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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.
The aim of this work was to characterize the distribution of TULV in European common vole populations, to clarify the host association of TULV and to investigate correlations between host population dynamics and changes in TULV prevalence. Furthermore, the potential of common voles as reservoir for other rodent-borne pathogens was examined in comparison to other rodent species.
Molecular and serological analysis of rodents captured at 87 locations in Germany, France, Luxembourg, and Austria revealed TULV infections at 53.6 % of all trapping locations. The seroprevalence in common voles was low with a mean of 8.5 % (range: 0 – 19 %). TULV RNA was more often detected (mean: 15.3 %, range 0 - 37.5 %). Field voles (Microtus agrestis) and water voles (Arvicola amphibius) were less often tested positive for TULV: mean seroprevalence was 7 % for field voles and 6.7 % for water voles. RNA could be detected in 5.4 % of all tested field voles and 3.2 % of water voles and with exception of a single field vole only when TULV-RNA-positive common voles were trapped at the same location. Those results indicate that TULV infections of field and water voles are spillover infections from sympatric TULV-infected common voles. Phylogenetic analysis revealed distinct genetic differences between TULV sequences of regions of greater geographical distance which were associated with different evolutionary common vole lineages. Furthermore, we could detect genetic differences between TULV strains from trapping sites close to each other (ca. 10 km).
In a capture-mark-recapture study 1042 common voles captured in live traps in Germany were sampled as well as 225 captured in snap traps. When analyzing the seroprevalence of fluctuating common vole populations over several years and seasons we found a negative correlation between prevalence and population density in the current season but a delayed density-dependent positive correlation between the current population density and seroprevalence in the next season. However, this trend varied geographically between the four trapping locations. Usually, population density as well as seroprevalence peaked at the end of the reproductive period in autumn with the exception of Weissach (2010-2012), Jeeser (2010) and Gotha (2012) where population peaks in summer were observed.
In a pilot study in Austria common voles were captured as well as three other rodent species. They were investigated not only for presence of different viruses (TULV, Dobrava- Belgrade orthohantavirus (DOBV), Puumala orthohantavirus (PUUV), Lymphocytic choriomeningitis mammarenavirus (LCMV), Cowpox virus (CPXV)) but also pathogenic bacteria and endoparasites (Leptospira spp., Toxoplasma gondii, Borrelia afzelii, Coxiella burnetii, Rickettsia spp. und Bartonella spp.). Of all four captured species, common voles were most often infected with at least one pathogen (66.7 %), followed by wood mice (Apodemus sylvaticus) (57.7 %), bank voles (Myodes glareolus) (35 %) and yellow-necked field mice (Apodemus flavicollis) (34.5 %). Common voles were also exceptionally susceptible to multiple infections: 66.7 % of them were infected with two or three different pathogens, compared to 6.9 % of yellow-necked field mice and 2.5 % of bank voles. No multiple infections could be detected in wood mice.
The broad geographic distribution of TULV in its reservoir host is in contrast to the rare reports of human infection but might be explained with a low pathogenicity for humans or with the low prevalence in host populations. In addition, the rare detection of human TULV infections could be a result of the used diagnostic methods. Since the reservoir population is known for its dramatic changes in population density and recurring superabundances which facilitates frequent contact to humans, TULV should more often be considered as cause for human disease in future analysis. In
addition, several other zoonotic pathogens could be detected in common voles which could influence TULV infections in the reservoir host but also TULV transmission to humans and therefore deserve more attention in future research.
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 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.
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.
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.
Compared to other human pathogens, S. aureus outstands with a remarkably broad spectrum of deseases: from minor skin infections over endocarditis, pneumoniae, and osteomyelitis, to septic shock. The prerequisite is an arsenal of adaptation strategies, encoded in the core and variable genome. It includes the coordinated expression of adhesins and toxins, evasion of the immune system, response to stress and starvation, adaptation of the metabolism, formation of biofilms and capsules, antibiotic resistance, and persistence on the skin, in nasal epithelial cells, and even in the inner of macrophages after phagocytosis. All these adaptation strategies enable S. aureus to colonize a diversity of niches within the human host. The inevitable requirement is the ability to activate the appropriate adaptation strategy at the right time and at the right place. S. aureus overcomes this challenge with a sophisticated regulatory network. This PhD thesis covers a broad spectrum of transcriptional regulators, involved in S. aureus pathogenesis: (1) the quorum sensing system Agr (regulation of early- and late stage virulence factors), (2) the Sar family (regulation of early- and late stage virulence factors), (3) SaeRS (regulation of accessory exotoxins and adhesins), (4) CodY (response to amino acid starvation, including extracellular proteases), (5) Sigma B (general stress response, including virulence factors), (6) Rex (anaerobic energy metabolism), (7) CtsR and HrcA (protein quality control), (8) PerR and Fur (oxidative stress response), and (9) antibiotic resistance. Traditionally, Proteomics constitute the long-lasting reputation of the Institute. In fact, the majority of investigations presented in this PhD thesis was initialized by proteomic analyses as the ultimate starting point. From the first day, a major goal of this PhD thesis was to add regulator-promoter interaction studies to the methodical spectrum. In particular, to complement transcriptomic and proteomic results by answering the logical follow-up question: Which regulator is responsible for the observed changes in gene expression and protein synthesis after application of a specific stimulus?
The first chapter provides specific analyses for three major regulators: Rex, CodY, and SarA. Publications were achieved for Rex (Hecker et al., 2009; Pagels et al., 2010). Results were mainly achieved by establishing regulator-promoter interaction methods (in particular EMSA and “footprinting”). Additionally, this chapter describes method development of a novel easy-to-apply method, named REPA (restriction endonuclease protection assay).
The second chapter presents method development for the genome-wide identification of regulator-promoter interactions, named “global footprinting”. This approach combines two already well-established methods: (A) Purification of a recombinant Strep-tagged regulator via Strep-tag affinity chromatography. The modification in “global footprinting” is to incubate the regulator with fragmented genomic S. aureus DNA, resulting in co-purification and enrichment of DNA streches with specific regulator binding sites. (B) Identification and quantification of these DNA streches via “next generation sequencing” (NGS). Using this combined approach, this PhD thesis was able to localize the most affine promoter binding site for the regulator Rex precisely down to one single base pair across the whole S. aureus genome.
The third chapter describes the assembly of a data library, collecting the majority of DNA microarray data and regulator-promoter interaction studies from the worldwide literature. This data library summarizes more than 50,000 regulatory events and more than 2,000 regulator binding sites. As published in the perspectives in Fuchs et al. (2018), this data library can be incorporated into the free-accessible online data base “Aureowiki” (provided and maintained by the Department of Functional Genomics, University of Greifswald). The major effort is the consolidation of these “big data” via in silico cluster analysis, comparing 282 different experimental conditions at once. The major finding of this analysis is the identification of seven functional and regulatory gene clusters in S. aureus pathogenesis that are conserved across S. aureus strain diversity. These findings allowed the creation of a prediction tool, to provide novel experimental starting points for the worldwide S. aureus research community. This prediction tool was successfully applied on several topics, and partially published: functional and regulatory prediction for a set of 20 selected lipoproteins as potential virulence factors (Graf et al., 2018), and prediciton of protein complexes (Liang et al., 2016).
Alltogether, this PhD thesis provides new insights into the molecular mechanisms of three pathogenesis-relevant regulators: Rex, CodY, and SarA. It describes the development of three novel experimental methods for wet and dry lab applications that can be used on research topics beyond S. aureus: REPA, “global footprinting”, and cluster analysis. Finally, cluster analysis identifies seven conserved fuctional and regulatory gene clusters, involved in S. aureus pathogenesis. This cluster anaysis is used as a prediction tool to provide novel experimental starting points, and to predict the physiological mode of action of newly discovered anti-staphylococcal agents.
A significant fraction of the decaying algal biomass in marine ecosystems is expected to be mineralized by particle-associated (PA) heterotrophic bacterial communities, which are thus greatly contributing to large-scale carbon fluxes. Whilst numerous studies have investigated the succession of free-living (FL) marine bacteria, the community structure and functionality of PA bacterial communities remained largely unexplored and knowledge on specific contributions of these microorganisms to carbon cycling is still surprisingly limited. This has mostly been due to technical problems, i.e., caused by the enormous complexity of marine particles and the high abundance of eukaryotic microorganisms within these particles. This thesis presents (a) an optimized metaproteomics protocol for an in-depth characterization of marine PA bacteria, (b) an application example with FL and PA communities sampled during a spring phytoplankton bloom in 2009 in the North Sea, which confirmed the reliability of the optimized metaproteomic workflow, (c) the metaproteomic analysis of particulate communities sampled during a spring phytoplankton bloom in 2018, resulting in an as yet unprecedented number of identified protein groups of the bacterial response bloom and (d) a proteomic analysis of a PA bacterial isolate grown on the two naturally abundant marine polysaccharides laminarin and alginate. The observed succession of bacterial clades during metaproteomic analyses of the investigated blooms highlights individual niche occupations, also visible on genus level. Additionally, functional data shows evidence for the degradation of different marine polysaccharides e.g., laminarin, alginate and xylan supporting the important role of PA bacteria during the turnover of oceanic organic matter. Furthermore, most of the identified functions fit well with the current understanding of the ecology of an algal- or surface-associated microbial community, additionally highlighting the importance of phytoplankton-bacterial interactions in the oceans. More detailed insights into the metabolism of PA bacteria were gained by the proteomic characterization of a selected PA bacterial isolate grown on laminarin and alginate. Functional analyses of the identified proteins suggested that PA bacteria employ more diverse degradation systems partially different from the strategies used by FL bacteria.
Gram-negative bacteria are known to naturally produce outer membrane vesicles (OMVs), which are closed nanoparticles (10 to 450 nm) containing virulence factors and pathogen associated molecular patterns (PAMPs). For over 20 years, OMVs of Neisseria meningitidis (N. meningitidis), in combination with three purified outer membrane proteins, have been successfully used as parts of human vaccines which illustrates the safety and potential of OMV based vaccines. So far only little is known about the OMVs of fish pathogenic bacteria. The production of OMVs has been described for the fish pathogenic gram-negative bacterium Aeromonas salmonicida (A. salmonicida) which is the causative agent of furunculosis resulting in high morbidity and mortality of salmonid fish. The immunostimulatory potential of OMVs derived from A. salmonicida as well as the possibility of establishing an oral vaccine model in Oncorhynchus mykiss (O.mykiss) (Rainbow trout) has been investigated in this study by conducting in vitro and in vivo experiments. Innate immune cells such as macrophages are one of the first cells to respond to pathogens once they breach the skin barrier, therefore the monocyte/macrophage cell line RTS-11 as well as leukocytes from the head kidney, consisting of a high percentage of phagocytic cells have been investigated. Additionally, leukocytes isolated from the peritoneal cavity as the main target for injectable vaccines have been studied in the in vitro experiments. These experiments indicate that OMVs derived from A. salmonicida are recognized by the monocyte/macrophage cell line RTS-11 as well as by leukocytes from the head kidney resulting in significant changes of the mRNA expression pattern of early inflammatory markers (IL-1β, IL-6, IL-8, IL-10, TGFβ). Having used the established peritoneal inflammation model of rainbow trout it could be shown that intraperitoneal (i.p.) vaccination of rainbow trout with OMVs results in a similar local immune response, especially in the recruitment of myeloid cells, compared to the injection of inactivated bacteria. The systemic cellular immune response differed between the two vaccine groups, even though a similar humoral immune response could be observed. Interestingly, i.p.vaccination with 10 µg of OMVs resulted in similar antibody titers as observed for fish, that were i.p. vaccinated with 108 CFU of inactivated A. salmonicida. The similar antibody titers after vaccination with OMVs might be explained by a stronger activation of CD8- T cells (likely CD4+ T cells) in the head kidney as well as in the blood in the OMV vaccinated group alone, which might result in an increased stimulation of B cells to produce antibodies.
Oral vaccination has been described as the ideal vaccination method for fish, but only few vaccines for oral application are licensed. Therefore, the established oral model for vaccination of rainbow trout with attenuated viral hemorrhagic septicemia virus (VHSV) was adapted to be used for inactivated A. salmonicida, even though initial trials indicated great similarities in the cellular response after i.p. and oral vaccination with inactivated strains of A. salmonicida, particularly in the response of the myeloid cells and lymphocytes in the target organs as well as the thrombocytes in the spleen. This could not be confirmed in a second oral vaccination trial. These results show how challenging the development of oral vaccines for fish is. The main challenge is the reproducibility of reliable results, since this is influenced by the difference in uptake of vaccine pellets or antigen degradation in the gut. Future oral vaccine trials should investigate different vaccination regimes, e.g., consecutive feeding, or a different composition of vaccine pellets, in order to further investigate the possibility of establishing an oral vaccine model for trout and so that future vaccine candidates, like OMVs, can be reliably tested in fish.