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Sonstige beteiligte Institutionen
- Genelux Corporation, San Diego Science Center, 3030 Bunker Hill Street, Suite 310, San Diego, California 92109, USA (1)
- MRB Forschungszentrum für Magnet-Resonanz-Bayern e.V., Am Hubland, D-97074 Würzburg (1)
- Research Center for Infectious Diseases (ZINF), University of Wuerzburg, Wuerzburg, Germany, (1)
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
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.
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.
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.
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.
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.
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.
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.
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.
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.