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Until today, more than 100 years after its first description in Italy, the highly pathogenic avian influenza virus (HPAIV) has not lost its fearsome character for wild birds, poultry and humans. On the contrary, the number of outbreaks with high casualty rates in wild birds and poultry has multiplied in recent years and cases of zoonotic infections are also increasingly reported from HPAI endemic areas. The epidemiology of these infections is complex and also involves surface water and possibly sediments of shallow standing waters, which could play a role as a vector medium and/or virus reservoir. The goal of this project was to expand current knowledge of the influence of water on the spread of AIV. As part of this project, we were able to ...
1. ...improve AIV detection methods using real time RT-PCR in terms of sensitivity and breadth of viruses detected. In addition, we succeeded in economizing the procedure so that fewer resources are required and results are obtained faster (publication I: [173]).
2. ...develop an ultrafiltration-based enrichment method for AIV from surface water and evaluate it with field samples from HPAI outbreak areas in wild bird habitats (Wadden Sea coast of Schleswig-Holstein) and previously unaffected regions (Antarctic Weddell Sea) (publication II: [174]). Furthermore, protocols for testing different environmental sample matrices for AIV screening were tested and compared to results of passive monitoring by dabbing diseased or dead wild birds. AIV was detected in more than half (61%) of 44 water samples. We received additional sediment samples from 36 of the 44 water samples. In 18 of 36 of the sediments tested, as well as in 4.16% of 1705 fecal samples tested AIV was detected. However, the studies of the environmental samples mostly yielded only generic AIV detections, with viral loads in the range of the detection limit. This massively hampered further investigations for sub- and pathotyping. In contrast, 79.41% of 68 samples from passive monitoring showed high to very high HPAIV viral loads which also allowed sub- and pathotyping.
3. ...demonstrate in animal experiments that even very low titers (0.1 TCID50 ml-1) of HPAI viral infectivity in water can induce productive infection in susceptible but clinically largely resistant mallard ducks (publication III: [175]). Furthermore, we were able to develop evidence that there is a difference in virus spread that depends on the type of (contaminated) water source. This means that infections on poultry farms with inverted or nipple drinkers may follow a different course than infections in the wild, which are mediated via larger surface waters.
Overall, the results of this project highlight the important role of surface and drinking water, as well as aquatic sediments, in the spread of AIV. The methods developed here for AIV detection extend the possibilities for surveillance of AIV infections; however, passive remains superior to active surveillance of HPAIV infections in several aspects. Examination of various environmental samples did not yield a significant advantage in terms of an early warning system that would indicate the presence or spread of HPAIV in wild bird habitats prior to the occurrence of lethal infections in wild birds.
Bacillus subtilis has been extensively used as a microbial cell factory for industrial enzymes due to its excellent capacities for protein secretion and large-scale fermentation. This bacterium is also an attractive host for biopharmaceutical production. However, the secretion potential of this organism is not fully utilized yet, mostly due to a limited understanding of critical rearrangements in the membrane proteome upon high-level protein secretion. Recently, it was shown that bottlenecks in heterologous protein secretion can be resolved by genome minimization. Here, we present for the first time absolute membrane protein concentrations of a genome-reduced B. subtilis strain (“midiBacillus”) expressing the immunodominant Staphylococcus aureus antigen A (IsaA). We quantitatively characterize the membrane proteome adaptation of midiBacillus during production stress on the level of molecules per cell for more than 400 membrane proteins, including determination of protein concentrations for ∼61% of the predicted transporters. We demonstrate that ∼30% of proteins with unknown functions display a significant increase in abundance, confirming the crucial role of membrane proteins in vital biological processes. In addition, our results show an increase of proteins dedicated to translational processes in response to IsaA induction. For the first time reported, we provide accumulation rates of a heterologous protein, demonstrating that midiBacillus secretes 2.41 molecules of IsaA per minute. Despite the successful secretion of this protein, it was found that there is still some IsaA accumulation occurring in the cytosol and membrane fraction, leading to a severe secretion stress response, and a clear adjustment of the cell’s array of transporters. This quantitative dataset offers unprecedented insights into bioproduction stress responses in a synthetic microbial cell.
Orthohantaviruses are rodent-borne pathogens distributed all over the world, which do not cause visible disease in their reservoir host. Puumala orthohantavirus (PUUV) causes most human hantavirus disease cases in Europe and is transmitted by the bank vole (Clethrionomys glareolus). Hantaviruses have a tri-segmented genome consisting of the large (L) segment, coding for the RNA-dependent RNA polymerase (RdRP), the medium (M) segment, encoding the glycoproteins, and the small (S) segment. The S-segment contains two major overlapping open reading frames (ORF) coding for the nucleocapsid (N) protein and a non-structural (NSs) protein, a putative type I interferon (IFN-I) antagonist. To date, pathogenesis and reservoir host adaptation of hantaviruses are poorly understood due to missing adequate cell culture and animal models.
In contrast to previous studies, in this work, data from spring and summer 2019 indicated a high vole abundance, a high PUUV prevalence in voles and high human incidence for some endemic regions in Germany, but elsewhere values were low to moderate. Regional and local human health institutions need to be aware about the heterogeneous distribution of human PUUV infection risk.
For a better understanding of virus-host associations, two novel cell lines from bank voles and common voles each were generated and their susceptibility and replication capacities for a variety of zoonotic and non-zoonotic viruses were analyzed. The PUUV strain Vranica/Hällnäs showed efficient replication in a new bank vole kidney cell line, but not in four other cell lines of bank and common voles. Vice versa, Tula orthohantavirus (TULV) replicated in the kidney cell line of common voles, but was hampered in its replication in other cell lines. Several viruses, such as Cowpox virus, Vaccinia virus, Rift Valley fever virus, and Encephalomyocarditis virus 1 replicated in all four cell lines. West Nile virus, Usutu virus, Sindbis virus and Tick-borne encephalitis virus replicated only in a part of the cell lines. These results indicate a tissue or species specific tropism for many of the tested viruses and the potential value of vole cell lines to address such questions in detail.
Using one of these new cell lines, the first German PUUV strains were isolated from bank voles caught in the highly endemic region around Osnabrück. Complete genomes were determined by target-enrichment-mediated high-throughput sequencing from original lung tissue, after isolation and after additional passaging in VeroE6 cells and a bank vole-derived kidney cell line. Different single amino acid substitutions were observed in the RdRP of the two stable PUUV isolates. The PUUV strain isolated on VeroE6 cells showed a lower titer when propagated on bank vole cells compared to VeroE6 cells. Additionally, glycoprotein precursor (GPC)-derived virus-like particles of a German PUUV strain from the same region allowed the generation of monoclonal antibodies that reacted with the isolated PUUV strains.
To investigate the role of PUUV and other vole-borne hantavirus NSs proteins, the evolution of the NSs and N encoding sequences was investigated by a field study in bank voles and the NSs sequences were characterized in vitro for their inhibitory effect on the human interferon-β promoter. Analysis of blood and lung samples of 851 bank voles trapped during 2010-2014 in Baden-Wuerttemberg and North Rhine-Westphalia resulted in detection of 27.8% PUUV-specific antibody positive bank voles, whereas in 22.3% PUUV-specific RNA was detected. In the hantavirus outbreak years 2010 and 2012 PUUV prevalence in bank voles was higher compared to 2011, 2013 and 2014. Sequences of the S segment of all positive bank voles showed amino acid and nucleotide sequence types of the NSs-ORF with temporal and/or local variation, whereas the N-ORF was highly conserved. One sequence type persisted over the whole observation period in both regions. The NSs coding sequence was highly divergent among regional bank vole populations in the outbreak year 2012.
Transfection experiments resulted in the detection of different products of the NSs-ORF of PUUV, TULV, Prospect Hill and Khabarovsk orthohantaviruses, due to translation initiation at different methionine codons along the coding sequence. Using luciferase reporter assays, the NSs proteins of PUUV, TULV, Prospect Hill and Khabarovsk orthohantaviruses showed inhibition of IFN-I induction of up to 70%, whereas Sin Nombre and Andes orthohantavirus NSs proteins showed a reduced effect compared to the other NSs proteins. The first 20 amino acids of the N-terminal region of PUUV NSs were found to be crucial for IFN-I promoter inhibition.
In conclusion, the newly established cell lines, antibodies, reporter assays and PUUV isolates are highly valuable tools for future hantavirus research. The activity of PUUV NSs protein in human cells contributes to our understanding of virus-host interactions and highlights the importance of corresponding future reservoir host studies. Hantavirus surveillance studies showed the necessity for timely information of the potential human PUUV infection risk to public health institutions in endemic areas to initiate appropriate actions.
LPAIV H9N2 and HPAIV H5N8 clade 2.3.4.4 viruses have been frequently isolated from domestic and wild birds in Germany and they are endemic in poultry worldwide. H9N2 is known to donate gene segments to other AIV with high case fatality rate in humans (e.g. H5N1, H7N9). Similarly, H5N8 devastated poultry worldwide since 2014 and has been recently isolated from humans. Therefore, it is important to understand the genetic predisposition for adaptation of H9N2 and H5N8 AIV in poultry and mammals. In the first publication, we focused on the variable hemagglutinin cleavage site (HACS) of European and Non-European H9N2 viruses, since the HACS is a main virulence determinant of AIV in birds. We found a preferential substitution of non-basic amino acids (G, A, N, S, D, K) in the HACS at position 319 of European H9N2 viruses compared to non-European H9N2 viruses. Recombinant viruses carrying different non-basic amino acids in the HACS modulated replication in vitro. While these non-basic amino acids did not affect virulence or transmission in chickens, they modulated virulence and replication in turkeys. Moreover, H9N2 viruses with non-basic amino acids in the HACS were able to replicate in mammalian brain cells for multiple cycles even without trypsin. In the second publication, we addressed the question whether reassortment between two recent German H9N2 and H5N8 clade 2.3.4.4. B viruses is possible and analysed the impact on virus fitness in mammals and birds. We found that H9N2 PB1 and NP segments were not compatible to generate infectious H5N8 viruses and this incompatibility was due to mutations outside the packaging region. However, H9N2 NS alone or in combination with PB2 and PA significantly increased replication of H5N8 in human cells. Moreover, H9N2 PB2, PA and/or NS segments increased virulence of H5N8 in mice. Interestingly, in chickens, reassortment with H9N2 gene segments, particularly NS, partially or fully impaired chicken-to-chicken transmission. These results indicate that the evolution of H9N2/H5N8 reassortants showing high virulence for mammals is unlikely to occur in chickens. In the third publication, we focused on the NS1 protein of different HPAIV H5N8 clade 2.3.4.4 viruses from 2013 to 2019 and studied the impact of its C-terminus (CTE) variation on virus fitness in chickens and ducks. Our findings revealed a preferential selection for a certain NS1 CTE length in 2.3.4.4. H5N8 clade A (237 aa) and B (217 aa) viruses over the common length of 230 aa. Indeed, the NS1 CTE can affect virus virulence and pathogenesis in a species and virus clade dependent manner. In chickens, although there was no impact on virulence, NS1 CTE of H5N8-A and H5N8-B, regardless of the length, have evolved towards higher efficiency to block the IFN response. In ducks, NS1 CTE contributed to efficient transmission, replication and high virulence of H5N8-B. In the fourth publication, we assessed the impact of variable length of NS1 on H5N8 virus replication in human cells and virulence in mice. We showed that NS1 of H5N8-B virus unlike the vast majority of NS1 of AIV, shared preferences for short NS1 similar to human and zoonotic influenza viruses. This virus (i) was able to efficiently block IFN and apoptosis induction which might be the first steps for efficient adaptation to human cells and (ii) without prior adaptation replicated at higher levels and was more virulent in mice than H5N8-A. The virulence of the latter virus increased after shortening the NS1 similar to H5N8-B virus. Therefore, it is conceivable that truncation in NS1 is a determinant for adaptation of H5N8 in mammals irrespective of its impact on virus fitness in poultry. Findings in this dissertation indicated that HA mutations in the European H9N2 and NS1 variations in H5N8 viruses play a role in virus fitness in poultry and/or mammals. These results improve our current understanding for AIV adaptation and are useful to assess the potential of these viruses to infect mammals.
Responses of bovine and human neutrophils to members of the Mycobacterium tuberculosis complex
(2023)
PMN are one of the most important cells of the innate immune system and are responsible for fast clearance of invading pathogens in most circumstances. The role of human PMN during mycobacterial infection have been widely studied. Nevertheless, there are contradicting results regarding their role in protection or pathology during TB. Similar studies focusing on bovine PMN and their role in M. bovis infection remain understudied. Also, not much is known about attenuation of M. tb in cattle and responses of PMN to this MTBC member.
The major aims of this study were to i) gain insights into bovine PMN biology and the cellular processes triggered by challenge with virulent mycobacteria and to ii) find out whether interspecies differences result in different outcomes upon in vitro challenge. In the first part of the work, a new isolation method for bovine PMN from whole blood was developed. Human and bovine PMN have different buoyant properties and hence need to be isolated using different procedures. The magnetic isolation method developed within this thesis is robust and results in very good yields of highly pure, viable bovine PMN populations. This is extremely advantageous and indispensable for downstream functional assays that are required to be performed on a single day.
The second goal of this study was to compare and contrast the functional differences between bovine and human PMN upon BCG infection. The findings reveal for the first time that human PMN phagocytose more BCG in comparison to bovine counterparts. Non-opsonized bacteria were internalized via the lectin-like C-domain, require cholesterol and an active cytoskeleton in human PMN, whereas opsonized bacteria entered cells via the CR3 and, in particular, CD11b. It remains unresolved why bovine PMN reacted differently, notably phagocytosis remained unaltered, to various treatments, including blocking monoclonal antibodies to CD11b and chemical inhibitors altering the cell membrane. Nonetheless, the increased uptake of BCG by human PMN correlates to more potent response of these cells in functional assays in comparison to bovine PMN. No PMN intrinsic differences were found in the basal cholesterol content. Comparative assays with the virulent strains would be essential in order to generalize these observations.
The third aim was to investigate the responses of bovine PMN to BCG, M. tb and M. bovis. While there was no difference in uptake between BCG and M. tb, serum opsonized BCG was taken up at a higher amount. This finding suggests differential binding of bacterial epitopes to host cell receptors which modulates mycobacteria uptake. However, between the virulent strains M. tb and M. bovis, the human-adapted bacillus was phagocytosed at a higher rate which hints towards the possibility of rapid recognition and clearance of M. tb in bovine host thereby possibly preventing pathology. The release of selective cytokines by PMN post infection with the virulent strains offers baseline information relevant for processes that probably occur in vivo. This work for the first time provides insights into responses of bovine PMN to mycobacteria in a two-tier approach: by cross-species analysis of PMN responses to selected mycobacterium and by head-to-head analysis of bovine PMN to animal-adapted and human-adapted mycobacteria.
As a prospect for future research in bovine PMN biology in the context of mycobacterial infection, it would be highly advantageous to compare the subcellular localization of M. tb and M. bovis in bovine PMN using confocal and/or electron microscopy. This analysis would confer proof on attachment or internalization of mycobacteria by PMN and identify the features of the mycobacteria-containing compartments. Also, in-depth investigations of additional entry pathways for the pathogen in bovine cells would be informative for unlocking downstream cell signaling events. In addition, PMN viability studies will be meaningful particularly in bovine PMN challenged with M. bovis and M. tb, given the impact of death patterns on tissue pathology. Current results and follow up studies will contribute to the understanding of the roles of PMN in controlling elimination or growth of M. bovis and M. tb in cattle.
Coding constraints imposed by the very small genome sizes of negative-strand RNA viruses (NSVs) have led to the development of numerous strategies that increase viral protein diversity, enabling the virus to both establish a productive viral replication cycle and effectively control the host antiviral response. Arenaviruses are no exception to this, and previous findings have demonstrated that the nucleoprotein (NP) of the highly pathogenic Junín virus (JUNV) exists as three additional N-terminally truncated isoforms of 53 kD (NP53kD), 47 kD (NP47kD), and 40 kD (NP40kD). The two smaller isoforms (i.e. NP47kD and NP40kD) have been characterized as products of caspase cleavage, which appears to serve a decoy function to inhibit apoptosis induction. However, whether they have additional functions in the viral replication cycle remains unknown. Further, the origin and function of NP53kD has not yet been described.
In order to first identify the mechanism responsible for production of the NP53kD variant, a possible role of additional caspase cleavage sites was first excluded using a site mutagenesis approach. Subsequently, alanine mutagenesis was then used to identify a region responsible for NP53kD production. As a result, three methionine residues were identified within the characterized sequence segment of NP, linking the production of NP53kD to an alternative in-frame translation initiation. Further site-directed mutagenesis of the previously identified putative in-frame methionine codons (i.e. M78, M80 and M100) finally led to the identification of translation initiation at M80 as being predominantly responsible for the production of NP53kD. Once the identity of all three NP isoforms was known, it was then of further interest to more deeply characterize their functional roles. Consistent with the N-terminal domain containing RNA binding and homotrimerization motifs that are relevant for the viral RNA synthesis process, it could be demonstrated that all three truncated NP isoforms lost the ability to support viral RNA synthesis in a minigenome assay. However, they also did not interfere with viral RNA synthesis by full-length NP, nor did they affect the ability of the matrix protein Z to inhibit viral RNA synthesis. Moreover, it was observed that loss of the oligomerization motifs in the N-terminus also affected the subcellular localization of all three NP isoforms, which were no longer localized in discrete perinuclear inclusion bodies, but rather showed a diffuse distribution throughout the cytoplasm, with the smallest isoform NP40kD also being able to enter the nucleus. Surprisingly, the 3'-5' exonuclease function of NP, which is associated with the C-terminal domain and plays a role in inhibiting interferon induction by digestion of double-stranded RNAs, was found to be retained only by the NP40kD isoform, despite that all three isoforms retained the associated domain. Finally, previous studies using transfected NP and chemical induction of apoptosis have suggested that cleavage of NP at the caspase motifs responsible for generating NP47kD and NP40kD plays a role in controlling activation of the apoptosis pathway. Therefore, to further characterize the connection between the generation of NP isoforms and the regulation of apoptosis in a viral context, recombinant JUNVs deficient in the respective isoforms were generated. Unlike infections with wild-type JUNV, mutations of the caspase cleavage sites resulted in the induction of caspases activation. Surprisingly, however, this was also the case for mutation of the alternate start codon responsible for NP53kD generation.
Taken together, the data from this study suggest a model whereby JUNV generates a pool of smaller NP isoforms with a predominantly cytoplasmic distribution. As a result of this altered localization, NP53kD appears to be able to serve as the substrate for further generation of NP47kD and NP40kD by caspase cleavage. Not only does this cleavage inhibit apoptosis induction during JUNV infection, it also results in a cytoplasmic isoform of NP that retains strong 3'-5' exonuclease activity (i.e. NP40kD) and thus may play an important role in preventing viral double-stranded RNA accumulation in the cytoplasm, where it can lead to activation of IFN signaling. Overall, such results emphasize the relevance of alternative protein isoforms in virus biology, and particularly in regulation of the host response to infection.
Ebolaviruses are zoonotic pathogens causing severe hemorrhagic fevers in humans
and non-human primates with high case fatality rates. In recent years, the number and
scope of outbreaks has increased, highlighting the importance of better understanding
the molecular aspects of ebolaviral infection and host cell interactions in order to be able to better control this virus.
To facilitate virus genome replication, transcription and protein expression,
ebolaviruses recruit and interact with specific host factors. These interactions play a key role in viral infection and influence virus survival and disease outcome. Based on a genome-wide siRNA screen, the three host factors CAD, NXF1 and UAP56 were
recently identified to be involved in ebolavirus genome replication and/or transcription
and/or mRNA-translation. However, mechanistical details of how these host factors
affect the ebolavirus lifecycle remained elusive.
In this thesis I analyzed the functional interactions between EBOV and these newly
identified host proteins in order to better understand the virus-host interface. To this
end I used siRNA knockdown as well as overexpression of these host proteins in
combination with different reverse-genetics based lifecycle modelling assays to
investigate the influence of CAD, NXF1 and UAP56 on individual aspects of the EBOV
lifecycle. Using these systems in relation with a host factor knockdown I was able to
show that the provision of pyrimidines by CAD plays an important role for both EBOV
genome replication and transcription, whereas NXF1 is predominantly required for
mRNA transport. I furthermore used immunofluorescence analysis to examine whether
these host factors are recruited by one or more EBOV proteins to inclusion bodies,
which represent physical sites of ebolavirus genome replication. During these
experiments, I was able to show that CAD and NXF1, and possibly also UAP56, are
recruited to EBOV inclusion bodies in order to fulfill their individual function for EBOV RNA synthesis or later steps in protein expression. Additionally, I was able to show that the uptake of NXF1 into NP-induced inclusion bodies is most likely mediated via the C-terminal domain of NP, and that the FG-repeat interaction domains of NXF1 are sufficient for recruitment. Further, my data indicate that RNA interaction of both NXF1 and NP is not required for this process, but rather important for exit of NXF1 from inclusion bodies. I therefore suggest that the viral mRNA is transferred in inclusionbodies from NP to NXF1, which leads to a rapid export of the NXF1 packed viral mRNA into the cytosol for mRNA translation.
The exact mechanism of how these host factors are recruited into inclusion bodies and whether they have similar functions in the lifecycle of other negative-sense RNA viruses still needs to be investigated. Nevertheless, this study increases our understanding of virus-host interaction of ebolaviruses, and thus helps to identify targets for the development of novel therapeutics against these viruses.
Abstract
DNA extraction and preservation bias is a recurring topic in DNA sequencing‐based microbial ecology. The different methodologies can lead to distinct outcomes, which has been demonstrated especially in studies investigating prokaryotic community composition. Eukaryotic microbes are ubiquitous, diverse, and increasingly a subject of investigation in addition to bacteria and archaea. However, little is known about how the choice of DNA preservation and extraction methodology impacts perceived eukaryotic community composition. In this study, we compared the effect of two DNA preservation methods and six DNA extraction methods on the community profiles of both eukaryotes and prokaryotes in phototrophic biofilms on seagrass (Zostera marina) leaves from the Baltic Sea. We found that, whereas both DNA preservation and extraction method caused significant bias in perceived community composition for both eukaryotes and prokaryotes, extraction bias was more pronounced for eukaryotes than for prokaryotes. In particular, soft‐bodied and hard‐shelled eukaryotes like nematodes and diatoms, respectively, were differentially abundant depending on the extraction method. We conclude that careful consideration of DNA preservation and extraction methodology is crucial to achieving representative community profiles of eukaryotes in marine biofilms and likely all other habitats containing diverse eukaryotic microbial communities.
Clostridioides difficile is the leading cause of antibiotic-associated diarrhea referring to infections of the gastrointestinal tract in the course of (broad-spectrum)antibiotic therapy. While antibiotic therapy, preferentially with fidaxomicin or vancomycin, often stops the acute infection, recurrence events due to remaining spores and biofilm-associated cells are observed in up to 20% of cases. Therefore, new antibiotics, which spare the intestinal microbiota and eventually clear infections with C. difficile are urgently required. In this light, the presented work aimed at the evaluation and characterization of three natural product classes, namely chlorotonils, myxopyronins and chelocardins, with respect to their antimicrobial activity spectrum under anaerobic conditions and their potential for the therapy of C. difficile infections. Briefly, compounds of all three classes were screened for their activity against a panel of anaerobic bacteria. Subsequently, the systemic effects of selected derivatives of each compound class were analyzed in C. difficile using a proteomics approach. Finally, appropriate downstream experiments were performed to follow up on hypotheses drawn from the proteomics datasets. Thereby, all three compound classes demonstrated significant activity against C. difficile. However, chelocardins similarly inhibited the growth of other anaerobes excluding chelocardins as antibiotic candidates for C. difficile infection therapy. In contrast, chlorotonils demonstrated significantly higher in vitro activity against C. difficile and close relatives compared to a small panel of other anaerobes. In addition, it could be shown that chlorotonils affect intracellular metal homeostasis as demonstrated in a multi-omics approach. The data led to speculate that chlorotonils eventually affect cobalt and selenate availability in particular. Moreover, a metaproteomics approach verified that oral chlorotonil treatment only marginally affected the intestinal microbiota of piglets on taxonomic and functional level. Furthermore, the proteome stress response of C. difficile 630 to myxopyronin B, which similarly showed elevated activity against C. difficile compared to a few other anaerobes, indicated that the antibiotic inhibited early toxin synthesis comparatively to fidaxomicin. Finally, evidence is provided that C. difficile 630 responds to dissipation of its membrane potential by production and accumulation of aromatic metabolites.
The anaerobic, gastrointestinal pathogen Clostridioides difficile can cause severe forms of enterocolitis which is mainly mediated by the toxins it produces. The RNA polymerase inhibitor Fidaxomicin is the current gold standard for the therapy of C. difficile infections due to several beneficial features including its ability to suppress toxin synthesis in C. difficile. In contrast to the Rifamycins, Fidaxomicin binds to the RNA polymerase switch region, which is also the binding site for Myxopyronin B. Here, serial broth dilution assays were performed to test the susceptibility of C. difficile and other anaerobes to Myxopyronin B, proving that the natural product is considerably active against C. difficile and that there is no cross-resistance between Fidaxomicin and Myxopyronin B in a Fidaxomicin-resistant C. difficile strain. Moreover, mass spectrometry analysis indicated that Myxopyronin B is able to suppress early phase toxin synthesis in C. difficile to the same degree as Fidaxomicin. Conclusively, Myxopyronin B is proposed as a new lead structure for the design of novel antibiotics for the therapy of C. difficile infections.
The anaerobic pathogen Clostridioides difficile is perfectly equipped to survive and persist inside the mammalian intestine. When facing unfavorable conditions C. difficile is able to form highly resistant endospores. Likewise, biofilms are currently discussed as form of persistence. Here a comprehensive proteomics approach was applied to investigate the molecular processes of C. difficile strain 630Δerm underlying biofilm formation. The comparison of the proteome from two different forms of biofilm-like growth, namely aggregate biofilms and colonies on agar plates, revealed major differences in the formation of cell surface proteins, as well as enzymes of its energy and stress metabolism. For instance, while the obtained data suggest that aggregate biofilm cells express both flagella, type IV pili and enzymes required for biosynthesis of cell-surface polysaccharides, the S-layer protein SlpA and most cell wall proteins (CWPs) encoded adjacent to SlpA were detected in significantly lower amounts in aggregate biofilm cells than in colony biofilms. Moreover, the obtained data suggested that aggregate biofilm cells are rather actively growing cells while colony biofilm cells most likely severely suffer from a lack of reductive equivalents what requires induction of the Wood-Ljungdahl pathway and C. difficile’s V-type ATPase to maintain cell homeostasis. In agreement with this, aggregate biofilm cells, in contrast to colony biofilm cells, neither induced toxin nor spore production. Finally, the data revealed that the sigma factor SigL/RpoN and its dependent regulators are noticeably induced in aggregate biofilms suggesting an important role of SigL/RpoN in aggregate biofilm formation.
Mussel farming, compared to marine finfish aquaculture, represents an environmentally friendly alternative for a high quality protein source and can at the same time be a measure to remove excess nutrients in eutrophic areas. As such, it is considered as a promising “blue growth” potential and promoted within the European Union. To expand mussel aquaculture, new regions have to be considered because there are multiple marine usages, and spatial limitations occur in coastal areas. The brackish Baltic Sea might be considered for expansion of mussel aquaculture. This study focusses on estimated production potential, economic profitability and nutrient remediation potential of mussel farming at different salinities. Four experimental mussel farms were set up along the German Baltic coast at salinities ranging from 7 to 17 psu. Collected growth data was used to calibrate and validate a Dynamic Energy Budget model and to predict the potential mussel production at 12 sites along the German coast. The estimated production and nutrient removal was used to assess economic profitability, assuming two usages of the harvest: human consumption and mussel meal production. Measured mussel specific growth rates increased with salinity from 0.05 mm d–1 in Greifswald Bay to 0.11 mm d–1 in Kiel Fjord. Within 6 months, a 1-ha farm could produce from 1 t (Darss-Zingst-Bodden-Chain) to 51 t (Flensburg) fresh mussels and remove 1.1 to 27.7 kg P and 24.7 to 612.7 kg N, respectively. Mussel farms at sites west of Rostock at salinities >10 psu could produce 5 cm mussels within 18 months, but only farms at Flensburg, Eckernförde and Kiel Fjord became profitable at a farm size of 4 ha (160,000 m3) at current market prices of 2.2 € kg–1. Regardless of the farm size, none of the farm sites could operate profitable if fresh mussels were sold for animal feeding at sales price of 0.06 € kg–1. Yearly nutrient removal costs at a small-scale farm (1 ha) ranged between 162 € (Flensburg) and 4,018 € (Darss-Zingst-Bodden-Chain) kg–1 nitrogen, and 3,580 € and 88,750 € kg–1 phosphorus, respectively.
Reactive oxygen species (ROS) can damage all cellular macromolecules and also produce secondary reactive intermediates, like reactive electrophilic species (RES) that include quinones or aldehydes. Low molecular weight (LMW) thiols are small thiol-containing compounds that play essential roles in the defense against ROS and RES in all organisms. The best studied LMW thiol is the tripeptide glutathione (GSH). Firmicutes bacteria including Bacillus und Staphylococcus species have been recently discovered to utilize the redox buffer bacillithiol (BSH). LMW thiols function as redox buffers to maintain the reduced state of the cytoplasm. Under conditions of oxidative stress, LMW thiols also react with protein thiols to form mixed LMW thiol – protein disulfides, termed S-thiolations, as major protection mechanism. Investigating the role of BSH in oxidative stress response and ROS-induced S-thiolations in Firmicutes bacteria was one subject of this PhD thesis. Specifically, the regulatory mechanisms and post-translational thiol-modifications in response to NaOCl stress were studied in the model bacterium for low-GC Gram-positive bacteria Bacillus subtilis. The transcriptome profile after NaOCl stress was indicative of disulfide stress and overlapped strongly with the response to diamide. NaOCl stress caused induction of the thiol- and oxidative stress-specific Spx, CtsR, PerR and OhrR regulons. Thiol redox proteomics identified only few NaOCl-sensitive proteins with reversible thiol-oxidations. Using mass spectrometry, eleven proteins were identified that were oxidized to mixed BSH protein disulfides (S-bacillithiolated) in B. subtilis cells after NaOCl-exposure. Methionine synthase MetE is the most abundant S-bacillithiolated protein in B. subtilis and other Bacillus species after NaOCl exposure. S-bacillithiolation of OhrR repressor leads to upregulation of the OhrA peroxiredoxin that confers together with BSH specific protection against NaOCl. S-bacillithiolation of MetE, YxjG, PpaC, and SerA causes hypochlorite-induced methionine starvation as supported by the induction of the S-box regulon. To further assess the conservation of targets for S-bacillithiolations in other Firmicutes bacteria, we studied the S-bacillithiolomes of Bacillus megaterium, Bacillus pumilus, Bacillus amyloliquefaciens, and Staphylococcus carnosus under NaOCl stress conditions. In total, 54 S-bacillithiolated proteins were identified, including 29 unique proteins and 8 conserved proteins involved in amino acid and cofactor biosynthesis, nucleotide metabolism, translation, protein quality control, redox and antioxidant functions. Together our data support a major role of BSH redox buffer in redox control and thiol protection of conserved and essential proteins against irreversible oxidation by S-bacillithiolations in Firmicutes bacteria. In response to ROS and RES, bacteria also activate the expression of antioxidant and detoxification enzymes, such as catalases, peroxidases, thiol-dependent peroxiredoxins and other specific oxidoreductases to detoxify ROS and RES. These defense mechanisms are often controlled by redox-sensitive transcription factors. B. subtilis encodes redox-sensing MarR-type regulators belonging to the OhrR and DUF24-families that are conserved among bacteria. Hence, we were further interested in this PhD thesis to study at the molecular and structural level the redox-sensing mechanisms of novel redox-sensing MarR/DUF24-type regulators in B. subtilis. We have characterized the regulatory mechanisms of HypR, YodB and CatR that sense and respond to hypochlorite, diamide and quinones stress. HypR is the first DUF24-family regulator whose crystal structure was resolved. HypR senses specifically disulfide stress and controls positively expression of the flavin oxidoreductase HypO after NaOCl and diamide stress. HypR resembles a 2-Cys-type regulator with a reactive nucleophilic N-terminal Cys14 and a second C-terminal Cys49. Besides HypR, B. subtilis encodes further MarR/DUF24-family members including the paralogous YodB and CatR repressors that sense quinones and diamide. YodB controls the azoreductase AzoR1, the nitroreductase YodC, and the Spx regulator. YodB resembles a 2-Cys-type MarR/DUF24-family regulator with three Cys residues (Cys6, Cys101, and Cys108) that form intermolecular disulfides in vivo under oxidative stress. YodB and its paralog CatR were further identified as repressors of the catDE operon encoding a catechol-2,3-dioxygenase that also contributes to quinone resistance. Although CatR is a 1-Cys-type regulator, our data showed that CatR also forms intermolecular disulfide in response to diamide and quinones in vitro. Thus, HypR, YodB and CatR are controlled by 2-Cys-type thiol-disulfide redox switches to sense disulfide and RES stress conditions, and to control specific RES detoxification enzymes.
This work focuses the glycoprotein H of PrV which was analysed by structure-based functional analyses by targeted site-directed mutagenesis. Disulfid bridges were introduced at specific sites and the effects on the fusion mechanism investigated. A revertant was obtained and characterised during the studies, as well as chimeric glycoprotein H proteins were constructed, combining the different domains of the glycoproteins Hs of PrV and HSV1.
Lyssaviruses, the causative agents of rabies, are a long-known threat for animals and humans. To date, terrestrial rabies still accounts for tens of thousands of human deaths annually, notwithstanding ambitious vaccination campaigns targeting susceptible dog and wildlife populations that act as reservoirs for the prototypic rabies virus. Moreover, the continuing discovery of newly emerging virus species in hitherto unconcerned chiropteran hosts and geographic regions drive the expansion of the Lyssavirus genus by unveiling its actual variety, host range and distribution.In this work, the genetic diversity of three distinct lyssaviruses, namely EBLV-1, KBLV and RABV, was elucidated by in-depth genomic analyses to provide further insight into lyssavirus evolution. The generation of full-genome sequences from primarily bat-associated Danish EBLV-1 samples significantly increased the number of available Danish EBLV-1 genome sequences while phylogenetic and phylogeographic analysis revealed a stronger phylogeographic structure for the cluster A1 of the sublineage EBLV-1a than it was postulated in previous studies. In addition, the acquisition of a nearly complete genome sequence for the Kotalahti bat lyssavirus provided the basis for the classification of this putative new lyssavirus species as a recognized member of the genus. Furthermore, phylogenetic analysis revealed the affiliation of KBLV to a group of Myotis-associated lyssaviruses giving a deeper insight into the shared evolutionary history of lyssaviruses co-evolving with particular bat species. Moreover, a deep-sequencing approach was utilized to assess the high genetic diversity of vaccine virus populations, uncovering three independent patterns of single nucleotide variants (SNVs) that became selected in ERA-related vaccine-induced cases. However, no apparent influence of the genetic diversity of vaccine viruses on microevolutionary processes like a potential reversion to virulence or a species-specific adaptation of the vaccine virus strains could be detected, leaving the question for the cause of rabies induction in the affected animals unanswered. Lastly, the successful implementation of a hybridization capturing system for the generation of full-genome sequences and deep-sequencing variant analyses of RABV and KBLV samples was demonstrated for a diagnostic bait set, highlighting the versatility and consistency of this approach to assess the genetic spectrum of known and novel lyssavirus species while setting the basis for its application and optimization in upcoming projects.In conclusion, as shown by the studies in this work, the investigation of lyssavirus genomes at the sub-consensus, full-genome and population level remains crucial to assess the complexity of lyssavirus evolution, as it provides an indispensable source of information to cover the diversity of the genus and understand evolutionary dynamics on a long-term and microevolutionary scale.
The following work is describing the development of two innovative biosensors for the detection of biologically relevant molecules in the field of ecology and medical diagnostics. Biosensors have the particularity to possess a biological partner which recognizes the target molecule and a physical detection method responsible for the transformation of this biological interaction into measurable information. In the present case, both biosensors are designed following the same strategy and use a recombinant produced human receptor as biological partner and the surface plasmon resonance (SPR) technique to transform the biological interaction in quantitative information. The progesterone biosensor is aimed to detect and quantify substances with affinity to the human progesterone receptor. The recent discoveries that some chemicals present in low quantities in the ecosystem called endocrine disrupting chemicals (EDCs) have a negative impact on the aquatic life fitness raised concerns about the effects of these same molecules to the human health. In order to assess the effects of these EDCs, the use of classical analytical detection methods like high performance liquid chromatography (HPLC) or gas chromatography (GC) is not sufficient as these techniques only quantify a defined molecule without giving information about its biological activity. By integrating a recombinant human progesterone receptor, the progesterone biosensor can determine the biological activity of an unknown molecule or of a mixture of molecules in a real sample. In this work, two different yeasts – one methylotrophic (Hansenula polymorpha) and one non-methylotrophic (Arxula adeninivorans) - were selected as host for the recombinant protein production and their performances were compared. Different purification strategies were assayed and the binding activity of the purified progesterone receptor was then confirmed by enzyme like receptor assay (ELRA) and SPR. This led to the design of a first version of the biosensor with the immobilization of a progesterone-BSA ligand to the surface of a SPR chip and the use of a progesterone receptor mixed with the target molecule as sample. This competitive assay format was successfully utilized with a commercial progesterone-BSA ligand as target molecule and the next step will be the adaptation of this biosensor for real samples measurements. The HER-2 biosensor was developed as an answer for one of the most critical issue in the field of breast cancer diagnostics. In approximately 30 % of cancer cases, the transmembrane protein HER-2 can be found in large amount at the surface of the carcinoma cells and these cases are known to be particularly aggressive. Based on the amount of HER-2 protein at the surface of the cells, the pathologists established a scale with four levels to adapt the treatment to each patient. Although effective therapies have been developed to treat the HER-2 positive breast cancer, one of the major challenges remains the classification of breast sample in this scale as the only accepted determination methods are immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) which are only qualitative. In this work, a biosensor has been designed to quantify the amount of the HER-2 protein in a crude cell extract from a breast cancer tissue sample. To achieve this, the strategy is to utilize an antibody specifically targeted against the HER-2 protein and bound to a SPR chip. As the development of this biosensor necessitated the use of large amount of purified HER-2 protein, it was decided to produce recombinant full-length HER-2 in two different yeasts and to purify it by chromatography. This recombinant protein production required particular attention due to the membrane localization of HER-2. The structural integrity of the recombinant protein was confirmed by Western Blot and ELISA and different antibodies were bound to SPR chips in order to detect the HER-2 protein. After finding the conditions giving an optimal SPR signal, a protocol was developed to extract native HER-2 from breast tissue sample and the biosensor was assayed with this crude cell extract.
Hepatitis E virus (HEV) is emerging worldwide as a zoonotic pathogen that has remained largely undetected for decades, if not centuries. Its enormous success can be attributed to the wide range of host species, which can transmit the virus to humans, depending on the viral genotype. As a result, HEV is likely to remain a challenge even when the remaining hepatitis viruses (HAV, HBV, HCV), which are transmitted exclusively between humans, are under control. Although millions of HEV infections occur each year, little is known about this puzzling pathogen. One major issue in HEV research is the lack of reliable model systems. Established animal models are inefficient, expensive, or simply not representative of human HEV. On the other hand, cell culture systems are limited by the slow growth of the virus and inefficient replication and infection. The aim of this work is to with deepen the understanding of zoonotic HEV in animal hosts in Germany. For this purpose, a molecular and phylogenetic characterization of HEV sequences from rabbits and swine was conducted. A novel subtype of the zoonotic genotype HEV-3 was identified in a rabbit sample, further emphasizing the role of rabbits as HEV host species and possible reservoir of zoonotic HEV infections in Germany. On the other hand, a molecular biological screening of pigs and wild boars in Mecklenburg-Western Pomerania indicates a wide range of HEV-3 subtypes circulating in swine in north-east Germany. Furthermore, an optimized replicon system was established in order to enable characterization of various HEV sequences by reverse genetics. As a proof of concept, two rabbit HEV derived replicons were compared with two established, cell culture adapted HEV strains. The influence of different regions of the nonstructural protein on HEV replication was determined and quantified. In particular, a system was established, to reproducibly compare different strains and genotypes. This refined replicon system will enable the characterization of further HEV sequences and thus expand the knowledge on the determinants of the viral life cycle.
Non-alcoholic fatty liver disease (NAFLD) is gaining in importance and is linked to obesity.
Especially, the development of fibrosis and portal hypertension in NAFLD patients requires treatment.
Transgenic TGR(mREN2)27 rats overexpressing mouse renin spontaneously develop NAFLD with
portal hypertension but without obesity. This study investigated the additional role of obesity in this
model on the development of portal hypertension and fibrosis. Obesity was induced in twelve-week
old TGR(mREN2)27 rats after receiving Western diet (WD) for two or four weeks. Liver fibrosis
was assessed using standard techniques. Hepatic expression of transforming growth factor-β1
(TGF-β1), collagen type Iα1, α-smooth muscle actin, and the macrophage markers Emr1, as well as
the chemoattractant Ccl2, interleukin-1β (IL1β) and tumor necrosis factor-α (TNFα) were analyzed.
Assessment of portal and systemic hemodynamics was performed using the colored microsphere
technique. As expected, WD induced obesity and liver fibrosis as confirmed by Sirius Red and Oil Red
O staining. The expression of the monocyte-macrophage markers, Emr1, Ccl2, IL1β and TNFα were
increased during feeding of WD, indicating infiltration of macrophages into the liver, even though this
increase was statistically not significant for the EGF module-containing mucin-like receptor (Emr1)
mRNA expression levels. Of note, portal pressure increased with the duration of WD compared
to animals that received a normal chow. Besides obesity, WD feeding increased systemic vascular
resistance reflecting systemic endothelial and splanchnic vascular dysfunction. We conclude that
transgenic TGR(mREN2)27 rats are a suitable model to investigate NAFLD development with liver
fibrosis and portal hypertension. Tendency towards elevated expression of Emr1 is associated with
macrophage activity point to a significant role of macrophages in NAFLD pathogenesis, probably
due to a shift of the renin–angiotensin system towards a higher activation of the classical pathway.The hepatic injury induced by WD in TGR(mREN2)27 rats is suitable to evaluate different stages of
fibrosis and portal hypertension in NAFLD with obesity
In temperate regions, climate warming alters temperature and precipitation regimes. During winter, a decline in insulating snow cover changes the soil environment, where especially frost exposure can have severe implications for soil microorganisms and subsequently for soil nutrient dynamics. Here, we investigated winter climate change responses in European beech forests soil microbiome. Nine study sites with each three treatments (snow exclusion, insolation, and ambient) were investigated. Long-term adaptation to average climate was explored by comparing across sites. Triplicated treatment plots were used to evaluate short-term (one single winter) responses. Community profiles of bacteria, archaea and fungi were created using amplicon sequencing. Correlations between the microbiome, vegetation and soil physicochemical properties were found. We identify core members of the forest-microbiome and link them to key processes, for example, mycorrhizal symbiont and specialized beech wood degraders (fungi) and nitrogen cycling (bacteria, archaea). For bacteria, the shift of the microbiome composition due to short-term soil temperature manipulations in winter was similar to the community differences observed between long-term relatively cold to warm conditions. The results suggest a strong link between the changes in the microbiomes and changes in environmental processes, for example, nitrogen dynamics, driven by variations in winter climate.
The role of cell-penetrating peptides in the induction of T cell responses by virus-like particles
(2023)
Many viral structural proteins can self-assemble into virus-like particles (VLPs). VLPs can serve as an effective vaccine or be used as a vaccine platform. One of these structural proteins is the hepatitis B virus core antigen (HBcAg), which appears to be suitable as an antigen carrier due to its high immunogenicity. HBcAg has a major immunodominant region (MIR) that is presented on the surface of the VLPs after self-assembly. Foreign antigens can be inserted into this region. Since HBcAg VLPs, unlike the Hepatitis B virus (HBV), do not have an envelope, they are not able to penetrate cell membranes efficiently. As an extracellular antigen, HBcAg VLPs primarily induce a strong humoral immune response.
In the present study, we investigated the extent to which HBcAg can be modified to also elicit an enhanced cellular, particularly a cytotoxic, immune response. A cytotoxic CD8+ T cell response is predominantly induced by intracellular antigens. Therefore, our goal was to increase the cell penetration capacity of VLPs. We aimed to achieve this by fusing cell-penetrating peptides (CPPs) to HBcAg. CPPs can spontaneously penetrate cell membranes to enter the cytoplasm of cells. To guarantee that the CCPs were localized to the surface of the VLPs, we fused CPPs to the N-terminus of HBcAg. The CCPs were followed by a tag to allow the purification of VLPs. The T cell epitopes, against which the induced CTL should be directed, were derived from the Large T antigen and inserted into the MIR of HBcAg. Finally, we fused fluorescent proteins to the C-terminus of HBcAg to track the entry of VLPs into cells.
Modifications of HBcAg may lead to reduced stability or altered structure of VLPs. To analyze the stability of VLPs, we used nanoscale differential scanning fluorimetry (nanoDSF) analysis. This revealed that the N-terminal fusion of CPPs or the tag to HBcAg does not reduce VLP stability. However, some peptides incorporated into the MIR had a significant effect on the structure and stability of the VLPs. While the incorporation of a Flag-tag or a peptide from ovalbumin had no negative effect on VLP stability, the incorporation of peptides representing T cell epitopes of Large T antigen interfered with VLP formation. Denaturation and reassembly of the aggregates significantly improved the homogeneity of the VLPs, and the C-terminal addition of arginine-rich domains enhanced stability.
Using live cell imaging and flow cytometry, we demonstrated that HBcAg VLPs functionalized with CPP exhibited up to 40% more efficient penetration into professional antigen-presenting cells (JAWS II) than HBcAg VLPs without CPP. This resulted in the increased presentation of integrated T cell epitopes by dendritic cells. In vivo, we detected significantly increased induction of SV40 Large T antigen-specific CTL in mice immunized with CPP-conjugated VLPs compared to unconjugated VLPs.
In this study, we demonstrated that a stronger cellular immune response can be induced by CPP-functionalized HBcAg VLPs than with the unmodified HBcAg VLPs in vitro as well as in vivo. This discovery may have positive implications for future vaccine development where an enhanced cellular component of the immune response is desirable.
Foot-and-mouth disease virus (FMDV) is a positive-sense RNA virus of the family Picornaviridae that comprises of seven serotypes and is distinguished by a high contagiosity with the ability of rapid spread. Strategies for abatement and control are based on an early detection, quick initiation of retaliatory actions and mass vaccinations. Therefore, aim of the study was the development of a fast and easy method for genome sequencing as well as an investigation into the causes, why some cell lines that are mainly used for vaccine production, are resistant towards FMDV infection. Finally, adaptive sequence changes in different cell culture systems and associated effects on particle stability and immunogenicity were examined.
In case of an outbreak it is of major importance to detect and rapidly characterize the circulating virus isolate to choose an appropriate vaccine to minimize the viral spread. In addition, comprehensive genome analysis of the outbreak strain provides information about the origin of the virus and allows molecular epidemiology. A universal primer set, covering most parts of the open reading frame of the viral genome, was developed to perform quick sequence analyses, independently of the viral serotype (Paper I). Especially in endemic regions, vaccination of susceptible animal species is the main action to combat foot-and-mouth disease (FMD) in an acute outbreak situation as well as a preventive measure. Reasons, why some baby hamster kidney (BHK) cell lines are resistant towards an infection with FMDV, were examined in a second study that narrowed down the cause for this phenomenon to an impaired attachment of the virus to the cell surface. Furthermore, an alternative approach could be developed to successfully adapt the virus to the resistant vaccine-production cell line by using a FMDV-sensitive “wet-nurse” cell line (Paper II). Adaptive changes in the capsid-coding region of the viral genome caused through cultivation and passaging of the virus in different BHK cell systems were the topics of the third study. It was shown that capsid alterations are rather serotype-specific and dependent on the cell line used than influenced by the cell media. Viral titers and neutralization profiles of the adapted isolates were not affected compared to the original viruses (Paper III).
Overall, this work expanded our knowledge on the control and eradication of FMD and will support the global effort to combat the disease.
Hantaviruses are enveloped viruses with a single-stranded RNA genome of negative polarity. The genome consists of three segments: small (S), medium (M) and large (L). As zoonotic pathogen, hantaviruses are worldwide responsible for 150,000 to 200,000 human disease cases per year. Two forms of human disease are currently distinguished: In the Americas the hantavirus cardiopulmonary syndrome (HCPS) and in Europe and Asia the hemorrhagic fever with renal syndrome (HFRS). Since the introduction of the German Protection against Infection Act in 2001 until now a total of 10,082 disease cases have been reported. As a result, hantavirus infections currently rank as the fifth frequent notifiable disease in Germany. More than 80% of these infections were caused by the hantavirus species Puumala virus (PUUV), transmitted by the bank vole Myodes glareolus. Besides temporal oscillations, an unequal geographical distribution of human PUUV cases was noticed in Germany and in other countries of Central Europe. This is reflected in the presence of endemic and non-endemic regions as well as of so-called outbreak years. Therefore, the overall objective of this study was to find out possible reasons for the inhomogeneous distribution of PUUV in Central Europe, in particular in Poland, Germany and certain districts of Baden-Wuerttemberg. The basic working hypothesis was that PUUV spread in Central Europe after the last glaciation with different evolutionary lineages of the bank vole and that the current emergence of PUUV in bank vole populations is determined by local geographical and ecological factors. Very little was known about the presence of PUUV in Poland. Earlier studies were based exclusively on serological detection of PUUV, but a molecular detection with subsequent phylogenetic investigation was missing so far. Therefore, 45 bank voles from the northeastern part of Poland were investigated by serological and molecular assays. In three animals from a forest region close to the city of Miko³ajki PUUV-reactive antibodies and/or PUUV RNA were detected. Phylogenetic analysis indicated the presence of a Latvian (LAT) PUUV strain. Viral RNA was detected in one bank vole of the Eastern evolutionary lineage and two animals of the Carpathian lineage. Thereby it could be demonstrated for the first time that the distribution of the LAT PUUV lineage ranges from Latvia south-west to the northeastern part of Poland. An inhomogeneous spatial distribution of human disease cases has been observed even for Baden-Wuerttemberg, a long time known endemic federal state of Germany. Therefore 660 bank voles were trapped during the outbreak and non-outbreak years 2012 and 2013 in four districts with high incidences (H) and in four districts with low incidences or lacking PUUV cases (L). During the outbreak year 2012 PUUV-positive bank voles were detected by serological and molecular investigations in seven of eight districts. In contrast, in the following year only in one district PUUV infected bank voles were detected. Furthermore, it was demonstrated that after a beech mast, i.e., a massive fructification of beech trees, in H districts with a higher percentage of beech forest coverage a higher number of human cases was notified, but not in L districts with a lower percentage of beech forest coverage. For the future development of early warning modules it is therefore necessary to have a long-term bank vole monitoring established that incorporates beech mast data and information on beech forest coverage. High endemic regions for PUUV are mainly located in the southern and western parts of Germany, whereas in the eastern and northern parts only low numbers or even no human cases are recorded. To find out possible reasons for this inhomogeneous distribution, 1,774 bank voles from different regions of Germany were investigated for PUUV infections and in parallel for the corresponding bank vole evolutionary lineage (Western, Eastern, Carpathian). The PUUV investigations indicated positive voles in the known endemic regions with an easternmost and northernmost occurrence in western Saxony-Anhalt, western Thuringia and in Osnabrück. In the northern and eastern part of Germany none of the 1,210 investigated bank voles showed a PUUV infection. In the southern and western parts of Germany only the Western bank vole lineage was identified, whereas the Eastern lineage was exclusively found in the eastern and northern part and the Carpathian lineage in the South-East and North-East of Germany. PUUV infections were found almost exclusively in bank voles of the Western lineage. Individuals of the other two vole lineages were found to be PUUV infected only in regions with sympatric occurrence of the Western lineage. The previously described contact zone of the different bank vole phylogroups ranges from Poland to the entire northern part of Germany. In conclusion, the results of this investigation indicate two potential major reasons for the inhomogeneous distribution of PUUV in Germany: First, PUUV of the CE lineage seems to be associated with the Western bank vole lineage. The current geographical distribution of virus and host might be explained by a post-glacial northern expansion of the bank vole starting at the western refuge. Second, the missing detection of PUUV in bank voles of the Western lineage in areas close to high endemic regions might be explained by the extinction of the virus due to a limited winter survival of infected animals during long and harsh winters. The virus stability outside the host or ecological barriers, such as isolated forest areas or broad rivers, might also influence the distribution of PUUV in bank vole populations.
The presented study was dedicated to outstanding issues in regard to the safety and efficacy of the LAV “CP7_E2alf”, during the final licensing process and towards its putative implementation in outbreak scenarios as emergency vaccine. (I) For application of a genetically engineered virus under field conditions, knowledge about its genetic stability is mandatory. Therefore, the genetic stability of “CP7_E2alf” needed to be assessed in vivo and in vitro. Mutation rates were compared to the parental pestivirus strains (BVDV-1 “CP7” and CSFV “Alfort/187”), and BVDV or CSFV field-strains. There was no indication that “CP7_E2alf” could be more prone to mutational events than its parental viruses or representative field-strains. Moreover, no recombination events were observed in in vitro experiments. In conclusion, the data obtained in this study confirm a strong genetic stability of “CP7_E2alf” as an important safety component. (II) Since vaccination of breeding animals is often discussed, this study was conducted to assess the safety of “CP7_E2alf” vaccination of breeding male pigs. The study with “CP7_E2alf” vaccinated boar demonstrated that the new CSFV marker vaccine is suitable for application in reproductive boar. Neither in organs of the uro-genital tract related to sperm production nor in urine or feces, vaccine virus genome was detectable. Dissemination of “CP7_E2alf” through semen, and shedding with urine and feces, is therefore highly unlikely. (III) In order to investigate the influence of pre-existing pestivirus antibodies of the efficacy of “CP7_E2alf”, a vaccination-challenge-trial was conducted with “CP7_E2alf” (Suvaxyn® CSF Marker) and the “gold-standard” of live-modified CSFV vaccines, the C-strain (RIEMSER® Schweinepestvakzine). Pre-existing antibodies against BVDV-1 were provoked through intramuscular inoculation of a recent field isolate from Germany. Seven days after the vaccination, all animals were challenged with highly virulent CSFV strain “Koslov”. It was demonstrated that pre-existing anti- BVDV-1 antibodies do not impact the efficacy of both live attenuated vaccines against CSFV. Both C-strain “Riems” and marker vaccine “CP7_E2alf” were able to confer full protection against the highly virulent challenge. However, slight interference was seen with serological DIVA diagnostics accompanying “CP7_E2alf”. Amended sample preparation and combination of test systems was able to resolve most cases of false positive reactions. However, in such a coinfection scenario, optimization and embedding in a well-defined surveillance strategy is clearly needed for marker vaccination scenarios. (IV) To supplement the data about the kinetic of maternally derived antibodies in piglets from sows vaccinated during outbreaks, a single “emergency-type” vaccination of two pregnant sows was done. Focus was laid on the kinetics of maternally derived antibodies (MDA) in the screening assays of their offspring with screening assays that would be used in case of CSFV outbreaks, i.e. CSFV E2 and Erns antibody ELISA. Upon vaccination with “CP7_E2alf” 21 days before farrowing, MDAs were measurable in all piglets born to vaccinated sows. The E2- ELISA reactivities showed an almost linear decrease over ten weeks after which all piglets were tested negative in the ELISA. Future studies should investigate, if MDA are able to protect offspring of vaccinated sows or whether the piglets should also be vaccinated.
Over thirty years have passed since the first description of ubiquitin-positive structures in the brain of patients suffering from Alzheimer’s disease. Meanwhile, the intracellular accumulation of ubiquitin-modified insoluble protein aggregates has become an indisputable hallmark of neurodegeneration. However, the role of ubiquitin and a fortiori the ubiquitin-proteasome system (UPS) in the pathogenesis of neurodevelopmental disorders (NDD) is much less described. In this article, we review all reported monogenic forms of NDD caused by lesions in genes coding for any component of the UPS including ubiquitin-activating (E1), -conjugating (E2) enzymes, ubiquitin ligases (E3), ubiquitin hydrolases, and ubiquitin-like modifiers as well as proteasome subunits. Strikingly, our analysis revealed that a vast majority of these proteins have a described function in the negative regulation of the innate immune response. In this work, we hypothesize a possible involvement of autoinflammation in NDD pathogenesis. Herein, we discuss the parallels between immune dysregulation and neurodevelopment with the aim at improving our understanding the biology of NDD and providing knowledge required for the design of novel therapeutic strategies.
Type I interferonopathies cover a phenotypically heterogeneous group of rare genetic diseases including the recently described proteasome-associated autoinflammatory syndromes (PRAAS). By definition, PRAAS are caused by inherited and/or de novo loss-of-function mutations in genes encoding proteasome subunits such as PSMB8, PSMB9, PSMB7, PSMA3, or proteasome assembly factors including POMP and PSMG2, respectively. Disruption of any of these subunits results in perturbed intracellular protein homeostasis including accumulation of ubiquitinated proteins which is accompanied by a type I interferon (IFN) signature. The observation that, similarly to pathogens, proteasome dysfunctions are potent type I IFN inducers is quite unexpected and, up to now, the underlying molecular mechanisms of this process remain largely unknown. One promising candidate for triggering type I IFN under sterile conditions is the unfolded protein response (UPR) which is typically initiated in response to an accumulation of unfolded and/or misfolded proteins in the endoplasmic reticulum (ER) (also referred to as ER stress). The recent observation that the UPR is engaged in subjects carrying POMP mutations strongly suggests its possible implication in the cause-and-effect relationship between proteasome impairment and interferonopathy onset. The purpose of this present review is therefore to discuss the possible role of the UPR in the pathogenesis of PRAAS. We will particularly focus on pathways initiated by the four ER-membrane proteins ATF6, PERK, IRE1-α, and TCF11/Nrf1 which undergo activation under proteasome inhibition. An overview of the current understanding of the mechanisms and potential cross-talk between the UPR and inflammatory signaling casacades is provided to convey a more integrated picture of the pathophysiology of PRAAS and shed light on potential biomarkers and therapeutic targets.
Protein quality control systems are essential for the viability and growth of all living organisms. They protect the cell from irreversible protein aggregation. Because the frequency of protein misfolding, which ultimately results in protein aggregation, varies with the environmental conditions, the amount and activity of protein quality systems have to be accurately adapted to the rate of protein misfolding. The main goal of this thesis was to gain detailed molecular insights into the transcriptional and post-translational regulation of these protein quality control networks in the ecologically, medically and industrially important phylum of low GC, Gram-positive bacteria. In these bacteria the core protein quality control systems are under the transcriptional control of the global repressor CtsR. In a first study it was demonstrated that the arginine kinase McsB is not responsible for the regulation of CtsR activity during heat stress, as was concluded by others on the basis of previous in vitro data. Rather, it was demonstrated that CtsR acts as an intrinsic thermosensor that adapts its activity to the surrounding temperature. CtsR displays a decreased DNA binding at higher temperatures, which leads to induction of transcription of the protein quality control systems under these conditions. This CtsR feature is conserved in all low GC, Gram-positive bacteria. However, the CtsR proteins of various low GC, Gram-positive species do not have the same temperature optima. CtsR responds to heat in a species-specific manner according to their corresponding growth temperature. Detailed analysis revealed that a highly conserved tetra-glycine loop within the winged helix-turn-helix domain of CtsR is responsible for thermosensing. Dual control of CtsR activity during different stresses was demonstrated for the first time in this work. In addition to heat-dependent de-repression, CtsR is inactivated by thiol-specific stress conditions. This latter de-repression depends on a molecular redox-switch that is independent of CtsR auto-regulation. In Bacillus subtilis and its closest relatives the McsA/McsB stress-sensing complex is responsible for CtsR de-repression during redox stress conditions. McsA is able to sense the redox state of the cell via its highly conserved cysteine residues. When these cysteines are reduced, McsA is able to bind and inhibit McsB. But when these cysteine residues are oxidized, McsB is released from McsA. Thereby, McsB is activated and removes CtsR from the DNA. However, the McsA/McsB complex is not present in all low GC, Gram-positive bacteria. In the species lacking this complex, ClpE is able to act as a redox-sensor probably via its highly conserved N-terminal zinc finger domain. When these cysteine residues are oxidized, ClpE is activated which results in CtsR de-repression. In addition to the transcriptional regulation of CtsR low GC, Gram-positive protein quality control systems are regulated post-transcriptionally. The expression of the McsA/McsB adaptor pair is regulated by CtsR. However, McsB activity is also tightly regulated by three different regulatory proteins (McsA/ClpC/YwlE). McsB is needed to target specific substrates to ClpC, either for refolding or degradation by the ClpCP protease. It was demonstrated that only the auto- phosphorylated form of McsB is able to bind to its substrates. This McsB function is inhibited in non-stressed cells by a direct interaction with ClpC. Consequently, McsB is activated by a release from ClpC during protein stress. In addition, McsB activation depends on the presence of its activator McsA. Accordingly, McsB cannot be activated as an adaptor protein during thiol-specific stress because McsA is no longer able to bind to McsB under these conditions. However, also active McsB is subject to post-translational control. Activated McsB is either de-phosphorylated by McaP or degraded by ClpCP ensuring an appropriate shut-down of the McsB adaptor. Both McaP and ClpC inhibit McsB activity with different intensities. ClpC possesses a stronger impact on McsB activity than McaP but both proteins are needed for an adequate silencing of McsB activity. In addition, it was shown for the first time that B. subtilis McsB is a global adaptor that influences the stability of multiple proteins. The B. subtilis ClpC protein is unlike most members of the Hsp100 family because it not only requires several adaptor proteins for substrate recognition but also for its general ATP- dependent activity. Biochemical analysis revealed how ClpC is activated by distinct adaptor proteins. McsB modulates ClpC activity by regulatory phosphorylation of arginine residues. Moreover, McaP (formerly YwlE) was identified as an arginine phosphatase that modulates the McsB mediated ClpC activity. MecA, another known adaptor protein for ClpC, activates ClpC independently of these arginine phosphorylations, which demonstrates the existence of multiple pathways for ClpC activation.
In this study the potential of molecular RT-PCR based methods for diagnostic or epidemiological investigations concerning negative-sense RNA viruses should be demonstrated exemplary for orthobunyaviruses (segmented genome) and lyssaviruses (non segmented genome). The recent discovery of a novel orthobunyavirus from the Simbu serogroup, Schmallenberg virus (SBV), via next generation sequencing and metagenome analysis led to the development of novel molecular detection methods. Due to the potential emergence of further orthobunyaviruses from the Simbu serogroup, a generic pan-Simbu real-time RT-PCR system was developed. This system was able to detect all tested Simbu serogroup viruses. As additional feature a species classification via sequencing is possible. Moreover, the novel pan-Simbu real-time RT-PCR system seems to offer a broad detection spectrum for orthobunyaviruses in general. Hence, this protocol allows a broad screening of samples predominantly for Simbu serogroup virus genomes but also might allow the identification of some related orthobunyaviruses in mammalian or insect samples. A comparison of the pan-Simbu real-time RT-PCR system with diagnostic real-time RT-PCRs revealed an overall higher sensitivity of the diagnostic assays for SBV detection. The diagnostic SBV-S3 assay convinced with the highest sensitivity and reliability for SBV detection. Additionally, the SBV-M1 assay turned out as highly specific for SBV and therefore is a valuable tool for a precise diagnosis in geographical regions where multiple orthobunyaviruses are endemic. Furthermore, the SBV genome diversity in Germany was investigated using a molecular epidemiological approach. Genome variability was extremely high in the N-terminal region of the putative envelope glycoprotein Gc which might have an impact on immunogenicity or host-cell infection. Phylogenetic analyses indicated that sequence variation is independent of host species and geographical distribution. In contrast to SBV as a novel pathogen, rabies encephalitis (caused by the prototype lyssavirus Rabies virus) is known for more than 4000 years. Thus numerous molecular techniques have been developed for lyssavirus detection, considering the diversity of this genus they all have certain limitations as regards their diagnostic range. Results of a lyssavirus ring trial among European laboratories indicate that RT-PCR could be a highly reliable diagnostic tool if at least two independent tests with broad diagnostic range are applied. Another approach suggested that a change from two-step to one-step PCR strategy or a variation of the RT-chemistry may have a remarkable influence on assay performance. However, no ultimate approach or strategy has been found yet, that would facilitate rabies routine diagnosis or epidemiological surveys on molecular grounds. Thus, there is a need for a potent, reliable and practical system for lyssavirus diagnosis and characterization, suitable as a second diagnostic line next to classical techniques like the fluorescent antibody test. For this purpose a diagnostic two level cascade protocol was developed with emphasis on the most relevant European lyssaviruses. On a first level two independent generic pan-lyssavirus screening assays, targeting different genomic regions, were applied. On a second level two probe-based species-specific multiplex PCR systems for the rapid classification of European lyssaviruses were used. All applied assays displayed an overall highly sensitive and specific detection with an excellent reproducibility and repeatability. Moreover, the diagnostic cascade protocol combines all known advantages of the real-time PCR technology including speed and reduced risk of cross-contamination with improved safety of molecular testing based on a double-check strategy for the screening as well as the confirmatory assays. In the frame of the second Bokeloh bat lyssavirus case in a German bat, the capability of real-time PCR for the quantification of viral loads was demonstrated. Another convenient example for the potential of molecular RT-PCR based methods is the epidemiological investigation of the rabies epizootic in Namibian kudu antelopes. Phylogenetic analyses of a 602 bp fragment of the nucleoprotein gene indicated a separate grouping of the Rabies virus (RABV) isolates from kudu apart from RABV isolates from jackals. Full genome sequencing revealed unique mutations in the glycoprotein gene of RABV isolates from kudu, suggesting an independent rabies cycle in Namibian kudu antelopes. All given examples were used to illustrate the application spectrum of molecular RT-PCR based methods for diagnostic or epidemiological purposes. The advantages of molecular techniques were emphasized and in particular real-time RT-PCR systems proved their fitness for purpose and appear to represent standard techniques for the next decade.
African swine fever virus (ASFV) is one of the most threatening animal viruses which has dramatically expanded its distribution range within the last years. ASFV was first described and is endemic in sub-Saharan Africa where it is transmitted in a sylvatic cycle between indigenous suids and Ornithodoros soft ticks. Therefore, ASFV is the only known DNA-arbovirus and, in addition to that, the only member of the genus Asfivirus within the family Asfarviridae. Being highly infectious to domestic pigs and wild boar, the virus was introduced into Georgia in 2007 and has subsequently spread throughout eastern Europe reaching the European Union in 2014. Despite almost 100 years of intensive research and the occurrence of African swine fever (ASF) on four continents including Europe, many aspects of its epidemiology, vector dynamics and virus evolution are unknown. In our study, first evidence is presented on endogenous ASFV-like (EASFL)- elements which are integrated into the genome of ASFV natural vectors, O. moubata soft ticks. Through a series of experiments including next-generation sequencing, infection experiments, phylogenetic and BEAST analyses as well as PCR-screening, evidence is provided that these elements belong to an ancestral ASFV strain that might have existed 50,000 to 30,000 years BCE. Further results suggest that the EASFL-elements are involved in protecting ticks against ASFV infection and might belong to a generalised tick defence mechanism. In order to evaluate factors influencing ASFV epidemiology in eastern Europe, experiments were conducted on possible indigenous vector species and circulating virus isolates. In the absence of the natural tick vector, blow fly larvae were considered as possible mechanical vectors involved in ASFV transmission and persistence. Results are presented that even after feeding on highly infectious wild boar tissue, fly larvae and pupae showed no contamination with infectious virus. On the contrary, the maggots appeared to have inactivated the virus in the organ tissue through their salivary secretions. Further experiments conducted on an ASFV-strain isolated from northeastern Estonia resulted in the first report of an ASFV-strain with attenuated phenotype isolated in Eastern Europe. Results from NGS-analyses provided evidence for a major genome reorganisation in that strain that included a large deletion and a duplication of multiple ASFV genes.
Taken together, this study provides novel insights into the epidemiology of ASF and evolution of ASFV one of the major threats to animal health worldwide and therefore does not only contribute significantly to basic research but possibly also to specific knowledge necessary for future disease management.
The advances in high-throughput sequencing technologies have revolutionized the possibilities for pathogen identification in cases of unknown disease origin. Diagnostic metagenomics allows the unbiased and simultaneous detection of almost all nucleic acids in a clinical sample, with the potential to provide pivotal insights into otherwise undeterminable causes of human or animal disease.
In this thesis, possibilities, pitfalls and the suitability of Ion Torrent and Illumina sequencing platforms for comprehensive use in diagnostic metagenomics were assessed and optimized procedures developed. Clinical field samples, undiagnosable by standard diagnostics, were taken as real-life examples for the investigations. The results show that cross-contamination due to index swapping and run-to-run-carryover constitute a major issue on Illumina platforms, severely compromising the correct interpretation of results for clinical specimens. In contrast, Ion Torrent platforms did not display any form of cross-contamination, however, the commercial library preparation method is less efficient. Combining the advantages of both platforms, customized Y adapters, facilitating highly efficient library preparation, were developed for Ion Torrent sequencing and applied in further experiments. The obstacles of strongly degraded RNA in formalin-fixed paraffin-embedded samples were identified and the workflow adapted to meet the requirements of smaller fragments. Additionally, it was shown that adequate sampling is a very important step, if not the most important step, in the workflow, as well as subsequent validation of the obtained results in terms of causation. The achievements in this study allow other researchers the application of a sensitive and optimized diagnostic metagenomics workflow.
Furthermore, the investigations on the clinical samples resulted in the discovery of a novel respirovirus with putative zoonotic potential, the first description of Borna disease virus 1 in human organ transplant recipients, and the discovery of a very distantly related novel ovine picornavirus. These discoveries build a basis for further research and expand the knowledge regarding new and emerging viruses.
The order of bats (Chiroptera) account for ~20% of all mammalian species and attracted immense global attention due to their identification as important viral reservoir. Bats can harbour a plethora of high-impact zoonotic viruses, such as filoviruses, lyssaviruses, and coronaviruses without displaying clinical signs of disease themselves. Given this striking diversity of the bat virome, their ability of self-powered flight, and global distribution, understanding chiropteran immunity is essential to facilitate assessment of future spillover events and risks.
However, scarcity of bat-specific or cross-reactive tools and standardized model systems impede progress until today. Furthermore, the richness of species led to generation of isolated datasets, hampering data interpretation and identification of general immune mechanisms, applicable for various chiropteran suborders/families. The key to unlocking bat immunity are coordinated research approaches that comprehensively define immunity in several species. In this work, an in-depth study of innate and adaptive immune mechanisms in the fructivorous Egyptian Rousette bat (Rousettus aegyptiacus, ERB) is presented.
Detailed stability analyses identified EEF1A1 as superior reference gene to ACTB, and GAPDH, which rendered unstable upon temperature increase or presence of type-I-IFN. Since the body core temperatures of pteropid bats reach from 35°C to 41°C and it has been postulated that bats display constitutive expression of IFNs, a suitable reference gene has to be stable under these physiologically relevant conditions. To study cellular innate immunity in detail, cell lines from the nasal epithelium, the olfactory compartment and the cerebrum were generated. To include immune responses of epithelia cells, essential for immunity at sites of primary viral infection, primary epithelia cells from the nasal epithelium, trachea, lung and small intestine were generated. Cellular identities were determined by comprehensive analyses of transcripts and proteins expressed by each cell line. The capacity of each cell line to produce type-I- and III-IFNs was assessed at 37°C and 40°C upon stimulation with viral mimetics. This revealed cell type-dependent differences is the capability to express IFNs upon stimulation. Furthermore, the constitutive expression of type-I- and III-IFNs was significantly elevated in higher temperatures and quantified at mRNA copy levels. To characterize ERB innate immunity upon infection with high-impact zoonotic viruses, cells from the nasal epithelium, the olfactory system, and the brain were infected with several lyssaviruses. This revealed striking differences in susceptibility: cells from the nasal epithelium rendered least whereas cells from the olfactory epithelium rendered most susceptible to viral infection and replication. Additionally, due to a lack of IFN expression in infected cells, it could be shown that LBV possibly possesses advanced strategies to ensure successful replication in ERB cells. Since the current SARS-CoV-2 pandemic put bats even further in the focus of zoonotic research, primary epithelial cells and animals were infected with this virus to monitor ERB-specific immune transcripts in cells and tissues. These studies revealed a notably early IFNG expression in the respiratory tract of infected individuals.
To understand immunomaturation in bats, the immune cell landscape in periphery and various tissue in adult and juvenile ERB was analyzed by flow cytometry and scRNA-seq, revealing intriguing, age-dependent variations in the abundance of granulocytes and lymphocytes. Flow cytometry revealed a significantly higher number of granulocytes in adults, as well as higher numbers of B cells in juveniles. scRNA-seq allowed detailed identification of different leukocyte subsets, uncovering the presence of highly-abundant NKT-like cells and a unique PLAC8 expressing B cell population. A functional characterization of phagocytic cells and lymphocytes derived from adult and juvenile ERB revealed no significant differences in cellular functionality.
In conclusion, the presented work demonstrated suitability of all established ERB cell lines to study bat immunity in vitro, which led to striking findings regarding IFN expression at steady state, or upon stimulation or viral infection. In addition, established qRT-PCR protocols allowed definition of constitutive and temperature-dependent elevation of IFN expression magnitudes, as well as insights into expression of immune-related transcripts in SARS-CoV-2 infected ERB. Finally, based on optimized scRNA-seq technologies and flow cytometry, frequencies and absolute cell counts could be determined in ERB of different ages, revealing e.g. age-dependent variations in leukocyte profile compositions.
The spatio-temporal reduction and oxidation of protein thiols is an essential mechanism in signal transduction inall kingdoms of life. Thioredoxin (Trx) family proteins efficiently catalyze thiol-disulfide exchange reactions andthe proteins are widely recognized for their importance in the operation of thiol switches. Trx family proteinshave a broad and at the same time very distinct substrate specificity–a prerequisite for redox switching. Despiteof multiple efforts, the true nature for this specificity is still under debate. Here, we comprehensively compare theclassification/clustering of various redoxins from all domains of life based on their similarity in amino acidsequence, tertiary structure, and their electrostatic properties. We correlate these similarities to the existence ofcommon interaction partners, identified in various previous studies and suggested by proteomic screenings. Theseanalyses confirm that primary and tertiary structure similarity, and thereby all common classification systems, donot correlate to the target specificity of the proteins as thiol-disulfide oxidoreductases. Instead, a number ofexamples clearly demonstrate the importance of electrostatic similarity for their target specificity, independent oftheir belonging to the Trx or glutaredoxin subfamilies
Swine are regarded as promising biomedical models, but the dynamics of theirgastrointestinal microbiome have been much less investigated than that of humans or mice. The aimof this study was to establish an integrated multi-omics protocol to investigate the fecal microbiomeof healthy swine. To this end, a preparation and analysis protocol including integrated samplepreparation for meta-omics analyses of deep-frozen feces was developed. Subsequent data integrationlinked microbiome composition with function, and metabolic activity with protein inventories, i.e.,16S rRNA data and expressed proteins, and identified proteins with corresponding metabolites.16S rRNA gene amplicon and metaproteomics analyses revealed a fecal microbiome dominated byPrevotellaceae,Lactobacillaceae,Lachnospiraceae,RuminococcaceaeandClostridiaceae.Similar microbiomecompositions in feces and colon, but not ileum samples, were observed, showing that feces can serveas minimal-invasive proxy for porcine colon microbiomes. Longitudinal dynamics in composition,e.g., temporal decreased abundance ofLactobacillaceaeandStreptococcaceaeduring the experiment,were not reflected in microbiome function. Instead, metaproteomics and metabolomics showed arather stable functional state, as evident from short-chain fatty acids (SCFA) profiles and associatedmetaproteome functions, pointing towards functional redundancy among microbiome constituents.In conclusion, our pipeline generates congruent data from different omics approaches on the taxonomyand functionality of the intestinal microbiome of swine.
Avian influenza viruses (AIVs) have their natural reservoir in wild aquatic birds but occasionally
spread to terrestrial poultry. While AIVs of subtypes H5 and H7 are well known to evolve highly
pathogenic avian influenza viruses (HPAIVs) during circulation in domestic birds, non-H5/H7
subtypes exhibit only a low to moderate pathogenicity. Furthermore, spillover events to a broad
range of mammalian hosts, including humans, with self-limiting to severe illness or even fatal
outcomes, were reported for non-H5/H7 AIVs and pose a pandemic risk. The evolution of high
virulent phenotypes in poultry and the adaptation of AIVs to mammalian hosts are predominantly
linked to genetic determinants in the hemagglutinin (HA). The acquisition of a polybasic cleavage
site (pCS) is a prerequisite for the evolution of HPAIVs in poultry, while changes in the receptor
binding preference and virus stability are essential for adaptation of AIVs to mammals.
In August 2012, an H4N2 virus with the pCS motif 322PEKRRTR/G329 but preserved trypsin
dependend replication and low pathogenicity in chickens was isolated on a quail farm in California.
In the first two publications, we followed different approaches to investigate virulence factors and
the potential risk for the transition of H4N2 to high virulence in chickens. The loss of N-terminal
glycosylations in the vicinity of the pCS resulted in decreased binding to avian-like receptors and
dramatically decreased virus stability. On the other hand, one deglycosylation increased virus
replication and tissue tropism in chicken embryos but did not alter virulence or excretion in
chickens. Furthermore, additional basic amino acids in the natural pCS motif improved the trypsin-independent
cleavage of HA and caused slightly increased tissue tropism in chickens. However,
the engineered motifs alone did not affect virulence in chickens. Intriguingly, they even had a
detrimental effect on virus fitness, which was restored after reassortment with segments of HPAIV
H5N1. Together, the results show the importance of HA glycosylations on the stability of H4N2 and
reveal the important role of non-HA segments in the transition of this virus to high virulence in
poultry.
The transmission of another non-H5/H7 AIV of subtype H10N7 from birds to seals resulted in mass
deaths in harbor seals in 2014 in northern Europe. The third publication describes nine mutations
in the HA1 subunit of seal isolates compared to avian H10Nx viruses. We found that some of these
mutations conferred a dual specificity for avian and mammalian receptors and altered
thermostability. Nevertheless, the H10N7seal remained more adapted to avian host cells, despite
of the alteration in the receptor binding specificity.
Altogether, this thesis demonstrates that naturally evolved AIVs beside H5 and H7 subtypes
support a highly pathogenic phenotype in the appropriate viral background and alter virulence and
host receptor specificity by few amino acid substitutions in the HA. These findings improve our
knowledge of the potential of non-H5/H7 AIVs to shift to high virulence in birds and the adaptation
in mammals.
For many years now, Bacillus megaterium serves as a microbial workhorse for the high-level production of recombinant proteins in the g/L-scale. However, efficient and stable production processes require the knowledge of the molecular adaptation strategies of the host organism to establish optimal environmental conditions. Here, we interrogated the osmotic stress response of B. megaterium using transcriptome, proteome, metabolome, and fluxome analyses. An initial transient adaptation consisted of potassium import and glutamate counterion synthesis. The massive synthesis of the compatible solute proline constituted the second longterm adaptation process. Several stress response enzymes involved in iron scavenging and reactive oxygen species (ROS) fighting proteins showed higher levels under prolonged osmotic stress induced by 1.8 M NaCl. At the same time, the downregulation of the expression of genes of the upper part of glycolysis resulted in the activation of the pentose phosphate pathway (PPP), generating an oversupply of NADPH. The increased production of lactate accompanied by the reduction of acetate secretion partially compensate for the unbalanced (NADH/NAD+) ratio. Besides, the tricarboxylic acid cycle (TCA) mainly supplies the produced NADH, as indicated by the higher mRNA and protein levels of involved enzymes, and further confirmed by 13C flux analyses. As a consequence of the metabolic flux toward acetyl-CoA and the generation of an excess of NADPH, B. megaterium redirected the produced acetyl-CoA toward the polyhydroxybutyrate (PHB) biosynthetic pathway accumulating around 30% of the cell dry weight (CDW) as PHB. This direct relation between osmotic stress and intracellular PHB content has been evidenced for the first time, thus opening new avenues for synthesizing this valuable biopolymer using varying salt concentrations under non-limiting nutrient conditions.
For many years now, Bacillus megaterium serves as a microbial workhorse for the high-level production of recombinant proteins in the g/L-scale. However, efficient and stable production processes require the knowledge of the molecular adaptation strategies of the host organism to establish optimal environmental conditions. Here, we interrogated the osmotic stress response of B. megaterium using transcriptome, proteome, metabolome, and fluxome analyses. An initial transient adaptation consisted of potassium import and glutamate counterion synthesis. The massive synthesis of the compatible solute proline constituted the second longterm adaptation process. Several stress response enzymes involved in iron scavenging and reactive oxygen species (ROS) fighting proteins showed higher levels under prolonged osmotic stress induced by 1.8 M NaCl. At the same time, the downregulation of the expression of genes of the upper part of glycolysis resulted in the activation of the pentose phosphate pathway (PPP), generating an oversupply of NADPH. The increased production of lactate accompanied by the reduction of acetate secretion partially compensate for the unbalanced (NADH/NAD+) ratio. Besides, the tricarboxylic acid cycle (TCA) mainly supplies the produced NADH, as indicated by the higher mRNA and protein levels of involved enzymes, and further confirmed by 13C flux analyses. As a consequence of the metabolic flux toward acetyl-CoA and the generation of an excess of NADPH, B. megaterium redirected the produced acetyl-CoA toward the polyhydroxybutyrate (PHB) biosynthetic pathway accumulating around 30% of the cell dry weight (CDW) as PHB. This direct relation between osmotic stress and intracellular PHB content has been evidenced for the first time, thus opening new avenues for synthesizing this valuable biopolymer using varying salt concentrations under non-limiting nutrient conditions.
For many years now, Bacillus megaterium serves as a microbial workhorse for the high-level production of recombinant proteins in the g/L-scale. However, efficient and stable production processes require the knowledge of the molecular adaptation strategies of the host organism to establish optimal environmental conditions. Here, we interrogated the osmotic stress response of B. megaterium using transcriptome, proteome, metabolome, and fluxome analyses. An initial transient adaptation consisted of potassium import and glutamate counterion synthesis. The massive synthesis of the compatible solute proline constituted the second longterm adaptation process. Several stress response enzymes involved in iron scavenging and reactive oxygen species (ROS) fighting proteins showed higher levels under prolonged osmotic stress induced by 1.8 M NaCl. At the same time, the downregulation of the expression of genes of the upper part of glycolysis resulted in the activation of the pentose phosphate pathway (PPP), generating an oversupply of NADPH. The increased production of lactate accompanied by the reduction of acetate secretion partially compensate for the unbalanced (NADH/NAD+) ratio. Besides, the tricarboxylic acid cycle (TCA) mainly supplies the produced NADH, as indicated by the higher mRNA and protein levels of involved enzymes, and further confirmed by 13C flux analyses. As a consequence of the metabolic flux toward acetyl-CoA and the generation of an excess of NADPH, B. megaterium redirected the produced acetyl-CoA toward the polyhydroxybutyrate (PHB) biosynthetic pathway accumulating around 30% of the cell dry weight (CDW) as PHB. This direct relation between osmotic stress and intracellular PHB content has been evidenced for the first time, thus opening new avenues for synthesizing this valuable biopolymer using varying salt concentrations under non-limiting nutrient conditions.
In vitro and in vivo analyses of mono- and mixed-species biofilms formed by microbial pathogens
(2022)
Microbial biofilms can be defined as multicellular clusters of microorganisms embedded in a self-produced extracellular matrix (ECM), which is primarily composed of polymeric biomolecules. Biofilms represent one of the most severe burdens in both industry and healthcare worldwide, causing billions of dollars of treatment costs annually because biofilms are inherently difficult to prevent, treat, and eradicate. In health care settings, patients suffering from cystic fibrosis, or patients with medical implants are highly susceptible to biofilm infections. Once a biofilm is formed, it is almost impossible to quantitatively eradicate it by mechanical, enzymatical, chemical, or antimicrobial treatment. Often the only remaining option to fully eradicate the biofilm is removing of the infected implant or body part. The primary reasons for the inherent resistance of biofilms against all forms of antimicrobial treatment are (I) a reduced metabolic activity of biofilm-embedded cells climaxing in the presence of metabolic inactive persister cells, as well as (II) the protective nature of the biofilm matrix acting as a (diffusion) barrier against antimicrobials and the host immune system. Consequently, there is an urgent need to better understand microbial biofilms from a structural and (patho-) physiological point of view in order to be able to develop new treatment strategies.
Therefore, the aims of this study were to investigate fundamental physiological properties of different clinically relevant single and multi-species biofilms, both in vitro and in vivo. Furthermore, the effectiveness of a novel treatment strategy using cold atmospheric pressure plasma was evaluated in vitro to treat biofilms of the pathogenic fungus C. albicans.
In article I, the intracellular and ECM protein inventory of Staphylococcus aureus during in vitro biofilm growth in a flow reactor was analyzed by liquid-chromatography coupled to tandem mass-spectrometry (LC-MS/MS) analysis combined with metabolic footprint analysis. This analysis showed that anaerobiosis within biofilms releases organic acids lowering the ECM pH. This, in turn, leads to protonation of alkaline proteins – mostly ribosomal proteins originating from cell lysis as well as actively secreted virulence factors – resulting in a positive net charge of these proteins. As a consequence, these proteins accumulate within the ECM and form an electrostatic network with negatively charged cell surfaces, eDNA, and metabolites contributing to the overall biofilm stability.
In article II, the in vivo metaproteome of the multi-species biofilm community in cystic fibrosis sputum was investigated. To this end, an innovative protocol was developed allowing the enrichment of microbial cells, the extraction of proteins from a small amount of cystic fibrosis sputum, and subsequent metaproteome analysis. This protocol also allows 16S sequencing, metabolic footprint analysis, and microscopy of the same sample to complement the metaproteome data. Applying this protocol, we were able to significantly enhance microbial protein coverage providing first insights into important physiological pathways during CF lung infection. A key finding was that the arginine deaminase pathway as well as microbial proteases play a so far underappreciated role in CF pathophysiology.
In articles III and IV, a novel treatment strategy for biofilms formed by the important fungal pathogen Candida albicans was evaluated in vitro. Biofilms were treated with two different sources of nonthermal plasma (with the Nonthermal Plasma Jet “kINPen09” as well as with the Microwave-induced plasma torch “MiniMIP”) and the effect on growth, survival, and viability was assessed by counting colony-forming units (CFU), by cell proliferation assays, as well as by live/dead staining combined with fluorescence microscopy, confocal laser scanning microscopy, (CLSM) and atomic force microscopy (AFM). These tests revealed that biofilms were effectively inactivated mostly on the bottom side of biofilms, indicating a great potential of these two plasma sources to fight biofilms.
An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum
(2021)
Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This facilitates the colonization of the lung by microbial pathogens and the concomitant establishment of chronic infections leading to tissue damage, reduced lung function, and decreased life expectancy. Although the interplay between key CF pathogens plays a major role during disease progression, the pathophysiology of the microbial community in CF lungs remains poorly understood. Particular challenges in the analysis of the microbial population present in CF sputum is (I) the inhomogeneous, viscous, and slimy consistence of CF sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. To address these challenges, we used 21 CF sputum samples to develop a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, protein extraction and subsequent metaproteomic analyses. As a proof of concept, we selected three sputum samples for detailed metaproteome analyses and complemented and validated metaproteome data by 16S sequencing, metabolomic as well as microscopic analyses. Applying our protocol, the number of bacterial proteins/protein groups increased from 199-425 to 392-868 in enriched samples compared to nonenriched controls. These early microbial metaproteome data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera could so far be underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo – an important prerequisite for the development of novel antimicrobial therapies to combat chronic recurrent airway infection in CF.
Lipoproteins of Staphylococcus aureus represent a major class of surface proteins, which are anchored to the outer leaflet of the cell membrane. Although they play a key role in the immune response and virulence, the majority of lipoproteins in this organism is still of unknown function. The aim of our study was to investigate the function of so far poorly or uncharacterized lipoproteins in S. aureus strain Newman. To this end, an integrated bioinformatical approach was applied to define the pan-lipoproteome of 123 completely sequenced S. aureus strains. In total, this analysis predicted 192 different potential lipoproteins, with a core lipoproteome of 39 and a variable lipoproteome of 153 lipoproteins. Out of those 192 lipoproteins, 141 are so far functionally uncharacterized. Primarily focusing on members of the core-lipoproteome with unknown or poorly characterized function, 24 lipoproteins or co-encoded neighbor proteins were selected for further characterization. Of those 24 proteins, 20 S. aureus markerless deletion mutants were constructed (S. aureus delta l01 - delta l20) and screened for an altered growth behavior under various conditions. Here, three mutants showed a temperature-sensitive phenotype, two mutants formed aggregates in the TSB of the manufacturer Merck (TSBMerck), and four mutants showed reduced growth under osmotic stress with 8% NaCl. An altered aggregation behavior was observed for four mutants in the presence of Triton X-100 and for eleven mutants in the presence of SDS. Furthermore, ten mutants revealed an impaired biofilm formation capacity as well as reduced hemolytic activity. Interestingly, S. aureus deletion mutants delta l14 (delta NWMN_1435) and delta l16 (delta NWMN_0646) showed an altered phenotype under nearly all tested growth and stress conditions. Most strikingly, both deletion mutants demonstrated dramatic defects in cell morphology and cell division during the transient growth phase in TSBMerck and were therefore selected for further detailed characterization. Electron microscopy imaging of the two mutants revealed an irregular cell shape, increased cell size, multiple displaced division septa, and incomplete separation of daughter cells resulting in the formation of cell aggregates in TSBMerck. Complementarily, microarray-based transcriptome analysis and whole-genome sequencing of S. aureus delta l14 and delta l16 suppressor mutants strongly point to a functional association of both lipoproteins with cell envelope- or cell division-related processes. Specifically, multiple hints suggest a functional connection of both lipoproteins with lipo- or wall teichoic acids. Of note, the phenotypes of S. aureus delta l14 and delta l16 are conditional and appear under some, but not all growth conditions. Thus, it is conceivable that the function of L14 and L16 is modulated by metabolic processes, or that the proteins might be part of a “backup system” becoming important only under certain conditions. Collectively, we propose that L14 and L16 fulfill a basic role in cell envelope- or cell division-related processes under specific growth conditions. Particularly, the activity of L14 and L16 might be necessary for the function or localization of lipo- or wall teichoic acids, and thus, might be linked to the regulation of autolysins. In conclusion, this study reveals important insights into the function of two so far uncharacterized but highly conserved lipoproteins in S. aureus.
Out of Control: The Role of the Ubiquitin Proteasome System in Skeletal Muscle during Inflammation
(2021)
The majority of critically ill intensive care unit (ICU) patients with severe sepsis develop ICU-acquired weakness (ICUAW) characterized by loss of muscle mass, reduction in myofiber size and decreased muscle strength leading to persisting physical impairment. This phenotype results from a dysregulated protein homeostasis with increased protein degradation and decreased protein synthesis, eventually causing a decrease in muscle structural proteins. The ubiquitin proteasome system (UPS) is the predominant protein-degrading system in muscle that is activated during diverse muscle atrophy conditions, e.g., inflammation. The specificity of UPS-mediated protein degradation is assured by E3 ubiquitin ligases, such as atrogin-1 and MuRF1, which target structural and contractile proteins, proteins involved in energy metabolism and transcription factors for UPS-dependent degradation. Although the regulation of activity and function of E3 ubiquitin ligases in inflammation-induced muscle atrophy is well perceived, the contribution of the proteasome to muscle atrophy during inflammation is still elusive. During inflammation, a shift from standard- to immunoproteasome was described; however, to which extent this contributes to muscle wasting and whether this changes targeting of specific muscular proteins is not well described. This review summarizes the function of the main proinflammatory cytokines and acute phase response proteins and their signaling pathways in inflammation-induced muscle atrophy with a focus on UPS-mediated protein degradation in muscle during sepsis. The regulation and target-specificity of the main E3 ubiquitin ligases in muscle atrophy and their mode of action on myofibrillar proteins will be reported. The function of the standard- and immunoproteasome in inflammation-induced muscle atrophy will be described and the effects of proteasome-inhibitors as treatment strategies will be discussed.
As the animal-to-human interface becomes increasingly narrow, transmission events of zoonotic pathogens between animals and humans become more and more probable. While SARS-CoV-2 already accomplished a spillover infection to humans and is responsible for the current pandemic, the bat H9N2 IAV with so far unknown zoonotic potential was only recently discovered. In order to identify I) the role and potential of a newly discovered, potentially pre-pandemic virus, such as the bat H9N2, or II) possible future prevailing virus mutant variants of an already known pandemic virus, such as SARS-CoV-2, it is important to characterize these emerging viruses in vivo as soon and as good as possible.
The first objective in this dissertation (Publications I and II) therefore deals with the characterization of bat H9N2 and the estimation of its zoonotic or even pandemic potential.
In Publication I, a general susceptibility of directly inoculated Egyptian fruit bats to bat H9N2 was confirmed by successful seroconversion, although exhibiting only moderate viral shedding. All three contact animals remained seronegative, though one contact bat showed slight lesions in the histopathological analysis.
Publication II further addressed the question of the zoonotic potential of this virus. Inoculation of day-old turkey hatchlings demonstrated moderate susceptibility to bat H9N2 infection with a measurable seroconversion, while day-old chicken hatchlings were not susceptible to bat H9N2. Ferrets proved to be highly susceptible to bat H9N2 with high viral shedding, a transmission efficiency rate of 100% to direct contact animals at 2 days post contact, but with only minimal clinical signs. Importantly, the virus demonstrated the ability to evade the MxA-restriction factor and to replicate efficiently in human lung tissue explants. Furthermore, seasonal IAV- and standard IAV-vaccines showed no cross reactivity against the bat-N2 protein in humans. Therefore, further research on such viruses is urgently needed in order to prevent a renewed pandemic situation in the future as caused by SARS-CoV-2.
The second objective in this dissertation dealt with the identification and characterization of emerging SARS-CoV-2 Variants of Concern (VOCs).
Therefore, in Publication III, competitive infection experiments were performed using the Syrian golden hamster, the ferret, and transgenic mouse models (K18-hACE2 and hACE2-KI). These studies revealed replicative and transmissive predominance of Alpha VOC over Beta VOC, but not over SARS-CoV-2 WT in the hamster model, although Beta VOC substantially replicated in the lungs of donor animals. In contrast, the Alpha VOC had an unambiguous replication and transmission advantage over WT SARS-CoV-2 in the ferret and both mouse models. A recombinant SARS-CoV-2 WT-SAlpha virus helped to assign the fitness advantage of this variant particularly to the spike protein-associated mutations.
In Publication IV, in vitro results inferred an early replicative fitness advantage of Omicron BA.1 over Delta VOC, although the opposite was observed in competitively inoculated hamsters, ferrets and naive hACE2-KI mice. In addition, Publication IV demonstrated a disadvantage in transmission for the VOC Omicron BA.1 over the Delta VOC and a lack of susceptibility of ferrets after a single infection with the VOC Omicron BA.1. An mRNA vaccination of K18-hACE2 mice caused a drastic reduction of infectious virus particles in organ material following an infection with a recombinant SARS-CoV-2 WT-SDelta, but not when challenged with the SARS-CoV-2 SOmicron BA.1 clone.
This dissertation includes numerous, comprehensive experimental studies that are generally important for the characterization of emerging, potentially pre-pandemic viruses and may provide crucial information about the future dominance of certain virus variants in an ongoing pandemic. Here, the need for the use of a variety of animal models becomes apparent. By characterizing and classifying potentially zoonotic strains, these methods will help to better prepare for potentially upcoming pandemics and, in the case of a zoonotic or even pandemic event, to better detect and understand the circulating strains and their evolution.
The here presented dissertation investigated the molecular mechanisms, by which the food industry model bacteria Pseudomonas fluorescens and Listeria monocytogenes, grown either as planktonic cultures, were inhibited by plasma treated water (PTW) produced by a microwave-induced plasma source (MidiPLexc). As a starting point, optimal operating parameters were determined with 5 standard liters per minutes(slm)compressed air during the treatment of 10 ml deionized water within a treatment time of up to 15 min (pre-treatment time). Treatment times of 1, 3 and 5 min were selected (post-treatment time). In addition to physical parameters, i.e. temperature measurements at different spots at the plasma source during the production of the PTW, the chemical composition of PTW was determined by pH measurements, chronoamperometry (determination of the H2O2 concentration), ion chromatography (determination of the NO2-, NO3- and ONOO- concentrations) and mass spectrometry (qualitative determination of the molecules). In addition, concentration changes of reactive species over a period of 3 h indicated a decrease of the NO2- concentration as well as an increase of the NO3- and ONOO- concentration in the PTW. Microbiological assays, i.e. quantification of colony-forming units (CFU), fluorescence and XTT assays, revealed a significant reduction of the proliferation ability of the cells, membrane damages and metabolic activity have been demonstrated for planktonic cultures as well as mono- and multispecies biofilms. PTW effects on biofilm structures were investigated using microscopic methods such as fluorescence microscopy, confocal laser scanning microscopy (CLSM), atomic force microscopy (AFM), and scanning electron microscopy (SEM), as well as physical methods such as contact angle measurements. Significant changes in the biofilm structure have been shown, which indicate an ablation of the biofilm mass from top to bottom by approximately 2/3 of the biofilm mass and a destruction of the extracellular matrix (ECM) by the reactive species within the PTW. Subsequently, fresh-cut lettuce has been treated with PTW produced by up-scaled plasma sources. Apart from qualitative parameters of the lettuce after PTW treatment such as texture and color, the concentration of PTW reactive species have been determined. These experiments showed that the composition of the reactive species were slightly different from that of the laboratory-scaled plasma source MidiPLexc. Notably, the PTW treatment did not cause significant changes in texture and color of the fresh-cut lettuce. Finally, a synergistic effect of PTW treatment followed by plasma-processed air (PPA) drying was demonstrated application-specific.
Summary
The susceptibility of Candida albicans biofilms to a non‐thermal plasma treatment has been investigated in terms of growth, survival and cell viability by a series of in vitro experiments. For different time periods, the C. albicans strain SC5314 was treated with a microwave‐induced plasma torch (MiniMIP). The MiniMIP treatment had a strong effect (reduction factor (RF) = 2.97 after 50 s treatment) at a distance of 3 cm between the nozzle and the superior regions of the biofilms. In addition, a viability reduction of 77% after a 20 s plasma treatment and a metabolism reduction of 90% after a 40 s plasma treatment time were observed for C. albicans. After such a treatment, the biofilms revealed an altered morphology of their cells by atomic force microscopy (AFM). Additionally, fluorescence microscopy and confocal laser scanning microscopy (CLSM) analyses of plasma‐treated biofilms showed that an inactivation of cells mainly appeared on the bottom side of the biofilms. Thus, the plasma inactivation of the overgrown surface reveals a new possibility to combat biofilms.
Members of the species Bacillus pumilus get more and more in focus of the biotechnological industry as potential new production strains. Based on secretome analysis, Bacillus pumilus strain Jo2, possessing high secretion capability, was chosen for an omics based investigation. The physiology of Bacillus pumilus cells growing either in minimal or complex medium was analyzed by a combination of proteomic and metabolomic methods. Master gels of the cytosolic and the secreted proteome covering major parts of the main metabolic pathways were created by means of 2D gel electrophoresis. Quantification of 2D gels allowed displaying the most abundant proteins in these sub-proteomes. Application of the GeLC-MS/MS technique tripled the number of identified proteins and enabled detection of many intrinsic membrane proteins. In total, 1542 proteins were identified in growing B. pumilus cells, among them 1182 cytosolic proteins, 297 membrane and lipoproteins and 63 secreted proteins. This accounts for about 43 % of the 3616 proteins encoded in the B. pumilus Jo2 genome sequence. By using GC-MS, IP-LC/MS and H-NMR methods numerous metabolites were analyzed and assigned to the reconstructed metabolic pathways. Our data indicate that applying a combination of proteomic and metabolomic techniques a comprehensive view of the physiology of growing B. pumilus cells can be gained. In addition, selected production-relevant genome features such as the restriction modification system, NRPS clusters and the secretory system of B. pumilus Jo2 are discussed. In their natural habitat, the soil, B. pumilus cells are often exposed to growth limiting conditions due to the lack of sufficient amounts of nutrients. Such limitations can also occur during fermentation conditions and will negatively influence the efficiency of the process. Glucose is the main carbon and energy source of B. pumilus. Thus, a deficiency of glucose has an enormous impact on cell growth. A 1D LC-MS/MS approach was performed to quantify the proteins using an N14/N15 labeling and to analyze the changes in the protein equipment when B. pumilus cells stop their exponential growth and become stationary due to limitation of glucose. 1033 proteins in the cytosolic fraction of B. pumilus cells were quantified and 272 of them appeared to be upregulated when the cells experience glucose starvation. 2D-PAGE was used to analyze the exoproteome of those cells. Glucose starving B. pumilus cells seemed to focus on usage of proteins and peptides as alternative carbon and energy sources instead of other carbohydrates. Especially the exoproteome of glucose starving cells is dominated by proteases and peptidases. Furthermore, cells used fatty acids as carbon source indicated by upregulation of enzymes involved in β-oxidation and the methylcitrate pathway. Bacillus pumilus is characterized by a higher oxidative stress resistance than other comparable industrially relevant Bacilli such as B. subtilis or B. licheniformis. In this study the response of B. pumilus to oxidative stress was investigated during a treatment with high concentrations of hydrogen peroxide at the proteome, transcriptome and metabolome level. Genes/proteins belonging to regulons, which are known to have important functions in the oxidative stress response of other organisms, were found to be upregulated, such as the Fur, Spx, SOS or CtsR regulon. Strikingly, parts of the fundamental PerR regulon responding to peroxide stress in B. subtilis are not encoded in the B. pumilus genome. Thus, B. pumilus misses the catalase KatA, the DNA-protection protein MrgA or the alkyl hydroperoxide reductase AhpCF. Data of this study suggests that the catalase KatX2 takes over the function of the missing KatA in the oxidative stress response of B. pumilus. The genome-wide expression analysis revealed an induction of bacillithiol (Cys-GlcN-malate, BSH) relevant genes. An analysis of the intracellular metabolites detected high intracellular levels of this protective metabolite, which indicates the importance of bacillithiol in the peroxide stress resistance of B. pumilus. Using the physiological knowledge gained during our studies, we analyzed samples taken during an industrial fermentation process. Five samples were taken during the processes using a protease overexpressing B. pumilus strain and a non-overexpressing B. pumilus reference strain. 2D-PAGE was employed to analyze the samples. 448 proteins could be identified in the samples from the protease overexpressing stain as well as 453 proteins in the reference strain. The proteins were quantified relatively comparing the different growth phases of each strain as well as comparing the strains to each other. The physiological knowledge gained from the shake flask studies enabled us to interpret the findings. Both strains showed an induction of proteins involved in acquisition of alternative carbon sources and of proteins involved in degradation and usage of fatty acids, e.g. the methylcitrate pathway, when they stop exponential growth. This is comparable to the results gained from the analysis of B. pumilus cells under glucose limitation, indicating similar conditions during the processes. Especially in the late phases of the fermentation processes the cells were obviously exposed to severe stress conditions. Our results demonstrated that overexpressing cells showed a significantly stronger oxidative stress response at the end of the fermentation process compared to non-overexpressing cells, which indicated that not only the high cell densities but also the overproduction of the target protein might be responsible for these conditions.
Bats are ancient mammals that evolved more than 50 million years ago. There are 1,240 different species (> 20% of mammalian species) described so far, which represent one of the most abundant, diverse and widely distributed mammalian groups. Bats are the only mammals which actively fly and therefore can migrate to different areas of the world. It has been increasingly recognized that bats are reservoirs for more than 100 virus species, and several are associated with animal and human epidemics. As natural hosts of rabies virus (RABV) and related lyssaviruses, bats have become a focus of research not only in South America and Africa, but also in Europe and North America. Bats are also considered to be unique in their potential to host emerging and re-emerging zoonotic viruses. To evaluate and reduce the potential risk of rabies transmission to humans or carnivore hosts (like fox, raccoon and dog etc.), active and passive surveillance studies of bat have been performed. Using these approaches diverse lyssaviruses have been detected in bats. However, these studies did not explain the rarely discovered epidemics and the underlying resistance or immune mechanisms in bats as natural hosts for lyssaviruses. Probably, bats are more resistant to lyssavirus infections than other animals. This hypothesis is introducing the research questions of the present thesis: (1) How do the innate immune responses protect bats from fatal outcome of lyssavirus infections? Interferon (IFN) responses which can be induced by the recognition of viruses by pattern recognition receptors act as the first line of defense against lyssavirus infections. Therefore, type I and type III IFNs from European bats were cloned and functionally characterized in this thesis (Chapter 2 and 4). (2) How do the lyssaviruses adapt to escape the host defenses by counteracting the IFN-mediated immune responses? And how do the bats control the viral replication via the IFN responses? To explore the complicated interactions and understand how European bats (Eptesicus serotinus, Myotis myotis and Nyctalus noctula) interact with European bat lyssaviruses (EBLV-1 and 2), a natural host related model for investigations of the bat´s immune system and the virus-host interactions has to be established. Since all of 52 identified European bats species are endangered and strictly protected, stable cell lines from different tissues of M. myotis for in vitro studies were developed and used for molecular and functional studies (Chapter 3 and 4). The data obtained from this thesis revealed that: (a) European bat IFNs do have similar but also distinct molecular characteristics compared with other mammalian IFNs (Chapter 2 and 4); (b) Both investigated bat type I IFNs, IFN-Kappa; and IFN-Omega; present potent anti-lyssaviral activities and display a pathogen associated pattern in the tested cell line (Chapter 2); (c) The established immortalized M. myotis cell lines are differently susceptible to lyssaviruses and contain a functional IFN-mediated signaling cascade (Chapter 3); (d) Bat type III IFN-Lambda;s display cell-type specific functions due to the distinct expression of the IFN-Lambda; receptor (Chapter 4); (e) In bat cell lines a possible evasive strategy of lyssavirus is based on the counteraction of IFN production and/or IFN-mediated defensive pathways (Chapter 3); (f) The higher resistance of brain derived cell line MmBr compared to other cell lines to lyssavirus infection indicates the natural ability of bat´s central nervous system to control the growth of neurotropic virus, which might be an essential reason for the nonclinical outcome (Chapter 3). Overall, the present thesis provides first insights into IFN-mediated innate immune responses against RABV and EBLVs infection in their natural reservoir hosts and an useful toolbox for comparative analysis of virus-host interactions.