Biology subjects

Salamaga, B.

Publications and source records attributed to Salamaga, B..

4 recordsLinked to original sources

bioRxiv2024

Exploring the role of E. faecalis Enterococcal Polysaccharide Antigen (EPA) and lipoproteins in evasion of phagocytosis

Enterococcus faecalis is an opportunistic pathogen frequently causing nosocomial infections. The virulence of this organism is underpinned by its capacity to evade phagocytosis, allowing dissemination in the host. Immune evasion requires a surface polysaccharide produced by all enterococci, known as the Enterococcal Polysaccharide Antigen (EPA). EPA consists of a cell wall-anchored rhamnose backbone substituted by strain-specific polysaccharides called "decorations", essential for the biological activity of this polymer. However, the structural determinants required for innate immune evasion remain unknown, partly due to a lack of suitable validated assays. Here, we describe a quantitative, in vitro assay to investigate how EPA decorations alter phagocytosis. Using the E. faecalis model strain OG1RF, we demonstrate that a mutant with a deletion of the locus encoding EPA decorations can be used as a platform strain to express heterologous decorations, thereby providing an experimental system to investigate the inhibition of phagocytosis by strain-specific decorations. We show that the aggregation of cells lacking decorations is increasing phagocytosis and that this process does not involve the recognition of lipoproteins by macrophages. Collectively, our work provides novel insights into innate immune evasion by enterococci and paves the way for further studies to explore the structure/function relationship of EPA decorations.

microbiology↗

bioRxiv2023

Determining the importance of the stringent response for methicillin-resistant Staphylococcus aureus virulence using a zebrafish model of infection

Staphylococcus aureus is a bacterial pathogen that poses a major threat to human health. The ability of this bacterium to adapt to stresses encountered in the host is essential for disease. The stringent response is a signalling pathway utilised by all bacteria to alarm cells when stressed, and has been linked to the virulence of a number of species. This signalling pathway is controlled by the nucleotide alarmones guanosine tetra-(ppGpp) and pentaphosphate (pppGpp: collectively termed (p)ppGpp), produced in S. aureus by three synthetase enzymes: Rel, RelP and RelQ. Here, we used a triple (p)ppGpp synthetase mutant ((p)ppGpp0) to examine the importance of this signalling network for the survival and virulence of S. aureus in vivo. Using an established zebrafish larval infection model, we observed that infection with (p)ppGpp0 resulted in attenuated virulence, which was not due to a reduced ability of the mutant to replicate in vivo. Of the three (p)ppGpp synthetases, Rel was established as key during infection, but roles for RelP and RelQ were also observed. Zebrafish myeloid cell depletion restored the virulence of (p)ppGpp0 during systemic infection, indicating that (p)ppGpp is important for survival within host phagocytes. Primary macrophages infection studies, followed by in vitro tolerance assays to key innate immune effectors, demonstrated that (p)ppGpp0 was more susceptible to stressors found within the intracellular macrophage environment, with roles for all three synthetases implicated. Lastly, the absence of CodY, a transcription factor linked to the stringent response, significantly increased the tolerance of S. aureus to phagolysosomal-like stressors in vitro, but had no impact in vivo. Taken together, these results define the importance of the stringent response for S. aureus infection, revealing that (p)ppGpp produced by all three synthetases is required for bacterial survival within the host environment by mediating adaptation to the phagolysosome.

microbiology↗

bioRxiv2023

The roles of GpsB and DivIVA in Staphylococcus aureus growth and division

The spheroid bacterium S. aureus is often used as a model of morphogenesis due to its apparent simple cell cycle. S. aureus has many cell division proteins that are conserved across bacteria alluding to common functions. However, despite intensive study we still do not know the roles of many of these components. Here we have examined the functions of the paralogues DivIVA and GpsB in the S. aureus cell cycle. Cells lacking gpsB display a more spherical phenotype than wild type, associated with a decrease in peripheral cell wall peptidoglycan synthesis. This correlates with an increased localisation of penicillin binding proteins at the developing septum, notably PBPs 2 and 3. Our results highlight the role of GpsB as an apparent regulator of cell morphogenesis in S. aureus.

microbiology↗

bioRxiv2018

Decoration of the enterococcal polysaccharide antigen EPA is essential for virulence, cell surface charge and sensitivity to effector molecules of innate immunity

Enterococcus faecalis is an opportunistic pathogen with an intrinsically high resistance to lysozyme, a key effector of the innate immune system. This high level of resistance requires several genes (oatA, pgdA, dltA and sigV) acting synergistically to inhibit both the enzymatic and cationic antimicrobial peptide activities of lysozyme. We sought to identify novel genes modulating E. faecalis resistance to lysozyme. Random transposon mutagenesis carried out in the quadruple oatA/pgdA/dltA/sigV mutant led to the identification of several independent insertions clustered on the chromosome. These mutations were located in a locus referred to as the enterococcal polysaccharide antigen (EPA) variable region located downstream of the highly conserved epaA-epaR genes proposed to encode a core synthetic machinery. The epa variable region was previously proposed to be responsible for EPA decorations, but the role of this locus remains largely unknown. Here, we show that EPA decoration contributes to resistance towards charged antimicrobials and underpins virulence in the zebrafish model of infection by conferring resistance to phagocytosis. Collectively, our results indicate that the production of the EPA rhamnopolysaccharide backbone is not sufficient to promote E. faecalis infections and reveal an essential role of the modification of this surface polymer for enterococcal pathogenesis.

microbiology↗