Interfakultäres Institut für Genetik und Funktionelle Genomforschung
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Teichonsäuren (TA) sind ein wesentlicher Bestandteil von Gram-positiven Bakterien und damit auch der Zellwand von Streptococcus pneumoniae. Die Glykopolymere werden in Peptidoglykan-verbundene Wandteichonsäuren (WTA) und Glykolipid-verbundene Lipoteichonsäuren (LTA) unterschieden. Anders als in anderen Gram-positiven Spezies weisen WTA und LTA von S. pneumoniae einen gemeinsamen zytoplasmatischen Biosyntheseweg auf, der in einer identischen Struktur beider Glykopolymere resultiert. Erst der finale Transfer der TA-Ketten an das Peptidoglykan oder den Glykolipid-Anker unterscheidet sich im Biosyntheseweg.
In der vorliegenden Arbeit wurde SPD_1672 von Stamm S. pneumoniae D39 (TIGR4: SP_1893) als putative Lipoteichonsäure Ligase (Teichoic acid Ligase = TacL) identifiziert. In experimentellen Maus-Infektionsmodellen der akuten Pneumonie und systemischen Infektion zeigte die tacL-Mutante eine verringerte Virulenz und in in vitro Zellkultur-basierten Infektionsanalysen eine abgeschwächte Adhäsion an Lungenepithelzellen. Obwohl die TacL-Defizienz einen signifikanten pathophysiologischen Einfluss in vivo zeigte, konnte bei in vitro Wachstumsanalysen in einem Komplexmedium sowie in chemisch definiertem Medium nur ein sehr geringer Effekt auf die Wachstumsrate gemessen werden. Die Feldemissions-Rasterelektronenmikroskopie (FESEM) und Transmissionselektronenmikroskopie (TEM) Aufnahmen zur Untersuchung der bakteriellen Morphologie sowie die Analyse der durchflusszytometrischen Bestimmungen des Kapselgehalts der tacL-Mutante zeigten, dass die Bakterienmorphologie mit dem typischen Erscheinungsbild als Diplokokken unbeeinflusst waren. Das an die TAs substituierte Phosphorylcholin (P-Cho) dient als Anker für sogenannte Cholin-Bindungproteine (CBP), die wiederum unter anderem eine Rolle in der Autolyse, Kompetenz und Virulenz von S. pneumoniae spielen. Unter Verwendung polyklonaler Antikörper gegen verschiedener CBPs wurde lediglich bei PspC, Pce und CbpJ eine veränderte Menge festgestellt, wohingegen bei PspA, CbpL, LytA und CbpG kein Unterschied in der Abundanz auf der Bakterienoberfläche in der tacL-Mutante im Vergleich zum isogenen Wildtyp gemessen werden konnte. Darüber hinaus gelang es im Rahmen dieser Arbeit weitere Deletionsmutanten in Genen zu generieren, die für Proteine kodieren, denen eine hypothetische Rolle in der Teichonsäurebiosynthese zugewiesen wird und deren Funktionen in einer nachfolgenden Arbeit charakterisiert werden sollte.
Im Rahmen dieser Arbeit konnte Ligase zu Assemblierung der LTA in S. pneumoniae identifiziert werden. Die beeinträchtigte Virulenz der tacL-Mutante im in vivo Maus-Infektionsmodell macht TacL zu einem potentiellen Ziel für antimikrobielle Substanzen.
Under hyperosmotic conditions, bacteria accumulate compatible solutes through synthesis or import. Bacillus subtilis imports a large set of osmostress protectants via five osmotically controlled transport systems (OpuA to OpuE). Biosynthesis of the particularly effective osmoprotectant glycine betaine requires the exogenous supply of choline. While OpuB is rather specific for choline, OpuC imports a broad spectrum of compatible solutes, including choline and glycine betaine. One previously mapped antisense RNA of B. subtilis, S1290, exhibits strong and transient expression in response to a suddenly imposed salt stress. It covers the coding region of the opuB operon and is expressed from a strictly SigB-dependent promoter. By inactivation of this promoter and analysis of opuB and opuC transcript levels, we discovered a time-delayed osmotic induction of opuB that crucially depends on the S1290 antisense RNA and on the degree of the imposed osmotic stress. Time-delayed osmotic induction of opuB is apparently caused by transcriptional interference of RNA-polymerase complexes driving synthesis of the converging opuB and S1290 mRNAs. When our data are viewed in an ecophysiological framework, it appears that during the early adjustment phase of B. subtilis to acute osmotic stress, the cell prefers to initially rely on the transport activity of the promiscuous OpuC system and only subsequently fully induces opuB. Our data also reveal an integration of osmostress-specific adjustment systems with the SigB-controlled general stress response at a deeper level than previously appreciated.
The M protein of Streptococcus canis (SCM) is a virulence factor and serves as a surface-associated receptor with a particular affinity for mini-plasminogen, a cleavage product of the broad-spectrum serine protease plasmin. Here, we report that SCM has an additional high-affinity immunoglobulin G (IgG) binding activity. The ability of a particular S. canis isolate to bind to IgG significantly correlates with a scm-positive phenotype, suggesting a dominant role of SCM as an IgG receptor. Subsequent heterologous expression of SCM in non-IgG binding S. gordonii and Western Blot analysis with purified recombinant SCM proteins confirmed its IgG receptor function. As expected for a zoonotic agent, the SCM-IgG interaction is species-unspecific, with a particular affinity of SCM for IgGs derived from human, cats, dogs, horses, mice, and rabbits, but not from cows and goats. Similar to other streptococcal IgG-binding proteins, the interaction between SCM and IgG occurs via the conserved Fc domain and is, therefore, non-opsonic. Interestingly, the interaction between SCM and IgG-Fc on the bacterial surface specifically prevents opsonization by C1q, which might constitute another anti-phagocytic mechanism of SCM. Extensive binding analyses with a variety of different truncated SCM fragments defined a region of 52 amino acids located in the central part of the mature SCM protein which is important for IgG binding. This binding region is highly conserved among SCM proteins derived from different S. canis isolates but differs significantly from IgG-Fc receptors of S. pyogenes and S. dysgalactiae sub. equisimilis, respectively. In summary, we present an additional role of SCM in the pathogen-host interaction of S. canis. The detailed analysis of the SCM-IgG interaction should contribute to a better understanding of the complex roles of M proteins in streptococcal pathogenesis.
Streptococcus pneumoniae is endowed with a variety of surface-exposed proteins representing putative vaccine candidates. Lipoproteins are covalently anchored to the cell membrane and highly conserved among pneumococcal serotypes. Here, we evaluated these lipoproteins for their immunogenicity and protective potential against pneumococcal colonisation. A multiplex-based immunoproteomics approach revealed the immunogenicity of selected lipoproteins. High antibody titres were measured in sera from mice immunised with the lipoproteins MetQ, PnrA, PsaA, and DacB. An analysis of convalescent patient sera confirmed the immunogenicity of these lipoproteins. Examining the surface localisation and accessibility of the lipoproteins using flow cytometry indicated that PnrA and DacB were highly abundant on the surface of the bacteria. Mice were immunised intranasally with PnrA, DacB, and MetQ using cholera toxin subunit B (CTB) as an adjuvant, followed by an intranasal challenge with S. pneumoniae D39. PnrA protected the mice from pneumococcal colonisation. For the immunisation with DacB and MetQ, a trend in reducing the bacterial load could be observed, although this effect was not statistically significant. The reduction in bacterial colonisation was correlated with the increased production of antigen-specific IL-17A in the nasal cavity. Immunisation induced high systemic IgG levels with a predominance for the IgG1 isotype, except for DacB, where IgG levels were substantially lower compared to MetQ and PnrA. Our results indicate that lipoproteins are interesting targets for future vaccine strategies as they are highly conserved, abundant, and immunogenic.
Staphylococcus aureus is a human pathogen that can cause a wide range of diseases. Although formerly regarded as extracellular pathogen, it has been shown that S. aureus can also be internalized by host cells and persist within these cells. In the present study, we comparatively analyzed survival and physiological adaptation of S. aureus HG001 after internalization by two human lung epithelial cell lines (S9 and A549), and human embryonic kidney cells (HEK 293). Combining enrichment of bacteria from host-pathogen assays by cell sorting and quantitation of the pathogen's proteome by mass spectrometry we characterized S. aureus adaptation during the initial phase between 2.5 h and 6.5 h post-infection. Starting with about 2 × 106 bacteria, roughly 1450 S. aureus proteins, including virulence factors and metabolic enzymes were identified by spectral comparison and classical database searches. Most of the bacterial adaptation reactions, such as decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied. However, differences were noted in central carbon metabolism including regulation of fermentation and threonine degradation. Since these differences coincided with different intracellular growth behavior, complementary profiling of the metabolome of the different non-infected host cell types was performed. This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate. With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and regulatory mutants.
To enable control of African swine fever (ASF) in Eastern and Southern Africa, prototype live vaccine candidates were generated by targeted gene deletions from a Kenyan genotype IX ASF virus (ASFV). It was attempted to delete known nonessential genes involved in virulence (encoding TK, dUTPase, CD2v, 9GL), possibly essential genes (p12, pA104R, ribonucleotide reductase), and genes with widely unknown functions (pK145R). Isolation of the desired virus recombinants by plaque assays or limiting dilutions on a wild boar lung cell line (WSL-HP) was facilitated by substitutive reporter gene insertions encoding fluorescent proteins (GFP, DsRed), or the human membrane protein CD4. The latter protein permitted enrichment of recombinant virus particles by magnetic activated cell sorting (MACS). The isolated ASFV recombinants were characterized by PCR and sequencing of the mutated genome parts, and replication kinetics and virus spread in cell culture were investigated. Deletion of TK, CD2v, or pK145R had no detectable effect on in vitro growth of ASFV Kenya. Interestingly, virus mutants lacking the DNA binding protein pA104R which has been considered to be essential for DNA replication, also exhibited almost wild type-like growth properties.
In contrast, ASFV mutants lacking ribonucleotide reductase or p12 could not be purified to homogeneity on WSL-HP cells, indicating these proteins are essential for virus replication in cell culture. Therefore, trans-complementing cells lines stably expressing ASFV p12 have been prepared which can now be used for mutant virus purification. If this approach is successful the resulting defective mutant ASFV Kenya-p12 might be suitable as a safe “disabled in second cycle” (DISC) live vaccine in swine.
In a novel approach to improve reverse genetics of ASFV the CRISPR/Cas9 cell line WSL-gRp30 (Hübner et al., 2018a) was co-transfected with genomic DNA of ASFV-KenyaCD2vDsRed, sgRNA plasmids targeting K145R or 9GL, and GFP-expressing recombination plasmids for homology-directed repair. For booting up of the noninfectious virus genome the cells were infected with phylogenetically distant helper virus (genotype II ASFV Armenia, 84% identity) which was selectively inhibited on the used cell line. The desired double-fluorescent double-deletion mutants could be isolated after few plaque purification steps on selective WSL-gRp30 cells. Next generation sequence (NGS) analyses of reconstituted ASFV Kenya genomes showed that no unwanted recombination with the helper virus occurred, indicating that the method might be also suitable for booting of synthetic ASFV genomes cloned and mutagenized in E. coli or yeast.
The modified CRISPR/Cas9 system of S. pyogenes might be also usable for generation of ASFV resistant pigs. To evaluate this alternative control measure WSL cell clones stably expressing Cas9 nuclease and single or multiple sgRNAs against essential ASFV proteins were prepared and tested for their susceptibility to infection. Strain specific sgRNAs targeting the p30 gene of ASFV Kenya or Armenia selectively inhibited the respective viruses, and a p12 gene-specific sgRNA abrogated replication of both genotypes almost completely. Interestingly, coexpression of four ASFV-specific sgRNAs did not enhance virus inhibition, but might help to reduce the frequency of escape mutants which were occasionally isolated from the single sgRNA-expressing cells, and exhibited silent base substitutions or in-frame deletions within the target genes. First attempts to express the in vitro tested CRISPR/Cas9 constructs in transgenic pigs are in progress.
CRISPR/Cas9 supported rescue of a defective BAC clone of pseudorabies virus (PrV) vaccine strain Bartha (Hübner et al., 2018b) was used to develop putative vectored vaccines against ASFV. In the present study expression cassettes for the codon-optimized p12 and p54 genes of ASFV were successfully inserted into the PrV genome. The insertions did not significantly affect PrV recombination in cell culture, and the transgenes were expressed at similar levels as in ASFV-infected cells. It has to be tested whether coinfection with vector constructs for these and other immunogenic ASFV proteins is able to protect pigs against a lethal challenge.
For characterization of the generated ASFV mutants and PrV vector constructs, monospecific antisera against several ASFV gene products (p11.5, p12, p54, pK145R, p285L) were prepared by immunization of rabbits with bacterial GST fusion proteins. The anti-p12 serum showed only weak and strain-specific reactions with the ASFV Kenya protein, but was nevertheless useful for identification of p12-expressing PrV recombinants and WSL cell lines. All other sera showed satisfying reactions in Western blot and mostly immunofluorescence analyses, and allowed i.a. precise localization of the pK145R and p285L proteins in ASFV-infected cells and virions (Hübner et al., 2019).
Bats (Chiroptera) form the second largest order of mammals and with over 1,250 species, they represent about 20% of all mammalian species worldwide. They are the only mammals with true and sustained flight and distributed all over the world except the arctic regions. Moreover, bats entered specific ecological niches and with their food spectra, they reduce different arthropod populations as well as disperse seeds and pollen of plant species in various regions and habitats.
Bats also have a crucial role in spreading high-pathogenic and zoonotic viruses, harbor in general more viruses (zoonotic and non-zoonotic), and, related to the species, number even more than rodents. However, clinical symptoms of viral diseases are rarely reported in bat communities. Also seroconversions after infection were not reported for a variety of viruses found in bats. Since the incidence of virus-positive bats estimated in passive surveillance studies is usually very low, it is a question how such viruses can use bats as reservoir hosts. There is obviously a special evolutionary relationship between the pathogens and bats as hosts, which are based on possibly physiologic adaptations also in resistance and immunity.
In this thesis, the two lyssaviruses, European Bat Lyssavirus 1 and 2 (EBLV-1 and -2) were chosen as a model to investigate the immune response of European bats against viral infection in vitro. Lyssaviruses are the causative agents of rabies, a fatal zoonotic disease with neurotropic characteristics.
One main question to investigate was in which way bats act as reservoir host and developed a high disease resistance. The present thesis is based on three hypotheses about innate immune response against lyssavirus infection:
A) In bats specific peripheral resistance mechanisms evolved which reduce the risk of systemic viral infection after a hypothesized airborne transmission and infection via nasal epithelium supported by the social structure of and communication within bat communities.
B) The co-evolution of EBLV and the innate resistance of bats resulted in a very effective type I interferon response to inhibit a systemic lyssavirus infection.
C) The specific physiology of body temperature of bats with daily torpor depresses the viral replication but favours the type I interferon response.
To analyze the interferon-based resistance mechanisms, the type I interferon (IFN) genes of two European bats species (Eptesicus serotinus and Myotis myotis) were cloned and sequenced. Using established cell lines from the respiratory nasal epithelium (MmNep), olfactory nasal epithelium (MmNol), and Bulbus olfactorius brain (MmBr), the type I IFN response along a possible airborne infection route was investigated. The anti-viral effects and induction of IFNs/interferon stimulated genes (ISGs) in each cell line were also investigated in detail after infection in vitro. Finally, the influence of different temperatures on lyssavirus replication was analyzed in cell culture experiments.
The results indicated that (a) along the hypothesized airborne infection route the susceptibility for lyssavirus infections is decreased, (b) the type I IFN activity in contrast is increased contributing to a limitation of lyssavirus replication and (c) an obvious influences of varying cultivation temperatures on the resistance against lyssavirus infections, which favor the IFN response and repressing lyssavirus replication.
The result from these in vitro studies supports the hypothesis of a special co-evolution between lyssaviruses and bats. However, in vivo studies on the relevance in infected animals are missing so far. This model could also explain the generally limited pathogenicity of bat-associated viruses.
Staphylococcus aureus is one of the commonly encountered bacteria of the human microbiome. Although mostly a seemingly harmless commensal microbe, S. aureus can act as an invasive pathogen with seriously devastating effects on its host’s health and wellbeing. A wide range of infections caused by this bacterium has been reported to affect diverse parts of the human body, including the skin, soft tissues and bones, as well as important organs like the heart, kidneys and lungs. Particularly, S. aureus is infamous for being a major causative agent of respiratory tract infections that may escalate up to necrotizing pneumonia. Due to its clinical relevance, this pathogen has been intensively studied for many years. Nonetheless, further research in this field is still needed, because of the high capacity of S. aureus to evolve drug resistance, its high genomic plasticity and adaptability and, not in the last place, the plethora of niches within the human body where it can thrive and survive. In this regard, there are still many uncertainties concerning the specific adaptations carried out by S. aureus during colonization and infection of the human body, the transition between both stages, and upon the invasion of different types of host cells. To shed more light on some of these adaptations, the research described in this thesis has employed in vitro models of infection that mimic particular conditions during the infectious process with special focus on the lung epithelium. The adaptations displayed by S. aureus were monitored using advanced proteomics. Furthermore, the analyses documented in this thesis included S. aureus strains with diverse backgrounds and epidemiology to take into account the genetic diversity encountered in this species.
Deciphering the influence of Streptococcus pneumoniae global regulators on fitness and virulence
(2019)
Streptococcus pneumoniae (S. pneumoniae; the pneumococcus) is a Gram-positive, aerotolerant, and opportunistic bacteria, which colonizes the upper respiratory tract of human. S. pneumoniae can further migrate to other sterile parts of the body, and causes local as well as fatal infections like, pneumonia, septicaemia and meningitis. Due to incomplete amino acid pathways, pneumococci are auxotrophic for eight different amino acids including glutamine and arginine. The pneumococcus has adapted to the various host environmental conditions and a number of systems are dedicated for the transport and utilization of nutrients such as monosaccharides, amino acids and oligopeptides.
In this study the amino acid metabolism was characterised by 15N-isotopologue profiling in two different pneumococcal strains, D39 and TIGR4. Efficient uptake of a labelled amino acids mixture of 15N-labelled amino acids showed that S. pneumoniae has a preference for the amino acids transport instead of a de novo biosynthesis. It is known that glutamine (Gln) serves as main nitrogen source for S. pneumoniae. The 15N-labelled Gln used in this study demonstrated an efficient 15N-enrichment of Glu, Ala, Pro and Thr. Minor enrichment was seen for the amino acids Asp, Ile, Leu, Phe, Tyr, and Val. Remarkably, labelled Gly and Ser could be determined in strain TIGR4, whereas for strain D39 these two labelled amino acids were not detected. This confirms earlier studies with 13C-labelled glucose, which showed the biosynthesis of Ser out of Gly. Strain TIGR4 was able to grow in chemically-defined medium depleted of Gly confirming that Gly can be synthesized out of serine by the action of the enzyme serine hydroxymethyltransferase (SHMT).
The transcriptional regulator GlnR controls the Gln and Glu metabolism in S. pneumoniae. Hence, the impact of the repressor GlnR on amino acids metabolism was also studied. An increased 15N-enrichment was determined for Ala and Glu in both used pneumococcal strains, while an increased level of Pro was only measured in the isogenic glnR-mutant of non-encapsulated D39.
Arginine can also serve as nitrogen source in strain TIGR4. The arginine deiminase system metabolizes Arg into ornithine, carbamoyl phosphate and CO2 by the generation of 1 ATP and 2 mol NH3. Because of the truncation of the arcA gene strain D39 lacks arginine deiminase activity and has thus no functional ADS system. When 15N-Arg was added for growth, only in strain TIGR4, thirteen (13) labelled amino acids were detected with the highest enrichment for Ala, Glu and Thr. Genes coding for the enzymes of the arginine metabolism and for arginine uptake are regulated by the activator ArgR2 in strain TIGR4. Inactivation of ArgR2 was not accompanied by an enrichment of labelled amino acids, when the argR2-mutant was grown with 15N-labelled Arg indicative of the important role of ArgR2.
The bicistronic operon arcDT encoding the arginine/ornithine transporter ArcD and a putative peptidase ArcT belong to the peptidase family M20. The in silico comparison of structures revealed a significant homology of ArcT to PepV of L. delbrueckii and to Sapep of S. aureus known as carboxypeptidase. ArcT was heterologously expressed in E. coli and purified under reducing conditions. An enzymatic reaction was established and several dipeptides like Ala-Arg, Arg-Ala, and Ala-Asp were used as substrates. In addition, the dependency on divalent cations was analysed. Cleavage of the dipeptide Ala-Arg was detected in the presence of Mn2+ as cofactor under reducing conditions. Reduced peptidase activity was observed when Zn2+ was added. No cleavage of the tripeptide Ala-Ala-Arg could be shown indicating that ArcT acts as dipeptidase with the preference to the Arg residue at the C-terminal end.
Bacterial meningitis caused by S. pneumoniae was studied in an in vivo proteomic analysis. In a mouse meningitis model S. pneumoniae was isolated from the cerebrospinal fluid (CSF) by a filter extraction step. The MS analysis identified AliB and ComDE only from CSF isolated pneumococci indicating that these proteins are expressed under infection conditions. Mice infected with D39 wild-type and isogenic aliB, comDE and aliB-comDE double knockout mutants showed significantly less number of pleocytosis in the CSF and lower bacterial load in the blood compared to the wild-type. The results indicate that AliB and ComDE play an important role during meningitis.
Phenotypic characterization was carried out to identify differences between the wild-type and the aliB-, comDE- and aliB-comDE double mutants. Oxidative stress conditions were induced by the application of hydrogen peroxide or paraquat during growth in a chemically-defined medium similar to the CSF. No alteration in growth and survival of these mutants compared to the wild-type was observed suggesting that oxygen radicals play not an important role during the progression of meningitis. In addition, no differences of AliB expression was detected in the ComDE deficient D39. No impact of aliB and comDE-mutation on the expression of different virulence factors like pneumolysin or proteins involved in capsular biosynthesis was detected.
In vitro proteome analysis was performed to compare the wild-type to the AliB, and ComDE deficient D39 in the early and mid logarithmic growth phase. More than 70 % of theoretically expressed proteins were identified. In the aliB-mutant 33 proteins were differentally expressed in the early growth phase and 50 proteins differed during mid log growth. For the comDE mutant 24 and 11 proteins differed in expression in these two growth phases. Interestingly, high level of AliA expression was identified in all samples. The aliB-mutant had a decreased abundance of the proteins resembling an oligopeptide ABC transporter (AmiA, AmiC, AmiD, AmiE). In addition, another ABC transporter for iron transport encoded by spd_1607 to spd_ 1610 was higher expressed in the aliB-mutant. In the ComDE deficient mutant lower abundance of the Ami transporter sytem was identified. An increased abundance of proteins involved in the pyrimidine metabolism (PyrF, PyrE, PyrDb, PyrB and PyrR) was recognized only in the early growth phase of the comDE-mutant. These analyses demonstrate the marginal changes in protein synthesis during growth of S. pneumoniae. These studies demonstrated the adaptation of the proteome of S. pneumoniae to different growth conditions and the impact of regulatory proteins on the availability of carbon and nitrogen sources.
