7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Prof. Dr.

Chair

Professor

A Novel Stx-phage-encoded RNA Methyltransferase Regulates Spontaneous Prophage Induction and STEC Virulence by Blocking Shiga Toxin Inactivation of Translation

Microbial Pathogenicity

Shiga toxin (Stx) released by Stx-producing E. coli (STEC), can cause life-threatening illness. The synthesis and release of Stx requires induction of Stx-encoding prophage present in all STEC genomes. The STEC strains PA2 and PA8 harbor nearly identical Stx-encoding prophages, yet exhibit differing spontaneous induction frequencies and virulence.ϕPA2 and ϕPA8 each bear an IS629 insertion element but in different locations. This element lies in an intergenic region in ϕPA8. In ϕPA2, it disrupts a gene predicted to encode a DNA adenine methyltransferase (M.EcoPA8). Expressing wild-type M.EcoPA8 in STEC::ϕPA2 restores both prophage induction frequency and virulence to STEC::ϕPA8 levels, but an active site mutant does not. Thus M.EcoPA8 methyltransferase activity governs prophage spontaneous induction and STEC virulence.Full, specific in vitro methyltransferase activity of M.EcoPA8 requires complexation with a second protein, PNB-2. Sequential deletion of the genes encoding these proteins leads to concomitant, progressive reductions in spontaneous induction and STEC virulence, showing that these phenotypes are interconnected.We found that despite its annotation as a DNA adenine Mtase, the M.EcoPA8-PNB-2 holoenzyme specifically methylates 16S rRNA, not DNA. A ΔM.ecoPA8 Δpnb-2 STEC strain has fewer ribosomes than the M.EcoPA8-PNB-2 replete STEC strain. The translation activity of extracts made from a ΔM.EcoPA8 ΔPNB-2 STEC strain is lower than that of extracts from STEC::ϕPA8 or from a strain without any Stx-encoding prophage. Significantly, translation extracts from M.EcoPA8 replete STEC exhibit greater resistance to Stx-mediated inactivation compared to STEC strains lacking M.EcoPA8. Collectively, these findings suggest that M.EcoPA8-mediated 16S rRNA methylation safeguards ribosomes from Stx inactivation, enhancing prophage and Stx production, resulting in PA8's enhanced virulence.16S rRNA sequencing identified 4 putative methylation sites all mapping onto the RNA polymerase-contacting face of the 30S ribosome subunit in the expressome. We suggest M.EcoPA8 -PNB-2 16S rRNA methylation protects the ribosome from Stx inactivation by stabilizing this complex.

Oral Presentation

The role of MerA in the defense against the thiol-specific oxidant hypothiocyanous acid (HOSCN) in Staphylococcus aureus

Introduction: Staphylococcus aureus colonizes the skin and the airways of the healthy human population, but can also lead to life-threatening infections. During colonization and phagocytosis by neutrophils, S. aureus encounters the oxidant HOSCN. Goals: Here, we aimed to characterize the defense mechanisms against HOSCN in S. aureus. Materials &amp; Methods: RNA-seq analyses, redox biosensor measurements, mutant phenotypes and crystal structure analysis were used to investigate the HOSCN defense mechanisms. Results: In the transcriptome, HOSCN caused a strong thiol-specific oxidative, electrophile and metal stress responses as well as protein damage in S. aureus, as indicated by the upregulation of the HypR, TetR1, PerR, QsrR, MhqR, CstR, CsoR, CzrA, AgrA, HrcA and CtsR regulons. Brx-roGFP2 biosensor measurements and Western blot analyses revealed a reversible oxidative shift of the bacillithiol redox potential (EBSH) and increased S-bacillithiolations under HOSCN in S. aureus, supporting its thiol-specific mode of action [1]. Using phenotype analyses, the HOSCN reductase MerA was shown to confer the highest resistance towards HOSCN stress in S. aureus [1, 2]. The crystal structure of the MerA dimer revealed its function as group I flavin disulfide reductase, with the FAD cofactor close to the C43XXXXC48 motif, which interacts with the H427XXXXE432 motif of the opposing subunit, facilitating deprotonation of the active site Cys43 in the HOSCN reduction cycle [2]. Summary: HOSCN causes a thiol-specific stress response, EBSH changes and S-bacillithiolation in S. aureus. MerA functions as HOSCN reductase and main protection mechanism against neutrophil oxidants.References:[1] Loi VV, Busche T, Schnaufer F, Kalinowski J, Antelmann H. 2023. The neutrophil oxidant hypothiocyanous acid causes a thiol-specific stress response and an oxidative shift of the bacillithiol redox potential in Staphylococcus aureus. Microbiol Spectr. 11:e0325223[2] Shearer HL, Loi VV, Weiland P, Bange G, Altegoer F, Hampton MB, Antelmann H, Dickerhof N. 2023. MerA functions as a hypothiocyanous acid reductase and defense mechanism in Staphylococcus aureus. Mol Microbiol. 119:456-470.

Type III secretion of transmembrane proteins and the case of the Salmonella´s chaperone, SscB

Type III secretion (T3S) systems are present in Gram-negative bacteria and consist of a needle-like structure that creates a direct channel for the delivery of bacterial proteins into host cells, ultimately promoting bacterial survival. Among the proteins delivered are those containing transmembrane domains (TMD). These can interact with a T3S chaperone (T3SC), which protects the more hydrophobic TMDs from erroneous recognition and targeting to bacterial membranes. This interaction should be critical for an efficient and correct T3S. Here, we aimed to unveil the key features and processes that underlie the T3S of TMD-effectors.In Salmonella, SscB/SseF are a chaperone/TMD-effector pair. Biophysical characterization of purified SscB and analysis of the predicted structure showed that SscB is structurally similar to T3SCs that bind translocators (TMD-proteins). Interestingly, a consensus amino acid sequence, which is conserved on the translocators of Salmonella and other bacterial species, was identified in SseF but also in the TMD-effector SseG. SscB interacts and promotes secretion of SseF, as seen previously, but also can interact and aid the secretion of SseG. We showed that mutagenesis of the consensus amino acid sequence abolished T3S of SseF and SseG. Next, it was observed that SscB and SseF co-stabilized but SseG was stable without the presence of the chaperone. This suggests that efficient SscB/SseF interaction is more critical than that of SscB/SseG. Moreover, since these three proteins are encoded adjacently in an operon, sscB-sseF-sseG, we have analyzed the effects of changing gene order on T3S. A decrease in the T3S of SseF was observed when sscB was relocated downstream of sseF.Overall, SscB shares features with chaperones of translocators, which challenges the current classification of T3SC. This suggests that the structure of the T3SC depends on the structural characteristics of their interacting partners rather than their timing of secretion. The gene order is important for efficient targeting to T3S, by possibly allowing for a more rapid interaction of SscB/SseF, which leads to protein co-stabilization and protection of the TMD of SseF.

Antigenic variation impacts gonococcal lifestyle and antibiotic tolerance by modulating interbacterial forces

Type 4 pili (T4P) are multifunctional filaments involved in adhesion, surface motility, biofilm formation, and horizontal gene transfer. These extracellular polymers are surface-exposed and, therefore, act as antigens. The human pathogen Neisseria gonorrhoeae uses pilin antigenic variation to escape immune surveillance, yet it is unclear how antigenic variation impacts other functions of T4P. Here, we addressed this question by replacing the major pilin of a laboratory strain of N. gonorrhoeae with pilins from clinical isolates. Structural predictions reveal filament features that vary from one strain to the next, with the potential to impact pilus:pilus interactions. Using a combination of laser tweezers, electron microscopy, and advanced image analysis, we explore the phenotypic consequences of these structural changes. We reveal that strains differing only in their major pilin sequence vary substantially in their attractive forces, which we attribute to variations in the stereochemistry of the T4P filament. In liquid culture, strongly interacting bacteria form microcolonies while weakly interacting bacteria retain a planktonic lifestyle. We show that lifestyle strongly affects growth kinetics and antibiotic tolerance. In the absence of external stresses, planktonic bacteria grow faster than aggregating bacteria. In the presence of the antibiotics ceftriaxone or ciprofloxacin, the killing kinetics indicate strongly increased tolerance of aggregating strains. We propose that pilin antigenic variation produces a mixed population containing variants optimized for growth, colonization, or survivability under external stress. Different environments select different variants, ensuring the survival and reproduction of the population as a whole.

Prof. Dr. med.

Analysis of the emerging global pathogen Stenotrophomonas maltophilia in single vs. multispecies biofilms

Stenotrophomonas maltophilia is an opportunistic pathogen causing various infections. Its prevalence has increased, especially in cystic fibrosis patients, and its multidrug resistance poses challenges in treatment. The bacterium forms multispecies biofilms with other pathogens and is known for its substantial role in pathogenicity in infected lungsOur goal is to understand the biofilm characteristics of S. maltophilia in single und mixed species biofilms as well as the differential gene expression patterns within these biofilmsIn this study over 200 clinical isolates of S. maltophilia as well as chromosomally labeled fluorescent strains of S. maltophilia K279a, P. aeruginosa PA01, S. aureus SH1000 and C. albicans SC5314 were used. Biofilms were formed in flow and static settings and were used for RNA seq analysis. Additionally, reporter fusion constructs of selected genes were generated and were further analyzed.Results and conclusionOur analysis revealed a strain-specific variability in biofilm formation and architecture among S.maltophilia isolates. RNA seq. analysis of S. maltophilia isolates identified shared and strain-specific genes, with iron uptake being a key factor in biofilm metabolism. LSM imaging showed that species interactions affect the structural composition of multispecies biofilms leading to a layer formation. S maltophilia showed alteration in lactate metabolism , Propionate degradation and a switch in cytochrome oxidases in our mixed biofilm models.The expression of virulence factors, QS signaling and cyclic diGMP was decreased in PA01 in coculture with K279a.Our data show that isolates of S. maltophilia are highly diverse on a phenotypic and genotypic level. Common genes were identified to play a crucial role in the biofilm formation of S. maltophilia. LSM imaging has shown a distinct distribution and layer formation of different species within mixed species biofilms. RNA seq analysis and the application of reporter fusion constructs showed specific and different expression patterns for each species as compared to single species biofilms, suggesting that each species acknowledge and respond to the presence of others in these biofilms.

Expression and activation control of the T6SS4 machinery in Yersinia pseudotuberculosis

The type VI secretion system (T6SS) is a contact dependent protein delivery system of Gram-negative bacteria to intoxicate prokaryotic or eukaryotic cells. The enteropathogen Yersinia pseudotuberculosis (Ypstb) encodes four complete clusters (T6SS1-4) with potentially different functions controlled by intricate, independent regulatory pathways. Under laboratory conditions, only T6SS4 is mildly expressed which makes thorough analysis of each system difficult. The recent identification of the T6SS4 activator RovC allows a more detailed investigation of the regulation of this cluster.Flow cytometry was used to analyse T6SS4 expression on a single cell level. We observed heterogenous expression of T6SS4 (T6SS4+ and T6SS4- subpopulations) within a Ypstb population, which strongly depends on temperature and growth phase. Heterogeneous expression is not only limited to T6SS4, as also rovC is heterogeneously expressed, indicating an unknown additional transcriptional regulator upstream of rovC. Although all T6SS4 genes are under the control of the same promoter, qRT-PCR revealed different amounts of gene transcripts within the cluster, suggesting additional post-transcriptional pathways. However, expression of T6SS4 compounds alone does not result in a firing event. Analysis with fluorescence microscopy could show that several additional triggers such as high cell density and osmotic pressure are required to activate the T6SS4.Our preliminary data show a tight regulation on transcriptional, post-transcriptional and post-translational levels, that differ from already known pathways in other organisms. However, as little is known about the T6SSs of Ypstb so far, further research is required to understand their function and the purpose of this heterogeneous expression.

Oral Session
The Bacterial Pathogen

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Oral SessionThe Bacterial Pathogen

Appointment

Chair

Topics

Programme

Oral Session
The Bacterial Pathogen