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The three closely related PUB proteins PUB22, PUB23 and PUB24 were described as important regulators for PTI signaling and plant immunity. To find cellular targets regulated by the action of the PUB triplet we performed a yeast two-hybrid screen to identify candidate target proteins of PUB22. We could identify Exo70B2 as a target protein of PUB22, which is ubiquitinated by the E3-ubiquitin ligase and consequently degraded in response to flg22 perception. The importance of Exo70B2 for immunity was shown by reverse genetics, demonstrating that exo70B2 mutants are impaired in PTI signaling and plant immunity.
Exo70B2 is one of 23 homologs of the yeast Exo70p in Arabidopsis thaliana, which is a subunit of an octameric protein complex, termed the exocyst. The exocyst complex is required for the tethering of post-Golgi vesicles to specific target membranes and thus an important component of intracellular vesicle trafficking. The elucidated function of Exo70B2 and its requirement for PTI signaling is a novel finding and similar functions had not yet been described for the exocyst complex or subunits thereof in plants. Additional target proteins of PUB22 are also predicted to be involved in vesicle trafficking processes, suggesting that PUB22 has specialized to regulate trafficking protein complexes required for PTI signaling.
Furthermore, the presented work suggests a mechanism for the regulation of Exo70B2 ubiquitination by PUB22. PUB22 was shown to be intrinsically instable due to its autocatalytic ubiquitination activity. Flg22 treatment induced the rapid post-translational stabilization of PUB22. This potentially enables the ligase to efficiently interact with Exo70B2, resulting in its polyubiquitination and 26S-proteasome-dependent turnover.
Staphylococcus aureus (SA) causes nosocomial infections including life threatening sepsis by multi-resistant strains (MRSA). It has the ability to form biofilms to protect it from the host immune system and from anti staphylococcal drugs. Biofilm and planctonic life style is regulated by a complex Quorum-Sensing (QS) system with agr as a central regulator. To study biofilm formation and QS mechanisms in SA a Boolean network was build (94 nodes, 184 edges) including two different component systems such as agr, sae and arl. Important proteins such as Sar, Rot and SigB were included as further nodes in the model. System analysis showed there are only two stable states biofilm forming versus planctonic with clearly different subnetworks turned on. Validation according to gene expression data confirmed this. Network consistency was tested first according to previous knowledge and literature. Furthermore, the predicted node activity of different in silico knock-out strains agreed well with corresponding micro array experiments and data sets. Additional validation included the expression of further nodes (Northern blots) and biofilm production compared in different knock-out strains in biofilm adherence assays. The model faithfully reproduces the behaviour of QS signalling mutants. The integrated model allows also prediction of various other network mutations and is supported by experimental data from different strains. Furthermore, the well connected hub proteins elucidate how integration of different inputs is achieved by the QS network. For in silico as well as in vitro experiments it was found that the sae-locus is also a central modulator of biofilm production. Sae knock-out strains showed stronger biofilms. Wild type phenotype was rescued by sae complementation. To elucidate the way in which sae takes influence on biofilm formation the network was used and Venn-diagrams were made, revealing nodes regulated by sae and changed in biofilms. In these Venn-diagrams nucleases and extracellular proteins were found to be promising nodes. The network revealed DNAse to be of great importance. Therefore qualitatively the DNAse amount, produced by different SA mutants was measured, it was tried to dissolve biofilms with according amounts of DNAse and the concentration of nucleic acids, proteins and polysaccharides were measured in biofilms of different SA mutants.
With its thorough validation the network model provides a powerful tool to study QS and biofilm formation in SA, including successful predictions for different knock-out mutant behaviour, QS signalling and biofilm formation. This includes implications for the behaviour of MRSA strains and mutants. Key regulatory mutation combinations (agr–, sae–, sae–/agr–, sigB+, sigB+/sae–) were directly tested in the model but also in experiments. High connectivity was a good guide to identify master regulators, whose detailed behaviour was studied both in vitro and in the model. Together, both lines of evidence support in particular a refined regulatory role for sae and agr with involvement in biofilm repression and/or SA dissemination. With examination of the composition of different mutant biofilms as well as with the examination of the reaction cascade that connects sae to the biofilm forming ability of SA and also by postulating that nucleases might play an important role in that, first steps were taken in proving and explaining regulatory links leading from sae to biofilms. Furthermore differences in biofilms of different mutant SA strains were found leading us in perspective towards a new understanding of biofilms including knowledge how to better regulate, fight and use its different properties.
Malignant melanoma is the most severe form of all skin cancers with a particular poor prognosis once metastases have developed. Angiogenesis, the formation of new blood vessels, is a prominent feature of human melanoma, which have angiogenic activity already early in development. This is at least partly ascribed to the action of MAPK- and PI3K pathways which are hyperactivated in most melanoma. Animal models which combine in depth in vivo examinations with the opportunity to perform small molecular screens are well suited to gain a more detailed insight into how this type of cancer modulates its angiogenic program. Here, a first transgenic melanoma angiogenesis model was established in the fish species Oryzias latipes (Japanese medaka). In this model, tumors are generated by the pigment cell-specific expression of the oncogenic receptor tyrosine kinase Xmrk. Xmrk is a mutated version of the fish Egfp. Furthermore, to get an angiogenesis model, a medaka line with endothelial cell specific GFP expression was used. By using crosses between these Xmrk- and GFP transgenic fishes, it was shown that angiogenesis occurs in a reactive oxygen species- and NF-κB-dependent manner, but was hypoxia-independent. It was observed that blood vessel sprouting and branch point formation was elevated in this model and furthermore that sprouting could even be induced by single transformed cells. The mouse melanocytes expressing the oncogenic receptor tyrosine kinase Xmrk as well human melanoma cells, which display various oncogenic alterations, produced pro-angiogenic factors, most prominently angiogenin, via NF-κB signaling. Furthermore, inhibiting NF-κB action prevented tumor angiogenesis and even led to the regression of existing tumor blood vessels. In summary, the present medaka melanoma angiogenesis model displays a high sensitivity for angiogenesis detection and is perfectly suited as in vivo model for the testing of anti-angiogenesis inhibitors, as exemplified by the NF-kappaB inhibitor.
Furthermore, results indicate that it might be a promising anti-tumor strategy to target signaling pathways such as the NF-κB pathway which are able to induce angiogenesis-dependent as well as -independent pro-tumorigenic effects.
Biological systems such as cells or whole organisms are governed by complex regulatory networks of transcription factors, hormones and other regulators which determine the behavior of the system depending on internal and external stimuli. In mathematical models of these networks, genes are represented by interacting “nodes” whose “value” represents the activity of the gene.
Control processes in these regulatory networks are challenging to elucidate and quantify. Previous control centrality metrics, which aim to mathematically capture the ability of individual nodes to control biological systems, have been found to suffer from problems regarding biological plausibility.
This thesis presents a new approach to control centrality in biological networks. Three types of network control are distinguished: Total control centrality quantifies the impact of gene mutations and identifies potential pharmacological targets such as genes involved in oncogenesis (e.g. zinc finger protein GLI2 or bone morphogenetic proteins in chondrocytes). Dynamic control centrality describes relaying functions as observed in signaling cascades (e.g control in mouse colon stem cells). Value control centrality measures the direct influence of the value of the node on the network (e.g. Indian hedgehog as an essential regulator of proliferation in chondrocytes). Well-defined network manipulations define all three centralities not only for nodes, but also for the interactions between them, enabling detailed insights into network pathways.
The calculation of the new metrics is made possible by substantial computational improvements in the simulation algorithms for several widely used mathematical modeling paradigms for genetic regulatory networks, which are implemented in the regulatory network simulation framework Jimena created for this thesis.
Applying the new metrics to biological networks and artificial random networks shows how these mathematical concepts correspond to experimentally verified gene functions and signaling pathways in immunity and cell differentiation. In contrast to controversial previous results even from the Barabási group, all results indicate that the ability to control biological networks resides in only few driver nodes characterized by a high number of connections to the rest of the network. Autoregulatory loops strongly increase the controllability of the network, i.e. its ability to control itself, and biological networks are characterized by high controllability in conjunction with high robustness against mutations, a combination that can be achieved best in sparsely connected networks with densities (i.e. connections to nodes ratios) around 2.0 - 3.0.
The new concepts are thus considerably narrowing the gap between network science and biology and can be used in various areas such as system modeling, plausibility trials and system analyses.
Medical applications discussed in this thesis include the search for oncogenes and pharmacological targets, as well their functional characterization.
The genetic information encoded with in the genes are transcribed and translated to give rise to
the functional proteins, which are building block of a cell. At first, it was thought that the
regulation of gene expression particularly occurs at the level of transcription by various
transcription factors. Recent discoveries have shown the vital role of gene regulation at the level
of RNA also known as post-transcriptional gene regulation (PTGR). Apart from non-coding RNAs
e.g. micro RNAs, various RNA binding proteins (RBPs) play essential role in PTGR. RBPs have
been implicated in different stages of mRNA life cycle ranging from splicing, processing,
transport, localization and decay. In last 20 years studies have shown the presence of hundreds
of RBPs across eukaryotic systems many of which are widely conserved. Given the rising number
of RBPs and their link to human diseases it is quite evident that RBPs have major role in cellular
processes and their regulation. The current study is aimed to describe the so far unknown
molecular mechanism of CCHC-type Zinc Finger Nucleic Acid Binding Protein (CNBP/ZNF9)
function in vivo.
CNBP is ubiquitously expressed across various human tissues and is a highly conserved RBP in
eukaryotes. It is required for embryonic development in mammals and has been implicated in
transcriptional as well as post-transcriptional gene regulation; however, its molecular function
and direct target genes remain elusive. Here, we use multiple systems-wide approaches to
identify CNBP targets and document the consequences of CNBP binding. We established CNBP as
a cytoplasmic RNA-binding-protein and used Photoactivatable Ribonucleoside Enhanced
Crosslinking and Immunoprecipitation (PAR-CLIP) to identify direct interactions of CNBP with
4178 mRNAs. CNBP preferentially bound a G-rich motif in the target mRNA coding sequences.
Functional analyses, including ribosome profiling, RNA sequencing, and luciferase assays
revealed the CNBP mode of action on target transcripts. CNBP binding was found to increase the
translational efficiency of its target genes. We hypothesize that this is consistent with an RNA
chaperone function of CNBP helping to resolve secondary structures, thus promoting
translation. Altogether this study provides a novel mechanism of CNBP function in vivo and acts
as a step-stone to study the individual CNBP targets that will bring us closer to understand the
disease onset.
Recent advances in the field of cancer immunotherapy have enabled this therapeutic approach to enter the mainstream of modern cancer treatment. In particular, adoptive T cell therapy (ACT) is a potentially powerful immunotherapy approach that relies on the administration of tumor-specific T cells into the patient. There are several strategies to obtain tumor-reactive cytotoxic T lymphocytes (CTLs), which have already been shown to induce remarkable responses in the clinical setting. However, there are concerns and limitations regarding the conventional approaches to obtain tumor-reactive T cells, such as accuracy of the procedure and reproducibility. Therefore, we aimed to develop two approaches to improve the precision and efficacy of tumor-reactive T cells therapy. These two techniques could constitute effective, safe and broadly applicable alternatives to the conventional methods for obtaining tumor-specific CTLs.
The first approach of this study is the so called “Doublet Technology”. Here, we demonstrate that peptide-human leukocyte antigen-T cell receptor (pHLA-TCR) interactions that involve immune reactive peptides are stable and strong. Therefore, the CTLs that are bound by their TCR to tumor cells can be selected and isolated through FACS-based cell sorting taking advantage of this stable interaction between the CTLs and the target cells. The CTLs from acute myeloid leukemia (AML) patients obtained with this technique show cytolytic activity against blast cells suggesting a potential clinical use of these CTLs. “Doublet Technology” offers a personalized therapy in which there is no need for a priori knowledge of the exact tumor antigen.
The second approach of this study is the Chimeric Antigen Receptor (CAR) Technology. We design several CARs targeting the B-Cell Maturation Antigen (BCMA). BCMA CAR T cells show antigen-specific cytolytic activity, production of cytokines including IFN-γ and IL-2, as well as productive proliferation. Although we confirm the presence of soluble BCMA in serum of multiple myeloma (MM) patients, we demonstrate that the presence of soluble protein does not abrogate the efficacy of BCMA CAR T cells suggesting that BCMA CAR T cells can be used in the clinical setting to treat MM patients. The high antigen specificity of CAR T cells allows efficient tumor cell eradication and makes CAR Technology attractive for broadly applicable therapies.
Desert ants of the genus Cataglyphis (Formicinae) are widely distributed in arid
areas of the palearctic ecozone. Their habitats range from relatively cluttered environments in the Mediterranean area to almost landmark free deserts. Due to their
sophisticated navigational toolkit, mainly based on the sky-compass, they were
studied extensively for the last 4 decades and are an exceptional model organism
for navigation. Cataglyphis ants exhibit a temporal polyethism: interior workers
stay inside the dark nest and serve as repletes for the first ∼2 weeks of their adult
life (interior I). They then switch to nursing and nest maintenance (interior II)
until they transition to become day-active outdoor foragers after ∼4 weeks. The
latter switch in tasks involves a transition phase of ∼2-3 days during which the
ants perform learning and orientation walks. Only after this last phase do the ants
start to scavenge for food as foragers.
In this present thesis I address two main questions using Cataglyphis desert ants
as a model organism:
1. What are the underlying mechanisms of temporal polyethism?
2. What is the neuronal basis of sky-compass based navigation in Cataglyphis
ants?
Neuropeptides are important regulators of insect physiology and behavior and as
such are promising candidates regarding the regulation of temporal polyethism in
Cataglyphis ants. Neuropeptides are processed from large precursor proteins and undergo substantial post-translational modifications. Therefore, it is crucial to biochemically identify annotated peptides. As hardly any peptide data are available
for ants and no relevant genomic data has been recorded for Cataglyphis, I started
out to identify the neuropeptidome of adult Camponotus floridanus (Formicinae)
workers (manuscript 1). This resulted in the first neuropeptidome described in an
ant species – 39 neuropeptides out of 18 peptide families. Employing a targeted
approach, I identified allatostatin A (AstA), allatotropin (AT), short neuropeptide
F (sNPF) and tachykinin (TK) using mass spectrometry and immunohistology to
investigate the distribution of AstA, AT and TK in the brain (manuscript 2). All
three peptides are localized in the central complex, a brain center for sensory integration and high-order control of locomotion behavior. In addition, AstA and
TK were also found in visual and olfactory input regions and in the mushroom
bodies, the centers for learning and memory formation. Comparing the TK immunostaining in the brain of 1, 7 and 14 days old dark kept animals revealed that
the distribution in the central complex changes, most prominently in the 14 day
old group. In the Drosophila central complex TK modulates locomotor activity
levels. I therefore hypothesize that TK is involved in the internal regulation of the
interior I–interior II transition which occurs after ∼2 weeks of age.
I designed a behavioral setup to test the effect of neuropeptides on the two traits:
’locomotor activity level’ and ’phototaxis’ (manuscript 3). The test showed that
interior I ants are less active than interior II ants, which again are less active
than foragers. Furthermore, interior ants are negatively phototactic compared to
a higher frequency of positive phototaxis in foragers. Testing the influence of AstA
and AT on the ants’ behavior revealed a stage-specific effect: while interior I behavior is not obviously influenced, foragers become positively phototactic and more
active after AT injection and less active after AstA injection. I further tested the
effect of light exposure on the two behavioral traits of interior workers and show that it rises locomotor activity and results in decreased negative phototaxis in
interior ants. However, both interior stages are still more negatively phototactic
than foragers and only the activity level of interior II ants is raised to the forager
level. These results support the hypothesis that neuropeptides and light influence
behavior in a stage-specific manner.
The second objective of this thesis was to investigate the neuronal basis of skycompass navigation in Cataglyphis (manuscript 4). Anatomical localization of the
sky-compass pathway revealed that its general organization is highly similar to
other insect species. I further focused on giant synapses in the lateral complex,
the last relay station before sky-compass information enters the central complex.
A comparison of their numbers between newly eclosed ants and foragers discloses
a rise in synapse numbers from indoor worker to forager, suggesting task-related
synaptic plasticity in the sky-compass pathway. Subsequently I compared synapse
numbers in light preexposed ants and in dark-kept, aged ants. This experiment
showed that light as opposed to age is necessary and sufficient to trigger this rise
in synapse number. The number of newly formed synapses further depends on the
spectral properties of the light to which the ants were exposed to.
Taken together, I described neuropeptides in C. floridanus and C. fortis, and provided first evidence that they influence temporal polyethism in Cataglyphis ants.
I further showed that the extent to which neuropeptides and light can influence
behavior depends on the animals’ state, suggesting that the system is only responsive under certain circumstances. These results provided first insight into the
neuronal regulation of temporal polyethism in Cataglyphis. Furthermore, I characterized the neuronal substrate for sky-compass navigation for the first time in
Cataglyphis. The high level of structural synaptic plasticity in this pathway linked
to the interior–forager transition might be particularly relevant for the initial calibration of the ants’ compass system.
Marine sponge-associated actinomycetes are reservoirs of diverse natural products with novel biological activities. Their antibiotic potential has been well explored against a range of Gram positive and negative bacteria. However, not much is known about their anti-infective or anti-virulence potential against human pathogens. This Ph.D. project aimed to investigate the anti-infective (anti-Shiga toxin and anti-biofilm) potential of sponge-derived actinobacteria through identification and isolation of their bioactive metabolites produced and characterizing their mechanism of action by transcriptomics. This thesis is divided into three studies with the overall objective of exploring the anti-infective efficacy of actinomycetes-derived extracts and compound(s) that could possibly be used as future therapeutics.
The first study deals with investigation on the anti-Shiga toxin effects of sponge-associated actinomycetes. Diarrheal infections pose a huge burden in several developing and developed countries. Diarrheal outbreaks caused by Enterohemorrhagic Escherichia coli (EHEC) could lead to life-threatening complications like gastroenteritis and haemolytic uremic syndrome (HUS) if left untreated. Shiga toxin (Stx) produced by EHEC is a major virulence factor that negatively affects the human cells, leading them to death via apoptosis. Antibiotics are not prescribed against EHEC infections since they may enhance the risk of development of HUS by inducing the production and release of Stx from disintegrating bacteria and thereby, worsening the complications. Therefore, an effective drug that blocks the Stx production without affecting the growth needs to be urgently developed. In this study, the inhibitory effects of 194 extracts and several compounds originating from a collection of marine sponge-derived actinomycetes were evaluated against the Stx production in EHEC strain EDL933 with the aid of Ridascreen® Verotoxin ELISA assay kit. It was found that treatment with the extracts did not lead to significant reduction in Stx production. However, strepthonium A isolated from the culture of Streptomyces sp. SBT345 (previously cultivated from the Mediterranean sponge Agelas oroides) reduced the Stx production (at 80 μM concentration) in EHEC strain EDL933 without affecting the bacterial growth. The structure of strepthonium A was resolved by spectroscopic analyses including 1D and 2D-NMR, as well as ESI-HRMS and ESI-HRMS2 experiments. This demonstrated the possible application of strepthonium A in restraining EHEC infections.
VI
In the second study, the effect of marine sponge-associated actinomycetes on biofilm formation of staphylococci was assessed. Medical devices such as contact lenses, metallic implants, catheters, pacemakers etc. are ideal ecological niches for formation of bacterial biofilms, which thereby lead to device-related infections. Bacteria in biofilms are multiple fold more tolerant to the host immune responses and conventional antibiotics, and hence are hard-to-treat. Here, the anti-biofilm potential of an organic extract derived from liquid fermentation of Streptomyces sp. SBT343 (previously cultivated from the Mediterranean sponge Petrosia ficiformis) was reported. Results obtained in vitro demonstrated its anti-biofilm (against staphylococci) and non-toxic nature (against mouse macrophage (J774.1), fibroblast (NIH/3T3) and human corneal epithelial cell lines). Interestingly, SBT343 extract could inhibit staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces without affecting the bacterial growth. High Resolution Fourier Transform Mass Spectrometry (HR-MS) analysis indicated the complexity and the chemical diversity of components present in the extract. Preliminary physio-chemical characterization unmasked the heat stable and non-proteinaceous nature of the active component(s) in the extract. Finally, fractionation experiments revealed that the biological activity was due to synergistic effects of multiple components present in the extract.
In the third study, anti-biofilm screening of 50 organic extracts generated from solid and liquid fermentation of 25 different previously characterized sponge-derived actinomycetes was carried out. This led to identification of the anti-biofilm organic extract derived from the solid culture of Streptomyces sp. SBT348 (previously cultivated from the Mediterranean sponge Petrosia ficiformis). Bioassay-guided fractionation was employed to identify the active fraction Fr 7 in the SBT348 crude extract. Further purification with semi-preparative HPLC led to isolation of the bioactive SKC1, SKC2, SKC3, SKC4 and SKC5 sub-fractions. The most active sub-fraction SKC3 was found to be a pure compound having BIC90 and MIC values of 3.95 μg/ml and 31.25 μg/ml against S. epidermidis RP62A. SKC3 had no apparent toxicity in vitro on cell lines and in vivo on the greater wax moth Galleria melonella larvae. SKC3 was stable to heat and enzymatic treatments indicating its non-proteinaceous nature. HR-MS analysis revealed the mass of SKC3 to be 1258.3 Da. Structure elucidation of SKC3 with the aid of 1D and 2D-NMR data is currently under investigation. Further, to obtain insights into the mode of action of SKC3 on S. epidermidis RP62A, RNA sequencing was done. Transcriptome data revealed that SKC3 was recognized by RP62A at 20 min and SKC3 negatively interfered with the central metabolism of staphylococci at 3 h. Taken
VII
together, these findings suggest that SKC3 could be a lead structure for development of new anti-staphylococcal drugs.
Overall, the results obtained from this work underscore the anti-infective attributes of actinomycetes consortia associated with marine sponges, and their applications in natural product drug discovery programs.
Shiga toxin producing E. coli strains (STEC) are a great concern to human health. Upon an infection with as few as 100 bacteria, humans can develop disease symptoms ranging from watery to bloody diarrhea or even develop the hemolytic uremic syndrome (HUS). The major factor contributing to the disease symptoms is Shiga toxin (Stx) which can bind to the eukaryotic cells in the intestine of the human and induce cell death via apoptosis. Based, among other things, on the microbiota composition, the impact of STEC can vary. Some bacteria of the microbiota can interfere with the colonization of STEC strains in the first place. Others cannot impair the colonization but interfere with the toxin production and there are still others which are even infected by stx encoding phages, being released from STEC strains. Those previously harmless bacteria subsequently contribute to the toxin increase and worsen the disease progression. Since the genetic information of Stx is encoded on a prophage, antibiotic treatment of patients can lead to an increased toxin and stx-phage release and is therefore not recommended. Several STEC epidemics in different countries, which even resulted in the death of some patients, demonstrated that there is an urgent need for alternative treatment strategies.
The E. coli strain Nissle 1917 (EcN) has been used as a probiotic to treat gastrointestinal infections for more than 100 years. It harbors several fitness factors which contribute to the establishment of an intact intestinal barrier in the human gut. Moreover, studies with EcN unraveled that the probiotic E. coli can interfere with the colonization of STEC strains and their toxin production. This study aimed to investigate if EcN could be a possible alternative or supplementary treatment strategy for STEC infected patients, or a preventive treatment for the patient’s close contact persons.
Therefore, EcN was firstly investigated for a possible stx-prophage integration into its’s genome which would eliminate it from being a potential treatment due to the possibility of disease worsening. Despite the presence of the stx-phage surface receptor YaeT, EcN demonstrated a complete resistance towards the lysis and the lysogeny by stx-phages, which was proven by PCR, phage-plaque assays and phage enrichment approaches. Transcriptome data could unravel that a lambdoid prophage in the genome of EcN is involved in the resistance towards the phage infection. Other commensal E. coli tested presented a stx-phage resistance as well and in silico analysis revealed that all of them harbor a complete lambdoid prophage besides the stx-phage susceptible K-12 strain MG1655. We assume that the resistance of EcN towards a stx-phage infection is connected to the presence of an intact lambdoid prophage which interferes with superinfection.
Further experiments regarding the impact of the microcin negative EcN mutant SK22D towards STEC strains depicted that SK22D did not only interfere with the toxin production but also negatively regulated the transcription of the entire stx-prophage in coculture with all STEC strains tested (O157:H7, O26:H11, O145:H25, O103:H2, O111:H- and two O104:H4 isolates from the 2011 outbreak in Germany). This influence on the pathogenic factor production was evinced to be cell contact independent as SK22D could even interfere with the pathogenic factor production when being separated from the STEC strain EDL933 by a Transwell membrane with the pore size of 0.4 µm. From this data we concluded, that factor(s) released by SK22D interfere with the lysis of STEC strains by stabilizing the lysogenic state.
Another positive aspect of EcN towards the pathogenicity of STEC strains was encountered when EcN was incubated with isolated stx-phages. The probiotic strain could reduce the infectivity of the phages towards a MG1655 lysis from ~ 1e7 pfus/ml to 0 after 44 h of incubation. Various approaches to determine the characteristics of the factor(s) of EcN which are involved in the phage inactivation depicted it to be a heat resistant stationary phase protein on the surface of EcN, which could be a component of its biofilm.
Regarding the protective role of EcN we could further evince that SK22D was capable of interfering with the lysogenic K 12 mediated increase of Stx and stx phages. Lysogenic K-12 strains were characterized by a huge increase of Stx and stx-phage production. The presence of SK22D anyhow, could interfere with this K-12 mediated pathogenic factor increase. Transwell and stx phage infection kinetics led to the proposal that SK22D interfered with the stx-phage infection of K-12 strains in the first place rather than disturbing the lysis of lysogenic K 12. The protection from the phage infection could be due to the growth of K 12 strains within the SK22D culture, whereby the phage susceptible strains are masked from phage detection.
Summarizing, this work could underline the beneficial attributes of EcN towards the STEC pathogenicity in vitro. These results should be considered as pioneers for future in vivo studies to enable EcN medication as a supportive STEC infection treatment strategy.
Humans are continuously exposed to airborne spores of the saprophytic fungus Aspergillus fumigatus. In healthy individuals, local pulmonary host defence mechanisms can efficiently eliminate the fungus without any overt symptoms. In contrast, A. fumigatus causes devastating infections in immunocompromised patients. However, local host immune responses against A. fumigatus lung infections in immunocompromised conditions have remained largely elusive.
Given the dynamic changes in immune cell subsets within tissues upon immunosuppressive therapy, we dissected the spatiotemporal pulmonary immune response after A. fumigatus infection to reveal basic immunological events that fail to effectively control the invasive fungal disease. In different immunocompromised murine models, myeloid but not lymphoid cells were strongly recruited upon infection. Notably, neutrophils and macrophages were recruited to infected lungs in different immunosuppressed regimens. Other myeloid cells, particularly dendritic cells and monocytes were only recruited in the corticosteroid model after infection. Lymphoid cells, particularly CD4+ or CD8+ T-cells and NK cells were highly reduced upon immunosuppression and were not recruited after A. fumigatus infection. Importantly, adoptive CD11b+ myeloid cell transfer rescued immunosuppressed mice from lethal A. fumigatus infection. These findings illustrate that CD11b+ myeloid cells are critical for anti-A. fumigatus defence under immunocompromised conditions.
Despite improved antifungal agents, invasive A. fumigatus lung infections cause a high rate morbidity and mortality in neutropenic patients. Granulocyte transfusions have been tested as an alternative therapy for the management of high-risk neutropenic patients with invasive A. fumigatus infections. To increase the granulocyte yield for transfusion, donors are treated with corticosteroids. Yet, the efficacy of granulocyte transfusion and the functional defence mechanisms of granulocytes collected from corticosteroid treated donors remain largely elusive.
We aimed to assess the efficacy of granulocyte transfusion and functional defence mechanisms of corticosteroid treated granulocytes using mouse models.
In this thesis, we show that transfusion of granulocytes from corticosteroid treated mice did not protect cyclophosphamide immunosuppressed mice against lethal A. fumigatus infection in contrast to granulocytes from untreated mice. Upon infection, increased levels of inflammatory cytokines helped to recruit granulocytes to the lungs without any recruitment defects in corticosteroid treated and infected mice or in cyclophosphamide immunosuppressed and infected mice that have received the granulocytes from corticosteroid treated mice. However, corticosteroid treated human or mouse neutrophils failed to form neutrophil extracellular traps (NETs) in in vitro and in vivo conditions. Further, corticosteroid treated granulocytes exhibited impaired ROS production against A. fumigatus. Notably, corticosteroids impaired the β-glucan receptor Dectin-1 (CLEC7A) on mouse and human granulocytes to efficiently recognize and phagocytize A. fumigatus, which markedly impaired fungal killing. We conclude that corticosteroid treatment of granulocyte donors for increasing neutrophil yields or patients with ongoing corticosteroid treatment could result in deleterious effects on granulocyte antifungal functions, thereby limiting the benefit of granulocyte transfusion therapies against invasive fungal infections.