Biology subjects

Urwin, L.

Publications and source records attributed to Urwin, L..

3 recordsLinked to original sources

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

CD9 co-operation with syndecan-1 is required for a major staphylococcal adhesion pathway

ObjectivesEpithelial colonisation is a critical first step in bacterial pathogenesis. Staphylococcus aureus can utilise several host factors to associate with cells, including 5{beta}l integrin and heparan sulphate proteoglycans, such as the syndecans. Here, we demonstrate that a partner protein of both integrins and syndecans, the host membrane adapter protein tetraspanin CD9, is essential for syndecan-mediated staphylococcal adhesion. Fibronectin is also essential in this process while integrins are only critical for post-adhesion entry into human epithelial cells. Methods and ResultsTreatment of epithelial cells with CD9-derived peptide or heparin caused significant reductions in staphylococcal adherence, dependent on both CD9 and syndecan-1. Exogenous fibronectin caused a CD9-dependent increase in staphylococcal adhesion whereas blockade of {beta}1 integrins did not affect adhesion but did reduce the subsequent internalisation of adhered bacteria. CD9 disruption or deletion increased {beta}1 integrin-mediated internalisation, suggesting that CD9 coordinates sequential staphylococcal adhesion and internalisation. ConclusionsCD9 controls staphylococcal adhesion through syndecan-1, using a mechanism that likely requires CD9-mediated syndecan organisation to correctly display fibronectin at the host cell surface. We propose that CD9-derived peptides or heparin analogues could be developed as anti-adhesion treatments to inhibit the initial stages of staphylococcal pathogenesis.

microbiology↗

bioRxiv2022

Purine nucleosides interfere with c-di-AMP levels and act as adjuvants to re-sensitize MRSA to β-lactam antibiotics

Elucidating the complex mechanisms controlling mecA/PBP2a-mediated {beta}-lactam resistance in methicillin resistant Staphylococcus aureus (MRSA) has the potential to identify new drug targets with therapeutic potential. Here, we report that mutations that interfere with de novo purine synthesis (pur operon), purine transport (NupG, PbuG and PbuX) and the nucleotide salvage pathway (DeoD2, Hpt) increased {beta}-lactam resistance in MRSA strain JE2. Extrapolating from these findings, exogenous guanosine and xanthosine, which are fluxed through the GTP branch of purine biosynthesis were shown to significantly reduce MRSA {beta}-lactam resistance. In contrast adenosine, which is fluxed to ATP, significantly increased oxacillin resistance, whereas inosine, which can be fluxed to ATP and GTP via hypoxanthine, only marginally reduced the oxacillin MIC. Increased oxacillin resistance of the nupG mutant was not significantly reversed by guanosine, indicating that NupG is required for guanosine transport, which in turn is required to reduce {beta}-lactam resistance. Suppressor mutants resistant to oxacillin/guanosine combinations contained several purine salvage pathway mutations, including nupG and hpt. Microscopic analysis revealed that guanosine significantly increased cell size, a phenotype also associated with reduced levels of c-di-AMP. Consistent with this, guanosine significantly reduced levels of c-di-AMP, and inactivation of GdpP, the c-di-AMP phosphodiesterase negated the impact of guanosine on {beta}-lactam susceptibility. PBP2a expression was unaffected in nupG or deoD2 mutants suggesting that guanosine-induced {beta}-lactam susceptibility may result from dysfunctional c-di-AMP-dependent osmoregulation. These data reveal the therapeutic potential of purine nucleosides as {beta}-lactam adjuvants that interfere with the normal activation of c-di-AMP required for high-level {beta}-lactam resistance in MRSA. ImportanceThe clinical burden of infections caused by antimicrobial resistant (AMR) pathogens is a leading threat to public health. Maintaining the effectiveness of existing antimicrobial drugs or finding ways to reintroduce drugs to which resistance is widespread is an important part of efforts to address the AMR crisis. Predominantly the safest and most effective class of antibiotics are the {beta}-lactams, which are no longer effective against methicillin resistant Staphylococcus aureus (MRSA). Here we report that the purine nucleosides guanosine and xanthosine have potent activity as adjuvants that can resensitise MRSA to oxacillin and other {beta}-lactam antibiotics. Mechanistically, exposure of MRSA to these nucleosides significantly reduced the levels of the cyclic dinucleotide c-di-AMP, which is required for {beta}-lactam resistance. Drugs derived from nucleotides are widely used in the treatment of cancer and viral infections highlighting the clinical potential of using purine nucleosides to restore or enhance the therapeutic effectiveness of {beta}-lactams against MRSA and potentially other AMR pathogens.

microbiology↗