraintestinal infections in humans and animals. APEC are the causative agent of localized colibacillosis or systemic infection in poultry. In this latter case, the syndrome starts as an infection of the upper respiratory tract and develops into a systemic infection. Generally, ExPEC are characterized by a broad variety of virulence-associated factors that may contribute to pathogenesis. Major virulence factors, however, that clearly define this pathotype, have not been identified. Instead, virulence-associated genes of ExPEC and thus also of APEC could be used in a mix-and-match-fashion. Both pathotypes could not be clearly distinguished by molecular epidemiology, and this suggested a hypothetical zoonotic risk caused by APEC. Accordingly, the main scientific question of this study was to characterize common traits as well as differences of APEC and human ExPEC variants that could either support the possible zoonotic risk posed by these pathogenic E. coli strains or indicate factors involved in host specificity. Comparative genomic analysis of selected APEC and human ExPEC isolates of the same serotype indicated that these variants could not be clearly distinguished on the basis of (i) general phenotypes, (ii) phylogeny, (iii) the presence of typical ExPEC virulence genes, and (iv) the presence of pathoadaptive mutations. Allelic variations in genes coding for adhesins such as MatB and CsgA or their regulators MatA and CsgD have been observed, but further studies are required to analyze their impact on pathogenicity. On this background, the second part of this thesis focused on the analysis of differences between human ExPEC and APEC isolates at the gene expression level. The analysis of gene expression of APEC and human ExPEC under growth conditions that mimick their hosts should answer the question whether these bacterial variants may express factors required for their host-specificity. The transcriptomes of APEC strain BEN374 and human ExPEC isolate IHE3034 were compared to decipher whether there was a specific or common behavior of APEC and human ExPEC, in response to the different body temperatures of man (37°C) or poultry (41°C). Only a few genes were induced at 41 °C in each strain relative to growth at 37 °C. The group of down-regulated genes in both strains was markedly bigger and mainly included motility and chemotaxis genes. The results obtained from the transcriptome, genomic as well as phenotypic comparison of human ExPEC and APEC, supports the idea of a potential zoonotic risk of APEC and certain human ExPEC variants. In the third part of the thesis, the focus was set on the characterization of Mat fimbriae, and their potential role during ExPEC infection. Comparison of the mat gene cluster in K-12 strain MG1655 and O18:K1 isolate IHE3034 led to the discovery of differences in (i) DNA sequence, (ii) the presence of transcriptional start and transcription factor binding sites as well as (iii) the structure of the matA upstream region that account for the different regulation of Mat fimbriae expression in these strains. A negative role of the H-NS protein on Mat fimbriae expression was also proven at 20 °C and 37 °C by real-time PCR. A major role of this fimbrial adhesin was demonstrated for biofilm formation, but a significant role of Mat fimbriae for APEC in vivo virulence could not yet be determined. Interestingly, the absence of either a functional matA gene or that of the structural genes matBCDEF independently resulted in upregulation of motility in E. coli strains MG1655 and IHE3034 by a so far unknown mechanism. In conclusion, the results of this thesis indicate a considerable overlap between human and animal ExPEC strains in terms of genome content and phenotypes. It becomes more and more apparent that the presence of a common set of virulence-associated genes among ExPEC strains as well as similar virulence gene expression patterns and phylogenetic backgrounds indicate a significant zoonotic risk of avian-derived E. coli isolates. In addition, new virulence factors identified in human ExPEC may also play a role in the pathogenesis of avian ExPEC.
Shigellosis, or bacillary dysentery, is a rectocolitis caused by the gram-negative, enteroinvasive bacteria of the genus Shigella. Shigellosis still remains a major public health burden with an estimated 80 million cases of bloody diarrhoea and 700.000 deaths per year, primarily in children under the age of 5. Shigella disrupts, invades, and causes inflammatory destruction of the colonic epithelium in humans through virulence effectors secreted by the type III secretion apparatus (TTSA). In contrast to the Shigella-induced manipulation of the host innate immune response, the impact of Shigella on the adaptive immunity has been poorly studied thus far. In order to understand why the naturally induced protective humoral response requires several infections to be primed and is of short duration, the work presented here investigates if Shigella is able to directly interact with T cells. Indeed, it has been shown that Shigella was able to invade and proliferate inside T cells. Furthermore, Shigella was able to inhibit T cell migration through a TTSA effector. Moreover, the Shigella effector IpgD, a phosphoinositide 4-phosphatase that specifically dephosphorylates phosphatidylinositol-(4,5)-bisphosphate (PIP2) into phosphatidylinositol-(5)-monophosphate (PI(5)P), was identified as the effector responsible for the observed inhibition. It could be demonstrated that IpgD was responsible for a reduction of intracellular PIP2 levels in T cells. Further experiments showed a reduced level of phosphorylated ezrin, radixin and moesin (ERM) proteins in infected, as well as with IpgD transfected, T cells. The ERM protein family plays an imported role in signal transduction and motility and their activity is closely related to the binding of PIP2. Therefore, the low level of PIP2 leads to a dephosphorylation of the ERM proteins which inhibits T cells response to chemokine stimulation. Indeed, IpgD transfected T cells show a reduced ability to re-localise the ERM proteins upon chemokine stimulation. Targeting T cell motility, via TTSA effectors, could explain the low level of specific T cell priming during Shigella infection. This is the first report of Shigella induced manipulation of T cell function and on the inhibition of T cell migration by a bacterial effector.
Schwämme (Phylum Porifera) sind der älteste rezente Tierstamm der Erde. Insbesondere marine Vertreter dieser sessilen Invertebraten sind oftmals mit einem mikrobiellen Konsortium assoziiert, welches hochgradig wirtsspezifisch und phylogenetisch divers ist. Die Biomasse dieser Mikroflora kann dabei rund die Hälfte der Masse eines Schwamms ausmachen. Die Komplexität des Konsortiums sowie der Mangel an kultivierbaren Vertretern der Schwamm-spezifischen Kladen erschwert dabei eine gezielte funktionelle Charakterisierung. Von besonderem Interesse hierbei ist das exklusiv in marinen Schwämmen vorzufindende Candidatus Phylum Poribacteria, für das bislang kein kultivierter Vertreter vorliegt. Die metabolisch aktiven und hochabundanten Poribakterien liegen in der extrazellulären Matrix des Schwammes vor und zeichnen sich durch das Vorhandensein einer Nukleoid-ähnlichen intrazellulären Struktur aus. Ziel dieser Promotionsarbeit war es, neue Einzelzell-basierte Methoden auf das Gebiet der funktionellen Charakterisierung von Bakterien anzuwenden, welche spezifisch mit dem mediterranen Schwamm Aplysina aerophoba assoziiert sind. Dabei wurden sowohl kultivierungs-abhängige, als auch kultivierungs-unabhängige Versuchsansätze verfolgt. Das Hauptaugenmerk dieser Studien lag dabei auf dem Candidatus Phylum Poribacteria. Während auf dem ‚dilution-to-extinction‘-Prinzip beruhende Hochdurchsatz-Kultivierungen nicht zum Erhalt einer Schwammsymbionten-Reinkultur führten, konnten durch eine Kombination aus FACS-Vereinzelung von Schwamm-assoziierten Bakterien und anschließenden Einzel-Genom-Amplifizierungen (‚whole genome amplifications‘) umfassende Einblicke in die metabolischen Kapazitäten von Schwammsymbionten gewonnen werden. Ferner gelang durch die Anwendung dieser neuen kultivierungs-unabhängigen Methode eine spezifische Verknüpfung von Phylogenie und Funktion Schwamm-assoziierter, nicht-kultivierbarer Bakterien. So konnte im Rahmen dieser Dissertation eine neue nicht-ribosomale Peptidsynthetase (NRPS) einem Vertreter einer Schwamm-spezifischen Chloroflexi-Klade zugewiesen werden. Ferner gelang die Zuordnung einer exklusiv in marinen Schwämmen vorgefundenen Polyketidsynthase (Sup-PKS) zu den Poribacteria. Die Klonierung von hochmolekularer, Einzel-Genom-amplifizierter DNA in Cosmide gewährte zudem Einblicke in den genomischen Kontext dieser, mit dem bakteriellen Sekundärmetabolismus assoziierten Gene. Die Pyrosequenzierung eines amplifizierten, von einem einzelnen Poribakterium abstammenden Genoms führte zudem zum Erhalt von rund zwei Megabasen an genetischer Information über diese Schwammsymbionten. Dadurch wurden detaillierte Informationen über den poribakteriellen Primär- und Sekundärstoffwechsel gewonnen. Die Auswertung der automatisch annotierten 454-Daten erlaubte die Rekonstruktion von Stoffwechselwegen, so z.B. der Glykolyse oder des Citratzyklus und bestätigte das Vorhandensein eines Sup-PKS-Gens im poribakteriellen Genom. Ferner konnten Gemeinsamkeiten mit den Schwesterphyla Planctomycetes, Chlamydiae und Verrucomicrobia gefunden werden. Zudem zeigte die vergleichende Analyse mit einem poribakteriellen Referenzklon aus einer bestehenden Metagenombank die genomische Mikroheterogenität innerhalb dieses Phylums. Nicht zuletzt konnte die Auswertung der poribakteriellen 454-Sequenzierung eine Reihe von möglichen Symbiose-Determinanten aufdecken, die beispielsweise am Austausch von Metaboliten zwischen den Interaktionspartnern beteiligt sind. Die Ergebnisse dieser Dissertationsarbeit stellen die Basis für eine gezielte und detaillierte funktionelle Beschreibung einzelner Bakterien innerhalb komplexer mikrobieller Konsortien dar, wie sie in marinen Schwämmen vorzufinden sind. Dieser Studie gewährte erstmalig umfassende Einblicke in das genomische Potential der nicht-kultivierten, Schwamm-assoziierten Poribacteria. Weiterführende Einzelzell-basierte Experimente werden in Zukunft dazu beitragen, das Bild von der Interaktion zwischen Bakterien und eukaryontischen Wirten zu komplettieren.
Um Änderungen in seiner Umwelt wahrnehmen zu können, benötigt S. aureus unterschiedliche Signaltransduktionssysteme. In dieser Arbeit wurde erstmals die Eukaryoten-ähnliche Serin/Threonin-Proteinkinase (STPK) PknB umfassend charakterisiert. Die posttranslationale Proteinmodifikation mittels Phosphorylierung spielt sowohl in Eukaryoten als auch in Prokaryoten eine wichtige Rolle. Man glaubte lange, dass die Phosphorylierung von Serin-, Threonin- und Tyrosinresten ein nur auf Eukaryoten beschränkter Regulationsmechanismus ist. Dagegen wurde die Phosphorylierung an Histidin- und Aspartatresten durch die Zweikomponenten-Systeme allein den Prokaryoten zugeordnet. Die Genomanalysen der letzten Jahre identifizierten jedoch STPKs und Serin/Threonin-Proteinphosphatasen (STPP) in nahezu allen prokaryotischen Genomen. Auch S. aureus codiert für eine STPK, die eine hohe Homologie zu den beschriebenen STPKs aufweist. In dieser Arbeit wurden mittels Microarray-Analyse einer ΔpknB-Mutante im Stamm 8325 erste Hinweise zur Funktion von PknB als Regulator der Zellwandsynthese sowie zentraler Stoffwechselwege gewonnen. Es wurden mittels Phosphopreoteom-Analysen in vivo-Substrate identifiziert und weiterhin die Kinase biochemisch charakterisiert.
Studien zur Verbreitung und genetischen Struktur des Colibactin-Genclusters in Enterobacteriaceae
(2009)
Horizontaler Gentransfer zwischen Bakterien – sogar zwischen verschiedenen Spezies – ist ein wichtiger Mechanismus für den Austausch genetischer Information. Dies kann dem Rezipienten einen selektiven Vorteil verleihen, z. B. durch die schnelle Aneignung von Genclustern, die für Pathogenitäts- oder Fitnessfaktoren kodieren. Die Variabilität bakterieller Genome durch Aneignung und Inkorporation genetischen Materials in das Genom trägt somit erheblich zur Evolution von Bakterien bei. Bakterielle Genome neigen allerdings dazu, nutzlose genetische Information zu verlieren und daher kann horizontal erworbener DNA häufig eine distinkte biologische Funktion zugeordnet werden. Das Colibactin-Gencluster, welches zuerst in Escherichia coli gefunden wurde, weist mehrere Charakteristika einer horizontal erworbenen genomischen Insel auf. Die Größe dieser genomischen Insel beträgt 54 kb und sie umfasst 20 offene Leseraster (ORFs), von denen acht für putative Polyketidsynthasen (PKS), nichtribosomale Peptidsynthasen (NRPS) und Hybride dieser kodieren. Colibactin übt einen zytopathischen Effekt (CPE) auf eukaryotische Zellen in vitro aus. Nach Kokultivierung Colibactin-Gencluster-positiven Bakterien mit eukaryotischen Zellen kommt es zu DNA Doppelstrang Brüchen, Zellzyklus-Arrest in der G2-Phase, Megalozytose und schließlich zum Zelltod. Diese Effekte sind mit denen des Zyklomodulins „Cytolethal Distending Toxin“ (CDT) vergleichbar, allerdings konnte die biologische Funktion des Colibactins in vivo bisher nicht aufgeklärt werden. Das Colibactin-Gencluster wurde bisher nur in Escherichia coli Stämmen der phylogenetischen Gruppe B2 als individuelle genomische Insel, integriert im tRNA-asnW-Gen, vorgefunden. Im Rahmen dieser Arbeit konnte das Colibactin-Gencluster auch in E. coli der phylogenetischen Gruppe B1 und in Citrobacter koseri, Enterobacter aerogenes und Klebsiella pneumoniae subsp. pneumoniae nachgewiesen werden. In diesen Bakterienstämmen ist das Colibactin-Gencluster Teil eines genetischen Elements, das Ähnlichkeit zu integrativen und konjugativen Elementen (ICE) aus E. coli und K. pneumoniae aufweist. Im Gegensatz zur hochkonservierten Integrationsstelle des Colibactin-Genclusters in tRNA-asnW in E. coli der phylogenetischen Gruppe B2 konnte die Integrationsstelle dieses ICE in E. coli der Gruppe B1 in tRNA-asnU bestimmt werden. In Bakterienstämmen der Spezies K. pneumoniae subsp. pneumoniae wurden vier verschiedene Integrationsstellen in fünf analysierten Stämmen identifiziert. Neben der Studien zur Verbreitung und chromosomalen Integration des Colibactin-Genclusters wurden Kolonisierungsstudien im murinen streptomycinbehandelten Intestinaltrakt mit E. coli Stamm Nissle 1917 durchgeführt, um eine mögliche Funktion des Colibactins im Darmtrakt näher zu untersuchen. Weder in nicht-kompetitiven noch in kompetitiven Versuchsdurchführungen konnte dabei ein Kolonisierungsvorteil durch Colibactin nachgewiesen werden. Die Ergebnisse dieser Arbeit haben gezeigt, dass das Colibactin-Gencluster in verschiedenen Spezies der Enterobacteriaceae vorhanden und funktional ist. Das Auftreten dieses sowohl als individuelle genomische Insel als auch als Teil eines ICE veranschaulicht die genetische Plastizität dieses Elements und die Bedeutung des horizontalen Transfers genetischen Materials. Die biologische Funktion des Colibactins in vivo bleibt weiterhin unklar und könnte sowohl die bakterielle Fitness als auch die Virulenz beeinflussen.
Der Erreger der Malaria tropica, Plasmodium falciparum, ist für eine jährliche Todesrate von über einer Million Menschen verantwortlich. Rasch zunehmende Erregerresistenzen gegen gängige Antimalariamedikamente und das Fehlen eines Impfstoffes machen die Suche nach neuen therapeutischen Ansätzen und Medikamenten unerlässlich. Sexualstadienspezifische Oberflächenproteine des Parasiten sind attraktive Zielstrukturen für die Entwicklung von TBV, welche eine Entwicklung von P. falciparum in der Mücke unterbrechen. Die Suche nach multiplen tier- oder bakterienähnlichen, extrazellulären Adhäsionsdomänen im Genom von P. falciparum führte zur Identifizierung einer Familie von sechs Proteinen mit hochkonservierten Adhäsionsmodulen, die vermutlich an Parasit-Parasit- oder Parasit-Wirtsinteraktionen beteiligt sind, was sie zu potentiellen Kandidaten für Komponenten von TBV macht. Aufgrund ihrer gemeinsamen LCCL-Domäne wurden diese Proteine PfCCp1 bis PfCCp5 sowie PfFNPA benannt. PfFNPA besitzt keine LCCL-Domäne, es ist jedoch ähnlich aufgebaut wie PfCCp5 und wurde daher mit in die PfCCp-Familie integriert. Die in der parasitophoren Vakuole reifer Gametozyten lokalisierenden PfCCp1- bis PfCCp3-Proteine werden während der Gametogenese teilweise freigesetzt und umgeben matrixähnlich entstehende Exflagellationszentren. In PfCCp2- und PfCCp3-defizienten Parasiten ist die Wanderung der Sporozoiten aus den Mitteldarmoozysten in die Speicheldrüsen der Mücke blockiert. Sexualstadien-spezifische Expression und eine wichtige Funktion bei der Entwicklung des Erregers in der Mücke sind die Hauptkriterien für potentielle TBV-Kandidaten. Diese viel versprechenden Daten waren Anlass, in der vorliegenden Arbeit, die bisher nur hypothetischen PfCCp5- und PfFNPA-Proteine genauer zu untersuchen. Expressionsstudien von PfCCp5 und PfFNPA mittels RT-PCR, Western-Blot-, Immunfluoreszenz- und Transmissionselektronenmikroskopischen-Analysen zeigten, dass sie sowohl plasmamembranassoziiert in der parasitophoren Vakuole als auch intrazellulär in reifen Gametozyten exprimiert werden. Beide Proteine sind in Gameto-zyten ab dem Stadium II detektierbar und weisen in unreifen Gametozyten ein punktiertes Expressionsmuster auf. In reifen Gametozyten konzentriert sich ihre Expression dagegen v. a. auf die Zellpole. Ferner werden PfCCp5 und PfFNPA auf der Oberfläche von Makrogameten, jedoch nicht in Mikrogameten und Ookineten exprimiert. Zusätzlich wird PfCCp5 in einem Teil reifer Schizonten eines gametozyten-bildenden Parasiten-Stammes exprimiert. Durch Integration eines Komplementations-Konstukts in die 3-untranslatierte Region von PfCCp5 bzw. PfFNPA konnte gezeigt werden, dass beide Gene genetisch manipulierbar sind. Mit PfCCp5- bzw. PfFNPA-KO-Konstrukten transfizierte WT-Parasiten wachsen nach erfolgter positiver Selektion jedoch nicht mehr. Diese Daten lassen vermuten, dass PfCCp5 und PfFNPA eine essentielle Funktion in den Blutstadien bzw. bei Gametozytenbildung haben. Zur weiteren Analyse von PfFNPA wurde ein verkürztes Protein durch Integration eines weiteren PfFNPA-KO-Konstrukts in den Locus von WT-Parasiten generiert. Erste Analysen des PfFNPA-KO-Phänotyps deuten darauf hin, dass durch die Ausschaltung der 3’-Region des Gens das Protein nicht mehr korrekt exprimiert wird, obwohl keine morphologischen Veränderungen der Blutstadien des Parasiten feststellbar sind. Außerdem werden PfCCp5 und PfFNPA ko-abhängig in PfCCp1-, PfCCp2- und PfCCp3-KO-Gametozyten exprimiert. Ko-Immunpräzipitationsstudien zeigten, dass beide Proteine mit den anderen PfCCp-Mitgliedern interagieren. Affinitätschromato-graphiestudien deckten dann direkte Interaktionen einzelner PfCCp-Domänen auf. Hierbei sind v. a. die LCCL-, die SR- und die NEC- Domäne an Proteininteraktionen beteiligt, was die Hypothese einer Komplexbildung der PfCCp-Familie während der Gametogenese des Erregers stützt. Transmissionsblockierungsstudien sollen nun die Eignung ausgewählter PfCCp-Proteine als TBV-Komponenten näher beleuchten. Zunehmende Resistenzen gegen gebräuchliche Malariamedikamente veranlassen zur Suche nach neuen Angriffspunkten zur Behandlung der Erkrankung. Die maßgeblich an der Hämoglobinhydrolyse beteiligten plasmodialen Cysteinproteasen Falcipain-2 und Falcipain-3 sind mögliche Ziele für die Entwicklung neuer Antimalariawirkstoffe. In der vorliegenden Arbeit wurden peptidomimetische 1,4-Benzodiazepin- und nicht-peptidische Etacrynsäurederivate in vitro auf ihre antiplasmodiale Wirkung an P. falciparum-Blutstadien getestet. Ein erstes Screening hatte gezeigt, dass die eine Vinylsulfonkopfgruppe tragenden 1,4 Benzodiazepinderivate rekombinant exprimiertes Falcipain-2 irreversibel hemmen. In vitro konnte dann auch eine antiplasmodiale Aktivität für diese Verbindungen festgestellt werden. Dockingstudien und HPLC-Assays mit den Etacrynsäurederivaten deckten eine Hemmung der Cysteinprotease Papain und der SARS-Mpro-Hauptprotease der Coronaviren auf. Weiterhin konnte in einem Screening an rekombinant exprimiertem Falcipain-2 und Falcipain-3 eine inhibitorische Wirkung für einen Teil dieser Etacrynsäurederivate festgestellt werden. Der In-vitro-Test an P. falciparum-Blutstadien deckte dann eine schwache antiplasmodiale Aktivität von fluorsubstituierten Etacrynsäurederivaten und von Derivaten mit einer modifizierten Etacrynsäurepartialstruktur auf. Der viel versprechendste Inhibitor dieser Studie wurde nun zur Identifizierung potentieller Bindungspartner mittels Affinitätsbindungsstudien biotyniliert. Zusammenfassend besitzen beide getesteten Wirkstoffklassen eine inhibierende Aktivität gegenüber Cysteinproteasen womit sie die Grundlage für die Entwicklung neuer, effektiverer plasmodialer Cysteinproteaseinhibitoren bieten.
The bacterial pathogen Legionella pneumophila replicates intracellularly in protozoa, but can also cause severe pneumonia, called Legionnaires' disease. The bacteria invade and proliferate in the alveolar macrophages of the human lung. L. pneumophila bacteria exhibit a biphasic life cycle: replicative bacteria are avirulent; in contrast, transmissive bacteria express virulence traits and flagella. Primarily aim of this thesis was to evaluate the impact of the regulatory proteins FleQ, FleR, and RpoN in flagellar gene regulation. Phenotypic analysis, Western blot and electron microscopy of regulatory mutants in the genes coding for FleQ, RpoN and FleR demonstrated that flagellin expression is strongly repressed and that these mutants are non-flagellated in transmissive phase. Transcriptomic studies of these putative flagellar gene expression regulators demonstrated that fleQ controls the expression of numerous flagellar biosynthetic genes. Together with RpoN, FleQ controls transcription of 14 out of 31 flagellar class II genes, coding for the basal body, hook, and regulatory proteins. Unexpectedly, 7 out of 15 late flagellar genes class III and IV) are expressed dependent on FleQ but independent of RpoN. Thus, in contrast to the commonly accepted view that enhancer binding proteins as FleQ always interact with RpoN to initiate transcription, our results strongly indicate that FleQ of L. pneumophila regulates gene expression RpoN-dependent as well as RpoN-independent. Moreover, transcriptome analysis of a fleR mutant strain elucidated that FleR does not regulate the flagellar class III genes as previously suggested. Instead FleR regulates together with RpoN numerous protein biosynthesis and metabolic genes. Based on these experimental results our modified model for the transcriptional regulation of flagellar genes in L. pneumophila is that flagellar class II genes are controlled by FleQ and RpoN, while flagellar class III and IV genes are controlled in a fleQ-dependent but rpoN-independent manner. Although all L. pneumophila strains share the same complex life style, various pathotypes have evolved. This is reflected by the genomes, which contain e.g. genomic islands. The genomic island Trb-1 of L. pneumophila Corby, carries all genes necessary for a type-IV conjugation system, an integrase gene and a putative oriT site. The second aim of this thesis was to investigate the implication of this genomic island in conjugative DNA transfer. Using conjugation assays we showed that the oriT site located on Trb-1 is functional and contributes to conjugation between different L. pneumophila strains. As this is the first oriT site of L. pneumophila known to be functional our results provide evidence that conjugation is a major mechanism for the evolution of new pathotypes in L. pneumophila.
Multizelluläre Gemeinschaften in Form bakterieller Biofilme stellen aus medizinischer Sicht ein großes klinisches Problem dar. Häufig lassen sich chronische oder rezidivierende Erkrankungen aber auch nosokomiale Infektionen auf die multizelluläre Lebensweise von humanpathogenen Erregern zurückführen. Sowohl fakultativ als auch obligat pathogene Escherichia coli-Stämme besitzen eine Vielzahl unterschiedlicher Faktoren, die die Biofilmbildung beeinflussen. Daran beteiligt sind unter anderem Flagellen, extrazelluläre polymere Substanzen, Adhäsine oder Oberflächen-assoziierte Proteine wie Autotransporter. Gegenstand der vorliegenden Arbeit ist die funktionale Charakterisierung des Proteins Antigen 43 (Ag43). Aufgrund seiner Autoaggregation-vermittelnden Eigenschaft trägt Ag43 ebenfalls zur Mikrokoloniebildung und Biofilmreifung bei. Antigen 43 ist ein Autotransporterprotein, welches innerhalb der Bakterienspezies Escherichia coli weit verbreitet ist. Interessanterweise besitzen viele E. coli-Isolate gleich mehrere identische oder ähnliche Kopien von agn43, die in der Regel von variablen Genombereichen (genomische Inseln, Plasmide) kodiert werden. Am Beispiel der Antigen 43-Varianten des uropathogenen Escherichia coli (UPEC)-Stammes 536 (O6:K15:H31), des kommensalen E. coli Isolats Nissle 1917 (O6:K5:H1) sowie des E. coli K-12-Laborstammes MG1655 (OR:H48:K-) ist die Bedeutung des Autotransporterproteins im Rahmen dieser Arbeit näher untersucht worden. Hierfür wurden die verschiedenen agn43-Allele in ein geeignetes Vektorsystem kloniert und im Adhäsin-freien Escherichia coli K-12-Stamm MG1655 ΔfimΔflu exprimiert. Da Antigen 43 in Wildtypstämmen posttranslational glykosyliert vorliegt, sind die Experimente zusätzlich unter Einfluß der heterologen, AIDA I-spezifischen Heptosyltransferase Aah (‘Autotransporter Adhesin Heptosyltransferase’) durchgeführt worden. Anhand von Bindungsstudien (intermolekulare Autoaggregation, Zelladhäsionstests) wurde gezeigt, daß sich einzelne Ag43-Varianten teilweise in ihren Eigenschaften unterscheiden. Im direkten Vergleich mit AIDA-I (‘Adhesin Involved in Diffuse Adherence’) enteropathogener Escherichia coli (EPEC) konnte für Ag43 nur eine Funktion als schwaches Adhäsin nachgewiesen werden. Die heterologe O-Glykosylierung beeinflußte die Funktionalität des Antigen 43 in unterschiedlichem Ausmaß. Je nach Autotransporter-Variante führte die Heptosylierung entweder zu signifikant reduzierten Affinitäten, oder sie hatte keinen Effekt auf die Bindungskapazität des Ag43. Antigen 43 weist zudem strukturelle Homologien zu vergleichbaren Domänen anderer Autotransporter auf. Für viele dieser Proteine konnte bereits eine adhäsive oder invasive Funktion nachgewiesen werden. Eine mögliche Interaktion von Ag43 mit eukaryontischen Rezeptoren ist hingegen noch nicht bzw. nur unvollständig untersucht worden. In Overlay assays und ELISAs wurde für Antigen 43 hier erstmals die spezifische Bindung an die extrazellulären Matrixkomponenten Kollagen und Laminin gezeigt. Zusammenfassend deuten die Ergebnisse dieser Arbeit darauf hin, daß das Escherichia coli spezifische Autotransporterprotein Antigen 43 nicht nur an der bakteriellen Biofilmbildung, sondern auch an der Besiedlung epithelialer Gewebe beteiligt sein kann. Seine Expression verschafft Bakterien einen Kolonisationsvorteil, der mit erhöhter Fitneß einhergeht. Die Aah-vermittelte O-Glykosylierung scheint für die Funktionalität von Ag43 nicht zwingend erforderlich zu sein. Des weiteren ist im Rahmen der vorliegenden Arbeit ein Testsystem entwickelt worden, das auf der Basis von Fluoreszenz-in-situ-Hybridisierungen (FISH) die Differenzierung von verschiedenen (uro-)pathogenen Mikroorganismen ermöglicht. Das etablierte Protokoll eignet sich nicht nur für die diagnostische Erregeridentifizierung, sondern auch in Abhängigkeit des Probenmaterials zur Untersuchung von (Multispezies-)Biofilmen.
Malaria stellt mit einer Mortalität von über einer Million Menschen pro Jahr die bedeutsamste Tropenkrankheit für den Menschen dar. Wachsende Resistenzen der Malariaerreger gegenüber den verfügbaren Medikamenten erhöhen mehr denn je den Druck, neue Therapiemöglichkeiten sowie einen Impfstoff gegen diese Krankheit zu entwickeln. Eine Unterbrechung des sexuellen Fortpflanzungszyklus im Laufe der Transmission von Mensch zu Stechmücke würde zu einem Verbreitungsstopp des Erregers führen. Sowohl die Identifizierung von molekularen Wechselwirkungen als auch die Erforschung von an Fertilisationsereignissen beteiligten Prozessen sind wichtige Schritte, um die Sexualphase des Erregers aufzuklären und neue Angriffspunkte für Medikamente oder Vakzine zu entwickeln. Dem Genom von P. falciparum konnten 92 putative Proteasen zugeordnet werden, von denen nur ein geringer Bruchteil charakterisiert worden ist. Unter Anwendung von Protease-Inhibitoren konnte in dieser Arbeit gezeigt werden, dass die Exflagellation der männlichen Gameten die Beteiligung von Proteasen verschiedener Kategorien benötigt. Die Ergebnisse belegten, dass die Aktivität von zwei oder mehr Serinproteasen, von Falcipain-ähnlichen Cysteinproteasen, von nicht-Thermolysin-ähnlichen Zink-Metalloproteasen und von Aspartatproteasen für den erfolgreichen Abschluss der männlichen Gametogenese eine wichtige Voraussetzung ist. Die Lokalisation des Cysteinproteasen- und Falcipain-hemmenden Inhibitors bADA konnte erstmals im Zytosol von Sexualstadien nachgewiesen werden. In dieser Arbeit wurden zusätzlich die Proteasen Calpain, DPAP2, GPI8, Metacaspase 2, Plasmepsin 6 und PfSub3 näher untersucht. RT-PCR-Analysen konnten die Transkription der sechs ausgesuchten Proteasen in gemischten asexuellen Parasiten sowie zum Großteil in Gametozyten, Gameten und Zygoten belegen. Die Transformation von asexuellen Parasiten mit entsprechenden knockout-Konstrukten deckte für Metacaspase 2 und PfSub3 auf, dass sie im asexuellen Vermehrungszyklus nicht essentiell und die entsprechenden Genloci für Rekombinationsereignisse zugänglich sind. Die Ergebnisse der übrigen Transformationen deuteten darauf hin, dass Calpain essentiell im asexuellen Vermehrungszyklus und dass der Genlocus von Plasmepsin 6 für Rekombinationsereignisse unzugänglich ist. Proteinexpressionsstudien anhand von Western-Blot-Analysen und Immunfluoreszenzstudien für PfSub3 konnten Hinweise darauf liefern, dass diese Serinprotease in asexuellen Parasiten, nicht-aktivierten sowie aktivierten Sexualstadien exprimiert wird. Aufgrund der in dieser Arbeit generierten Ergebnisse konnten im Laufe der Gametogenese auftretende Gametenfilamente morphologisch beschrieben sowie Hinweise auf ihre mögliche Funktion erlangt werden. Durch die Anwendung von Immunfluoreszenzstudien, rasterelektronenmikroskopischen Aufnahmen sowie die Analyse lebender Gameten konnte gezeigt werden, dass die bis zu 180 µm langen Filamente am Ende geschlossen sind und einen Durchmesser von ca. 200 nm aufweisen. Die tubulären Zellausläufer konnten weiterhin als verzweigte sowie nicht-verzweigte Ausläufer der parasitären Plasmamembran dargestellt werden, die mit Zytoplasma gefüllt sind. Es konnte belegt werden, dass die Aktin-assoziierten Filamente in periodischen Abständen von beulenartigen Auswölbungen unterbrochen werden und dass sie in rasterelektronenmikroskopischen Analysen ein perlschnurartiges Erscheinungsbild aufweisen. Weiterhin wurde dokumentiert, dass die Zellausläufer mit typischen sexualstadienspezifischen Proteinen wie Pfs25, Pfs230, Pfs48/45 und PfCCp4 assoziiert vorliegen, wobei das Fehlen einzelner dieser Proteine jedoch nicht das Ausbilden der Gametenfilamente verhinderte. Als typisches Charakteristikum der Filamente konnte ihre Eigenschaft beschrieben werden, mehrere Makrogameten und zum Teil Gametozyten in einem Zellkluster miteinander netzartig zu verbinden, wobei bis zu neun Filamente von einem Makrogameten ausgehend beobachtet werden konnten. Die Gametenfilamente zeigten ebenfalls die Fähigkeit, an umliegende nicht-infizierte Erythrozyten sowie mit asexuellen Parasiten infizierte Erythrozyten zu adhärieren. Die Filamente waren bereits fünf Minuten nach der Aktivierung der Gametozyten und im Laufe der Gametogenese bei 33 bis 73 % der Zellen nachweisbar. Die Gametenfilamente blieben bis zu 12 Stunden nach Aktivierung der Gametozyten mit der Zelloberfläche verbunden. Der aktive Einzug eines Zellfilaments sowie die Bildung der Gametenfilamente im Mitteldarm der Stechmücke konnte ebenfalls demonstriert werden. Die in dieser Arbeit dargestellten Ergebnisse lieferten unter anderem den Grundbaustein einer formulierten Funktionshypothese für diese Gametenfilamente. Es wird angenommen, dass die Filamente aufgrund ihrer adhäsiven Eigenschaften im Laufe der Befruchtung von Plasmodium im Mitteldarm der Stechmücke auftreten. Möglicherweise bedienen sich vitale Gameten dieser Strukturen, um andere Sexualstadien zu finden und sie zu verbinden.
Recent progresses and developments in molecular biology provide a wealth of new but insufficiently characterised data. This fund comprises amongst others biological data of genomic DNA, protein sequences, 3-dimensional protein structures as well as profiles of gene expression. In the present work, this information is used to develop new methods for the characterisation and classification of organisms and whole groups of organisms as well as to enhance the automated gain and transfer of information. The first two presented approaches (chapters 4 und 5) focus on the medically and scientifically important enterobacteria. Its impact in medicine and molecular biology is founded in versatile mechanisms of infection, their fundamental function as a commensal inhabitant of the intestinal tract and their use as model organisms as they are easy to cultivate. Despite many studies on single pathogroups with clinical distinguishable pathologies, the genotypic factors that contribute to their diversity are still partially unknown. The comprehensive genome comparison described in Chapter 4 was conducted with numerous enterobacterial strains, which cover nearly the whole range of clinically relevant diversity. The genome comparison constitutes the basis of a characterisation of the enterobacterial gene pool, of a reconstruction of evolutionary processes and of comprehensive analysis of specific protein families in enterobacterial subgroups. Correspondence analysis, which is applied for the first time in this context, yields qualitative statements to bacterial subgroups and the respective, exclusively present protein families. Specific protein families were identified for the three major subgroups of enterobacteria namely the genera Yersinia and Salmonella as well as to the group of Shigella and E. coli by applying statistical tests. In conclusion, the genome comparison-based methods provide new starting points to infer specific genotypic traits of bacterial groups from the transfer of functional annotation. Due to the high medical importance of enterobacterial isolates their classification according to pathogenicity has been in focus of many studies. The microarray technology offers a fast, reproducible and standardisable means of bacterial typing and has been proved in bacterial diagnostics, risk assessment and surveillance. The design of the diagnostic microarray of enterobacteria described in chapter 5 is based on the availability of numerous enterobacterial genome sequences. A novel probe selection strategy based on the highly efficient algorithm of string search, which considers both coding and non-coding regions of genomic DNA, enhances pathogroup detection. This principle reduces the risk of incorrect typing due to restrictions to virulence-associated capture probes. Additional capture probes extend the spectrum of applications of the microarray to simultaneous diagnostic or surveillance of antimicrobial resistance. Comprehensive test hybridisations largely confirm the reliability of the selected capture probes and its ability to robustly classify enterobacterial strains according to pathogenicity. Moreover, the tests constitute the basis of the training of a regression model for the classification of pathogroups and hybridised amounts of DNA. The regression model features a continuous learning capacity leading to an enhancement of the prediction accuracy in the process of its application. A fraction of the capture probes represents intergenic DNA and hence confirms the relevance of the underlying strategy. Interestingly, a large part of the capture probes represents poorly annotated genes suggesting the existence of yet unconsidered factors with importance to the formation of respective virulence phenotypes. Another major field of microarray applications is gene expression analysis. The size of gene expression databases rapidly increased in recent years. Although they provide a wealth of expression data, it remains challenging to integrate results from different studies. In chapter 6 the methodology of an unsupervised meta-analysis of genome-wide A. thaliana gene expression data sets is presented, which yields novel insights in function and regulation of genes. The application of kernel-based principal component analysis in combination with hierarchical clustering identified three major groups of contrasts each sharing overlapping expression profiles. Genes associated with two groups are known to play important roles in Indol-3 acetic acid (IAA) mediated plant growth and development as well as in pathogen defence. Yet uncharacterised serine-threonine kinases could be assigned to novel functions in pathogen defence by meta-analysis. In general, hidden interrelation between genes regulated under different conditions could be unravelled by the described approach. HMMs are applied to the functional characterisation of proteins or the detection of genes in genome sequences. Although HMMs are technically mature and widely applied in computational biology, I demonstrate the methodical optimisation with respect to the modelling accuracy on biological data with various distributions of sequence lengths. The subunits of these models, the states, are associated with a certain holding time being the link to length distributions of represented sequences. An adaptation of simple HMM topologies to bell-shaped length distributions described in chapter 7 was achieved by serial chain-linking of single states, while residing in the class of conventional HMMs. The impact of an optimisation of HMM topologies was underlined by performance evaluations with differently adjusted HMM topologies. In summary, a general methodology was introduced to improve the modelling behaviour of HMMs by topological optimisation with maximum likelihood and a fast and easily implementable moment estimator. Chapter 8 describes the application of HMMs to the prediction of interaction sites in protein domains. As previously demonstrated, these sites are not trivial to predict because of varying degree in conservation of their location and type within the domain family. The prediction of interaction sites in protein domains is achieved by a newly defined HMM topology, which incorporates both sequence and structure information. Posterior decoding is applied to the prediction of interaction sites providing additional information of the probability of an interaction for all sequence positions. The implementation of interaction profile HMMs (ipHMMs) is based on the well established profile HMMs and inherits its known efficiency and sensitivity. The large-scale prediction of interaction sites by ipHMMs explained protein dysfunctions caused by mutations that are associated to inheritable diseases like different types of cancer or muscular dystrophy. As already demonstrated by profile HMMs, the ipHMMs are suitable for large-scale applications. Overall, the HMM-based method enhances the prediction quality of interaction sites and improves the understanding of the molecular background of inheritable diseases. With respect to current and future requirements I provide large-scale solutions for the characterisation of biological data in this work. All described methods feature a highly portable character, which allows for the transfer to related topics or organisms, respectively. Special emphasis was put on the knowledge transfer facilitated by a steadily increasing wealth of biological information. The applied and developed statistical methods largely provide learning capacities and hence benefit from the gain of knowledge resulting in increased prediction accuracies and reliability.
Asymptomatische Bakteriurie (ABU) stellt eine bakterielle Infektion der Harnblase über einen langen Zeitraum dar, die häufig von Escherichia coli hervorgerufen wird, ohne dass typische Symptome einer Harnwegsinfektion auftreten. Um die Charakteristika von ABU E. coli Isolaten genauer zu untersuchen, wurden die Geno- und Phänotypen von 11 ABU-Isolaten verglichen. Außerdem wurden in mehreren aufeinanderfolgenden in vivo-Reisolaten des Modell-ABU Stammes 83972 die Veränderungen im Transkriptom, Proteom und Genom während einer langfristigen Persistenz in der menschlichen Blase charakterisiert. Schließlich wurde der Effekt des menschlichen Wirtes auf die bakterielle Adaptation durch einen Vergleich von in vitro- mit in vivo-kultivierten Stämmen abgeschätzt. ABU-Isolate stellt eine heterogene Gruppe von Organismen dar. Diese können den vier phylogenetischen Hauptgruppen von E. coli sowie unterschiedlichen klonalen Gruppen zugeordnet werden. Dementsprechend unterscheiden sie sich erheblich bezüglich der Zusammensetzung des Genomes, der Genomgröße und auch der Ausstattung mit UPEC-typischen Virulenz-assoziierten Genen. Multi-Lokus-Sequenz-Typisierung legt nahe, dass bestimmte ABU Stämme sich durch Genomreduktion aus UPEC Stämmen entwickelt haben, die eine Harnwegsinfektion mit charakteristischen Symptomen auslösen konnten. Folglich erlaubt die hohe Genomplastizität von E. coli keine generalisierte Betrachtung einzelner Isolate eines Klons. Genomreduktion über Punktmutationen, Genom-Reorganisation und Deletionen resultierte in der Inaktivierung einiger Gene, die für einige UPEC Virulenz-Faktoren kodieren. Dies stützt die Vorstellung, dass eine verminderte bakterielle Aktivierung der Entzündung der Wirtsschleimhaut den Lebensstil von ABU (bei diesen E. coli-)Isolaten fördert. Genregulation und genetische Diversität sind Strategien, die es Bakterien ermöglichen unter sich fortlaufend ändernden Bedingungen zu leben bzw. zu überleben. Um die anpassungsbedingten Veränderungen bei einem langfristigen Wachstum in der Blase zu untersuchen, wurden aufeinanderfolgende Reisolate, denen eine langfristige in vivo-Kolonisierung im menschlichen Wirt beziehungsweise eine in vitro-Kultivierung vorausgegangen ist, im Hinblick auf Veränderungen Genexpression und Genomorganisation analysiert. In diesem Zusammenhang konnte gezeigt werden, dass E. coli in der Lage ist, seine metabolischen Netzwerke verschiedenen Wachstumsbedingungen anzupassen und individuelle bakterielle Kolonisierungsstrategien entwickeln kann. Transkriptom- und Proteom-Analysen zeigten verschiedene metabolische Strategien zur Nährstoffbeschaffung und Energieproduktion bei untersuchten in vivo-Reisolaten vom Stamm 83972, die es ihnen ermöglichen, den Wirt zu kolonisieren. Das Zurückgreifen auf D-Serin, Deoxy- und Ribonucleoside sowie die bidirektionale Umwandlung zwischen Pentose und Glucuronat waren hoch-regulierte Stoffwechselwege, die die in vivo-Reisolate mit zusätzlicher Energie für ein effizientes Wachstum in der Blase versorgen. Zudem wurden in dieser Studie die Netzwerke für eine Reaktion auf Abwehrmechanismen des Wirtes erforscht: Erstmals wurde hier die Rolle der Klasse-III-Alkoholdehydrogenase AdhC, bekannt durch ihre Bedeutung bei der Entgiftung von Stickstoffmonoxid, bei der Wirtsantwort während einer asymptomatischen Bakteriurie gezeigt. Aufeinanderfolgende in vivo- und in vitro-Reisolate vom Stamm 83972 wurden ebenfalls bezüglich ihrer Genomstruktur analysiert. Einige Veränderungen in der Genomstruktur der aufeinanderfolgenden Reisolate, die von einer humanen Kolonisierungsstudie stammen, implizieren die Bedeutung einer Interaktion der Bakterien mit dem Wirt bei der Mikroevolution der Bakterien. Dagegen war die Genomstruktur von Reisolaten eines langfristigen in vitro-Kultivierungsexperiments, bei dem sich der Stamm 83972 ohne Wirtskontakt vermehrt hat, nicht von Veränderungen betroffen. Das legt nahe, dass die Immunantwort eine Genomplastizität fördert und somit eine treibende Kraft für den ABU Lebensstil und die Evolution im Harnwegstrakt ist.
Streptococcus pneumoniae (pneumococci) are Gram-positive bacteria and commensals of the nasopharyngeal cavity. Besides colonization, pneumococci are responsible for severe local infections such as otitis media, sinusitis and life-threatening invasive diseases, including pneumonia, sepsis and meningitis. The surface of pneumococci is decorated with proteins that are covalently or non-covalently anchored to the cell wall. The most unique group of cell wall associated proteins in pneumococci are the choline-binding proteins (CBPs). PspC, also known as SpsA or CbpA, is a multifunctional choline-binding protein that plays an essential role in pneumococcal pathogenesis by functioning as an adhesin. PspC promotes adherence of pneumococci to mucosal epithelial cells by interacting in a human specific manner with the free secretory component (SC) or to SC as part of the secretory IgA (SIgA) or polymeric immunoglobulin receptor (pIgR). PspC also interacts specifically with the soluble complement Factor H. Apparently, PspC uses two different epitopes for binding the soluble host protein Factor H and SC of pIgR. However, the mechanism by which these independent interactions facilitate pneumococcal infections under physiological and host specific conditions have not yet been completely elucidated. This study aims to explore the impact of the PspC interaction with human pIgR (hpIgR) or complement regulator Factor H on pneumococcal virulence. Here the cellular and molecular basis of PspC-mediated adherence to and invasion of host epithelial and endothelial cells was demonstrated. The genetic approach, specific pharmacological inhibitors and immunoblot analysis demonstrated the complexity of the induced signal transduction pathways during PspC-hpIgR mediated pneumococcal uptake by host cells. Inhibition studies with specific inhibitors of actin cytoskeleton and microtubules demonstrated that the dynamics of host cell cytoskeleton are essential for pneumococcal uptake by mucosal epithelial cells. Moreover, this study reports for the first time that the small GTPase Cdc42 is essential for pneumococcal internalization into epithelial cells via the PspC-hpIgR mechanism. In addition, in infection experiments performed in presence of specific inhibitors of PI3-kinase/Akt and protein tyrosine kinase (PTKs), hpIgR-mediated pneumococcal uptake by host cells was significantly blocked. Amongst PTKs the Src kinase pathway, ERK1/2 and JNK pathways were implicated during pneumococcal ingestion by hpIgR expressing cells. In addition, inhibition experiments performed in the presence of individual inhibitors or with a combination of inhibitors suggested the independent activation of PI3-kinase/Akt and Src kinase pathways during pneumococcal infections of hpIgR expressing cells. By employing specific inhibitors and siRNA in cell culture infection experiments it was further demonstrated that pneumococcal endocytosis by host epithelial cells via the PspC-hpIgR mechanism depends on clathrin and dynamin. PspC recruits also Factor H to the pneumococcal cell surface. Consequently, the impact of pneumococcal cell surface bound Factor H on adherence to host cells and the molecular mechanism facilitating the uptake of Factor H bound pneumococci by epithelial cells was investigated. Flow cytometry and immunoblots revealed that S. pneumoniae has evolved the ability to recruit both purified Factor H as well as Factor H from human plasma or serum. Moreover, it was demonstrated that the recruitment of Factor H is independent of the PspC-subtypes and that capsular polysaccharide (CPS) interferes with its recruitment. Factor H bound to pneumococci significantly increased bacterial attachment to and invasion of host epithelial cells including nasopharyngeal cells (Detroit562), lung epithelial cells (A549), and human brain-derived endothelial cells (HBMEC). Blocking experiments demonstrated that bacteria bound Factor H interacts via the heparin binding sites on Factor H with eukaryotic cell surface glycosaminoglycans and that this interaction promotes pneumococcal adherence to host cells. In addition, inhibition studies with mAbs recognizing specifically different short consensus repeats (SCR) of Factor H suggested that SCR 19-20 of Factor H are essential for the pneumococcal interaction with host epithelial cells via Factor H. In the presence of Factor H, attachment of pneumococci to human polymorphonuclear leukocytes (PMNs) is enhanced. The integrin CD11b/CD18 was identified as the cellular receptor on PMNs. By using pharmacological inhibitors the impact of host cell cytoskeleton and signalling molecules, such as PTKs and PI3-kinase, for Factor H-mediated pneumococcal internalization into eukaryotic cells was shown. Taken together, the results revealed that Factor-H mediated pneumococcal infection requires a concerted role of host epithelial cell surface glycosaminoglycans, integrins and host cell signalling pathways.
In dieser Arbeit wurde in Legionella pneumophila Corby ein bislang nicht in L. pneumophila beschriebener Bereich im Genom kloniert und sequenziert, der für ein putatives Konjugations- Typ IV Sekretionssystem kodiert. Alle für ein Typ IV Sekretionssystem notwendigen Gene sind vorhanden. Zum einen kodieren diese ein „mating pair formation“ System, also Proteine für die Pilusgenese und energieabhängigen Transport von Substraten aus der Bakterienzelle. Konjugationsexperimente zeigen, dass es sich bei den „DNA transfer and replication“ Genen trb/tra System um einen funktionierenden Mechanismus zur Mobilisierung von DNA handelt.
Marine Schwämme (Phylum Porifera) sind sessile Invertebraten, deren Biomasse bis zu 60% aus Mikroorganismen bestehen kann. Während die mikrobielle Diversität in Schwämmen in den letzten Jahren recht gut beschrieben wurde, weiß man noch sehr wenig über mögliche Funktionen und Interaktionen zwischen Schwamm-assoziierten Mikroorganismen mit ihren Wirten. Das Ziel dieser Promotionsarbeit war es, den Prozess der mikrobiellen Nitrifikation im bakterienhaltigen Mittelmeerschwamm Aplysina aerophoba nachzuweisen und im Kontext der Symbiose näher zu untersuchen. Die Nitrifikation beschreibt die zweistufige Oxidation von Ammoniak zu Nitrit und weiter zu Nitrat und wird von bestimmten Mikroorganismen zur Energiegewinnung durchgeführt. Um dieser Fragestellung nachzugehen, wurden physiologische Untersuchungen an lebenden Schwämmen während Freilandexkursionen nach Rovinj (Kroatien) durchgeführt. Frisch gesammelte Schwämme wurden zu unterschiedlichen Jahreszeiten in experimentellen Aquarien jeweils über einen Zeitraum von über 24 Stunden gehältert. Die Konzentrationen von Ammonium, Nitrit und Nitrat wurden in Zeitintervallen mittels photometrischer Nachweise gemessen und die Aufnahme- und Exkretionsraten berechnet. Nitrit wurde in keinem der Experimente messbar ausgeschieden. Ammonium, als natürliches Stoffwechselendprodukt mariner Schwämme, wurde von A. aerophoba in Raten ausgeschieden, die saisonal variabel waren. Im Frühjahr wurde keine Ammonium-ausscheidung beobachtet während die Exkretionsrate zum Sommer hin stetig anstieg. Nitrat, welches natürlicherweise nur durch mikrobielle Nitrifikation entstehen kann, wurde saisonunabhängig konstant ausgeschieden. Ammoniumaufnahme-Experimente zeigten auf, dass Ammonium im Frühjahr rasch aufgenommen wurde und dass Ammonium die Nitratexkretionsrate bis zu vierfach stimulierte, wohingegen im Sommer keine Ammoniumaufnahme und keine Stimulation der Nitratexkretion stattfanden. Durch Zugabe des spezifischen Inhibitors der Nitrifikation, Nitrapyrin, konnte die Nitratexkretion in A. aerophoba vollständig gehemmt werden. Im Gegensatz zu bakterienhaltigen Schwämmen zeigten sogenannte bakterienfreie Schwämme erwartungsgemäß keine Nitratausscheidung. Das 16S rRNA- und das amoA-Gen wurden als molekulare Marker verwendet, um nitrifizierende Mikroorganismen in Schwämmen phylogenetisch zu identifizieren. Es konnten zahlreiche 16S rRNA-Gene aus insgesamt sechs Schwammarten inklusive Aplysina aerophoba amplifiziert und dem marinen Nitrosospira Cluster 1 zugeordnet werden. Aus A. aerophoba konnten auch Nitrosospira amoA-Gensequenzen gewonnen werden. Archaeale 16S rRNA- und amoA-Gensequenzen wurden ebenfalls aus A. aerophoba gewonnen, wobei die 16S rRNA-Gene mit anderen aus Schwämmen stammenden Sequenzen ein Schwamm-spezifisches Cluster innerhalb der Crenarchaea Gruppe I.1A bildeten. Unter Verwendung spezifischer Fluoreszenz-markierter 16S rRNA Sonden konnten den Nitrosospira Cluster 1 und Crenarchaea Gruppe 1 zugehörige Zellen innerhalb des mikrobiellen Konsortiums aus A. aerophoba nachgewiesen werden. Basierend auf der geschätzten Menge nitrifizierender Mikroben in der Schwammmesohylmatrix und den Nitratexkretionsraten wurde eine zellspezifische Ammoniakoxidationsrate von 1,6 fmol Zelle-1 h-1 errechnet. Der Nachweis von 16S rRNA- oder funktionellen Genen des anaeroben mikrobiellen N-Kreislaufs in A. aerophoba verlief negativ. Darüber hinaus wurde eine in vorherigen Arbeiten aus dem mit A. aerophoba assoziierten mikrobiellen Konsortium erstellte Metagenombank auf das Vorhandensein von funktionellen (amoA) Nitrosospira- und Crenarchaea-Genen untersucht. Aus der Sequenzierung des archaealen Metagenomklons 58F6 resultierte die Sequenz des kompletten AMO-Operons eines möglicherweise Schwamm-spezifischen Crenarchaeoten. Diese Ergebnisse liefern erste funktionelle Einblicke in die komplexen Stoffflüsse und Wechselwirkungen zwischen Schwämmen und den mit ihnen assoziierten mikrobiellen Konsortien. Aufgrund dieser Arbeit wurde ein Modell des Stickstoffkreislaufs in A. aerophoba erstellt, welches die Mikroorganismen mit möglichen Stoffwechselfunktionen in dem Wirtsschwamm verknüpft. Diese Arbeit trägt zu dem Informationsstand über die Interaktionen zwischen Schwämmen und Mikroorganismen bei und leistet einen Beitrag zur Aufklärung des Stickstoffkreislaufs in A. aerophoba.
Die sekretorischen Aspartatproteasen (Saps) des Hefepilzes Candida albicans gelten als wichtiger Virulenzfaktor dieses opportunistischen Krankheitserregers. Die zehn Sap-Isoenzyme werden von einer Genfamilie codiert, deren Vertreter (SAP1-SAP10) in der Vergangenheit bereits intensiv untersucht wurden. SAP-Expressionsanalysen und die Charakterisierung von sap-Deletionmutanten, die in einem auxotrophen Laborstamm hergestellt wurden, führten aber zu teilweise widersprüchlichen Ergebnissen, weshalb die Rolle der einzelnen Proteine bis heute nicht zweifelsfrei aufgeklärt ist. Eine differentielle Expression der SAP-Gene in unterschiedlichen Stadien einer Infektion wurde jedoch in vielen unabhängigen Studien gezeigt. In der vorliegenden Arbeit wurde zunächst die Expression der Gene SAP1-SAP6 in einem etablierten in vitro-Modell einer Candida-Vaginitis untersucht, das auf der Infektion von rekonstituiertem humanem Epithel (RHE) basiert. Dazu wurden Reporterstämme verwendet, die bereits früher für Expressionsanalysen in verschiedenen in vivo-Infektionsmodellen in Mäusen eingesetzt worden waren. Dies erlaubte es einerseits unterschiedliche Nachweismethoden zur SAP-Genexpression in einem standardisierten Modell zu vergleichen und andererseits das Expressionsmuster der SAP-Gene in einem in vitro-Infektionsmodell mit den Ergebnissen von in vivo-Infektionsexperimenten zu korrelieren. Es zeigte sich in Übereinstimmung mit Ergebnissen früherer in vivo-Experimente in Mäusen, dass bei der Infektion des vaginalen Gewebes vor allem das SAP5-Gen induziert wurde. Allerdings weicht dieses Ergebnis deutlich von Ergebnissen anderer Studien ab, die mit Hilfe anderer Methoden ein ungleiches Muster der SAP-Expression detektierten. Um die Rolle der Gene SAP1-SAP6 bei der Infektion genauer zu untersuchen, wurden deshalb Mutanten hergestellt, in denen einzelne oder mehrere SAP-Gene mit Hilfe einer neuartigen Mutagenesestrategie erstmals aus dem Genom eines wildtypischen C. albicans-Stammes deletiert wurden. Überraschenderweise konnte sowohl in Einzelmutanten der Gene SAP1-SAP6 als auch in sap1 sap2 sap3- und sap4 sap5 sap6-Triplemutanten keine verminderte Fähigkeit zur Invasion und Schädigung von humanem Gewebe in RHE festgestellt werden. Eine in früheren Arbeiten beschriebene abgeschwächte Virulenz solcher Mutanten konnte auch in einem murinen Modell für eine disseminierende Infektion nicht beobachtet werden. Die Sekretion von Aspartatproteasen ermöglicht es C. albicans Proteine als alleinige Stickstoffquelle zum Wachstum zu verwenden. Unter diesen Bedingungen wird spezifisch die Expression des SAP2-Gens induziert; jedoch ist über die Mechanismen dieser Regulation noch wenig bekannt. Aus diesem Grund wurden in dieser Arbeit Promotor-Deletionsanalysen des SAP2-Gens und des mit diesem co-regulierten Oligopeptidtransportergens OPT1 durchgeführt. Es zeigte sich, dass unterschiedliche Bereiche innerhalb der 3,5 kb großen regulatorischen Region von SAP2 gemeinsam eine Expression dieses Gens unter induzierenden Bedingungen ermöglichen. Für das OPT1-Gen konnte eine regulatorische Region eingegrenzt werden, die für die Expression dieses Gens essentiell ist. In den für die Expression von SAP2 und OPT1 wichtigen Regionen wurden ähnliche Sequenzen gefunden, die als Bindungsstellen für regulatorische Faktoren dienen könnten. In dieser Arbeit wurden neue Erkenntnisse zur Regulation und Bedeutung der sekretierten Aspartatproteasen von C. albicans erhalten. Um eine endgültige Bewertung der Rolle dieser Enzyme in der Virulenz des Erregers zu ermöglichen, sind jedoch noch weitere detaillierte Studien unter Verwendung verschiedenster Infektionsmodelle nötig.
Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are exploited by human-specific pathogens to anchor themselves to or invade host cells. Interestingly, human granulocytes express a specific isoform, CEACAM3, that can direct efficient, opsonin-independent phagocytosis of CEACAM-binding Neisseria, Moraxella and Haemophilus species. As opsonin-independent phagocytosis of CEACAM-binding Neisseria depends on Src-family protein tyrosine kinase (PTK) phosphorylation of the CEACAM3 cytoplasmic domain, we hypothesized that an SH2-containing protein might be involved in CEACAM3-initiated, phagocytosis-promoting signals. Accordingly, we screened glutathione-S-transferase (GST) fusion proteins containing SH2 domains derived from a panel of signaling and adapter molecules for their ability to associate with CEACAM3. In vitro pull-down assays demonstrated that the SH2 domain of the adapter molecule Nck (GST-Nck SH2), but not other SH2 domains such as the Grb2 SH2 domain, interact with CEACAM3 in a phosphotyrosine-dependent manner. Either deletion of the cytoplasmic tail of CEACAM3, or point-mutation of a critical arginine residue in the SH2 domain of Nck (GST-NckSH2R308K) that disrupts phosphotyrosine binding, both abolished CEACAM3-Nck-SH2 interaction. Upon infection of human cells with CEACAM-binding Neisseria, full-length Nck comprising an SH2 and three SH3 domains co-localized with tyrosine phosphorylated CEACAM3 and associated bacteria as analyzed by immunofluorescence staining and confocal microscopy. In addition, Nck could be detected in CEACAM3 immunoprecipitates confirming the interaction in vivo. Importantly, overexpression of a GFP-fusion protein of the isolated Nck SH2 domain (GFP-Nck-SH2), but not GFP or GFP-Nck SH2 R308K reduced CEACAM3-mediated phagocytosis of CEACAM-binding Neisseria suggesting that the adaptor molecule Nck plays an important role in CEACAM3-initiated signaling leading to internalization and elimination of human-specific pathogens.
Cutaneous leishmaniasis is an infectious disease that is endemic especially in tropical and desert regions with an incidence of 1.5 million cases per year and a prevalence of 12 million people infected worldwide. The infection can be caused by the intracellular parasite Leishmania major. The disease has been studied extensively in the murine model. It has become apparent that the induction of a class of interferon (IFN)--producing CD4+ T helper cells (TH1 cells) that activate macrophages to kill the parasites they harbor is desicive for the establishment of immunity. The redirection of the host’s immune response towards a protective TH1 phenotype will also be the key to an effective vaccine. Dendritic cells (DC) loaded with leishmanial antigens ex vivo were lately described as vaccines against L. major infections. One single recombinant Leishmania antigen, LeIF (Leishmania homologue of eukaryotic ribosomal initiation factor 4a), which was identified as a protein that stimulates DC to secrete interleukin (IL)-12 and discussed as a pattern-associated molecular pattern (PAMP), was found to mediate a protective TH1-dependent effect when used for pulsing of DC. The application of recombinant proteins is tied to many disadvantages, which is why other methods of antigen administration have been developed. RNA electroporation of DC has recently emerged from tumor research as a safe and versatile method of antigen delivery, by which a large number of RNA molecules encoding a specific antigen gains access to the cytosol of DC by an electrical impulse. The present study describes, for the first time, transfection of DC with RNA encoding a molecularly defined parasite antigen. Initially, a standardized protocol for RNA transfection was established, using the enhanced green fluorescent protein (EGFP) as reporter antigen. EGFP-RNA was well translatable in an in vitro translation system, and both a DC cell line (fetal skin-derived DC; FSDC) and murine primary bone marrow-derived DC (BMDC) could be transfected efficiently, with a yield of up to 90% and 75%, respectively. In both cell types, maximal transfection efficiency was attained with 20 µg RNA and could not be further increased with larger amounts of RNA. The level of antigen expression, measured as the mean fluorescence intensity (MFI) by flow cytometry, was directly proportional to the amount of RNA used for transfection. In FSDC, transfection efficiency and MFI were generally higher than in BMDC when the same amounts of RNA were used. Furthermore, the kinetics was shown to be sensitive to treatment with lipopolysaccharide (LPS): the expression peak was higher and was reached sooner, followed by a more rapid decline. In transfection experiments with LeIF, two variants of LeIF-RNA were used: LeIF(fl)-RNA, encoding the complete LeIF sequence, and LeIF(226)-RNA, encoding only the aminoterminal half of the LeIF sequence (226 amino acids), the immunogenic part of LeIF. Only LeIF(fl) was detectable by Western Blot in whole cell lysates of BMDC after LeIF(fl)-RNA transfection, whereas LeIF(226) could never be detected in LeIF(226)-transfected BMDC. However, as both constructs were well translatable in a cell-free system, the failure to detect LeIF(226) in BMDC lysates did not represent a failure in RNA translation, but rather a rapid antigen degradation. It was therefore expected that LeIF(226)-transfected BMDC should nevertheless be able to present LeIF(226)-derived antigenic peptides to T cells from BALB/c mice primed with recombinant LeIF (rLeIF). This hypothesis was confirmed by measuring IFN- production in BMDC-T cell co-incubation assays, showing that rLeIF-pulsed, LeIF(226)- and LeIF(fl)-transfected day 7 BMDC did indeed activate T cells from LeIF-immunized mice in an antigen-specific manner. In contrast, IL-4 was not produced, which was consistent with the fact that T cells found in lymph nodes from LeIF-primed mice are primarily of the TH1 type. In the supernatants of LeIF-transfected BMDC cultures, in contrast to rLeIF-pulsed BMDC, the proinflammatory cytokines IL-1β, IL-6, IL-10 and IL-12 were not detected. This effect was not due to the electroporation procedure, as cytokine production by BMDC electroporated with rLeIF was only partially impaired. Also, the expression levels of CD86 were lower upon LeIF transfection than after pulsing with rLeIF. Thus, LeIF transfection did not induce maturation of DC. In conclusion, LeIF-transfected BMDC may have acted as semi-mature antigen-specific tolerance inducers, with regulatory T cells as responders. The effect of LeIF transfection on the immunostimulatory capacity of BMDC was not significantly increased when day 8 or 9 BMDC were used. However, day 8, and even more day 9 BMDC pulsed with rLeIF mounted a vigorous T cell response. Day 9 BMDC were able to activate naïve T cells. In conclusion, before a strong T cell response against LeIF can be induced, DC need to – besides presenting antigen and expressing co-stimulatory molecules – exhibit a susceptibility to the innate signaling molecule LeIF which is linked to their maturation age. This third signal is provided by extracellular rLeIF, but it is not conveyed – or is suppressed – by intracellular LeIF after LeIF-RNA transfection. Furthermore, electroporation of rLeIF abrogated IL-12 production by BMDC completely, the production of IL-1 was reduced with higher antigen doses, and the production of IL-10 was partially increased. The IL-6 production was unaffected. This altered cytokine profile suggests that LeIF as a PAMP might have a bipartite nature: besides exhibiting the capacity to stimulate IL-12 production upon extracellular presence, thereby enhancing host resistance against L. major, LeIF could also contribute to parasitic host evasion mechanisms from intracellular compartments of DC, possibly by interfering with mitogen-activated protein (MAP) kinase signaling pathways. Thus, the adjuvant properties of LeIF depend both on its mode of delivery (transfection with RNA vs. pulsing with the recombinant protein) and the targeted compartment (extra- vs. intracellular). From this work, it can be summarized that BMDC are well transfectable with a parasite antigen. The antigen is processed and presented, but it is not recognized as a PAMP by DC. Hence, transfection with antigen-encoding mRNA by itself does not convey all necessary signals for the elicitation of a potent immune response.
Ziel dieser Arbeit war es, eine Reihe von 17 ausgewählten Kohlehydraten auf deren Fähigkeit zu bewerten, die Adhärenz humanpathogener Enteritis-/Diärrhoe-Erreger zu reduzieren oder gänzlich zu verhindern. Gestestet wurde die Adhärenz an Kryoschnitten humaner Darmbiopsien. Hierbei wurde die bakterielle Adhäsion ohne Zusatz der Kohlehydrate bestimmt und als Vergleichswert gegenüber dem Ansatz mit Zusatz der Kohlehydrate herangezogen. Dabei kamen Stämme folgender Spezies zum Einsatz: entero-aggregative E. coli, entero-hämorrhagische E. coli, entero-pathogene E. coli, entero-toxigene E. coli, Salmonella enterica Serovar Typhimurium und Shigella flexneri. Als Positiv-Kontrolle wurde Citrobacter freundii verwendet.
Neisseria meningitidis und Neisseria gonorrhoeae sind humanpathogene Vertreter der Gattung Neisseria. Diese Erreger verfügen über eine Reihe von Virulenzfaktoren, um erfolgreich den menschlichen Körper zu besiedeln. Dabei spielen die neisseriellen OpaCEA-Proteine eine wichtige Rolle. Diese Adhäsine binden an bestimmte Rezeptoren der humanen CEACAM-Familie, die auf unterschiedlichen Zelltypen im menschlichen Körper exprimiert werden. Die Interaktion der OpaCEA-Proteine mit der stark konservierten aminoterminalen Domäne von bestimmten CEACAMs führt zu einer Aufnahme der Bakterien in die Zellen. Dort können sie, vor der humoralen Immunantwort geschützt, persistieren oder sich durch Transzytose in tiefer gelegene Gewebe weiter ausbreiten und letztendlich eine disseminierte Krankheit auslösen. Da CEACAMs eine entscheidende Rolle bei der Infektion mit pathogenen Neisserien spielen, wurde in der vorliegenden Arbeit die Interaktion dieser zellulären Rezeptoren mit pathogenen Neisserien und die daraus resultierenden molekularen Ereignisse auf bakterieller bzw. zellulärer Seite näher untersucht. Zunächst wurde eine neuartige Methode entwickelt, die im Gegensatz zu herkömmlichen Strategien eine schnelle und quantitative Erfassung von Neisserien-CEACAM-Interaktionen ermöglicht. Bei dieser Methode wurden GFP-fusionierte, lösliche aminoterminale CEACAM-Domänen eingesetzt, die spezifisch an OpaCEA-exprimierende Bakterien binden. Einzelne Bakterien einer Population, die mit den fluoreszierenden Rezeptordomänen assoziierten, konnten aufgrund ihrer erhöhten Fluoreszenz im Durchflußzytometer sehr schnell und quantitativ bestimmt werden. Diese Rezeptordomänen wurden außerdem als molekulares Werkzeug zur Erstellung eines OpaCEA-induzierten Transkriptionsprofils von Opa-positiven Meningokokken verwendet. Transkriptomanalysen mittels Mikroarrays zeigten, dass die Interaktion OpaCEA-exprimierender Meningokokken mit der löslichen, aminoterminalen Domäne von CEACAM1 eine Herrunterregulation von 56 Genen sowie eine Hochregulation von sieben Genen in Neisseria meningitidis MC58 zur Folge hatte. Dabei konnte ein hochreguliertes Gen identifiziert werden, dessen Genprodukt aufgrund seiner Homologie zu einem bakteriellen -Hämolysin möglicherweise virulenz-assoziiert ist. Die Erstellung dieses Transkriptionsprofils beruhte auf der Interaktion zwischen der aminoterminalen Domäne von CEACAM1 und seinen bis heute einzig bekannten neisseriellen Liganden, den OpaCEA-Proteinen. Bemerkenswerterweise konnte im Rahmen der vorliegenden Arbeit ein Opa-negativer Meningokokkenstamm isoliert werden, der ebenfalls an CEACAM1 bindet und von CEACAM1-exprimierenden Zellen internalisiert wird. Da dieser Meningokokkenstamm keine Opa-Proteine exprimierte, muß er über ein weiteres Adhäsin verfügen, das mit CEACAM1 assoziiert. Interessanterweise konnte gezeigt werden, dass für die CEACAM1-vermittelte Aufnahme dieser Opa-negativen Meningokokken mehrere extrazelluläre Domänen des Rezeptors notwendig sind. Im Gegensatz zur Aufnahme OpaCEA-exprimierender Bakterien war die aminoterminale Domäne essentiell, aber nicht ausreichend für diesen phagozytischen Vorgang, der unabhängig vom Aktinzytoskelett erfolgte. Auch bei Bindungsstudien mit löslichen CEACAM1 Konstrukten gab es Differenzen zwischen den opaquen und nicht-opaquen Bakterienstämmen. So zeigte sich, dass im Gegensatz zu OpaCEA-exprimierenden Meningokokken, die mit monomeren Formen des löslichen Rezeptors assoziieren konnten, Opa-negativen Meningokokken nur mit multimerisierten Formen des Rezeptors interagierten. CEACAM1 stellt den einzigen Rezeptor aus der CEACAM-Familie dar, mit dem Opa-negative Meningokokken interagieren können. Demgegenüber assoziieren OpaCEA-exprimierende Neisserien mit mehreren Mitgliedern der CEACAM-Familie, unter anderem mit CEACAM3. In der vorliegenden Arbeit konnte gezeigt werden, dass die c-Jun N-terminale Kinase an Signaltransduktionswegen, die durch Interaktion von OpaCEA-exprimierenden Neisserien mit CEACAM3 ausgelöst wurden, beteiligt ist. Erstaunlicherweise konnte durch Inhibition der c-Jun N-terminalen Kinase CEACAM3-vermittelte Aufnahme der opaquen Bakterien in die Zelle reduziert werden. Da die Aktivierung der c-Jun N-terminalen Kinase unabhängig von der Phosphorylierung der ITAM-ähnlichen Sequenz erfolgte, scheint dieses Molekül an einem neuartigen Signalweg beteiligt zu sein, der komplementär zu bereits bekannten CEACAM3-vermittelten Signalprozessen abläuft. Die in der vorliegenden Arbeit zusammengefassten Befunde liefern neue Einblicke in die Wechselwirkung zwischen pathogenen Neisserien und ihren Wirtszellen und können als Ausgangspunkt für interessante weiterführende Analysen dienen.