Biology subjects

Corrigan, R. M.

Publications and source records attributed to Corrigan, R. M..

6 recordsLinked to original sources

bioRxiv2026

Non-canonical stringent response signaling mediates antimicrobial fatty acid sensitivity in Staphylococcus aureus

Staphylococcus aureus is a leading cause of skin and soft tissue infections, yet colonization of healthy skin is limited by multiple defenses, including antimicrobial fatty acids (AFAs) produced by host cells and the resident microbiota. The mechanisms by which S. aureus overcomes these lipid-based defenses remain incompletely understood. Here, we show that mutations truncating the essential stringent response regulator Rel confer broad tolerance to both host-and microbially-derived AFAs in diverse S. aureus strains. Unlike classical stringent response activation, C-terminal Rel truncations do not induce typical (p)ppGpp-dependent transcriptional changes but instead enhance activity of the alternative sigma factor SigB and the staphylococcal accessory regulator SarA. This SigB-SarA regulatory cascade promotes transcriptional remodeling, including upregulation of pyrimidine biosynthesis genes, and coincides with alterations to cell envelope structure. Moreover, in the absence of SigB or SarA, tolerance can be restored through mutations in the serine/threonine phosphatase Stp1, highlighting additional pathways that modulate cell envelope-mediated resistance to AFAs. These findings identify a previously unrecognized consequence of Rel mutation in S. aureus; whereby small truncations may promote survival in AFA-rich host environments facilitating skin colonization and infection. ImportanceStaphylococcus aureus is a major cause of skin and soft tissue infections, and persistent skin colonization is a significant risk factor for infection. Antimicrobial fatty acids (AFAs), produced by microbes and host cells on human skin, normally limit S. aureus colonization. The mechanisms that allow this pathogen to overcome these lipid defenses are incompletely understood. We show that mutations truncating the essential stringent response regulator Rel enable S. aureus to tolerate both host-and microbially derived AFAs. These Rel variants, which have been identified in clinical isolates, alter cell envelope properties through the transcriptional regulator SarA rather than activating a classical stringent response. Our findings reveal a previously unrecognized adaptation that may facilitate S. aureus survival on the skin and promote infection.

microbiology↗

bioRxiv2024

Adaptation to skin mycobiota promotes antibiotic tolerance in Staphylococcus aureus

The microbiota can promote host health by inhibiting pathogen colonization, yet how host-resident fungi, or the mycobiota, contribute to this process remains unclear. The human skin mycobiota is uniquely stable compared to other body sites and dominated by yeasts of the genus Malassezia. We observe that colonization of human skin by Malassezia sympodialis significantly reduces subsequent colonization by the prominent bacterial pathogen Staphylococcus aureus. M. sympodialis secreted products possess potent bactericidal activity against S. aureus and are sufficient to impair S. aureus skin colonization. This bactericidal activity requires an acidic environment and is exacerbated by free fatty acids, demonstrating a unique synergy with host-derived epidermal defenses. Leveraging experimental evolution to pinpoint mechanisms of S. aureus adaptation in response to the skin mycobiota, we identified multiple mutations in the stringent response regulator Rel that promote survival against M. sympodialis. Similar Rel alleles have been reported in S. aureus clinical isolates, and natural Rel variants are sufficient for tolerance to M. sympodialis antagonism. Partial stringent response activation underlies tolerance to clinical antibiotics, with both laboratory-evolved and natural Rel variants conferring multidrug tolerance. These findings demonstrate the ability of the mycobiota to mediate pathogen colonization resistance, identify new mechanisms of bacterial adaptation in response to fungal antagonism, and reveal the potential for microbiota-driven evolution to shape pathogen antibiotic susceptibility. Highlights- M. sympodialis reduces colonization of human skin by S. aureus - Bactericidal activity of M. sympodialis is exacerbated by features of the skin niche - S. aureus Rel variants are sufficient for tolerance to Malassezia antagonism - Evolved tolerance to yeast antagonism coincides with S. aureus multidrug tolerance

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↗

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↗

bioRxiv2021

(p)ppGpp inhibits 70S ribosome formation in Staphylococcus aureus by impeding GTPase-ribosome interactions

During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of the stress signalling network termed the stringent response. Screening for (p)ppGpp-binding targets within Staphylococcus aureus identified four ribosome-associated GTPases (RA-GTPases), RsgA, RbgA, Era and HflX, each of which are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) states. Entry into the OFF-state from the ON-state occurs upon hydrolysis of GTP, with GTPase activity increasing substantially upon ribosome association. When bound to (p)ppGpp, GTPase activity is inhibited, reducing 70S ribosome assembly. Here, we sought to determine how (p)ppGpp impacts RA-GTPase-ribosome interactions by examining the affinity and kinetics of binding between RA-GTPases and ribosomes in various nucleotide-bound states. We show that RA-GTPases preferentially bind to 5'-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a regulatory mechanism within the cell. Binding to (p)ppGpp reduces stable association of RA-GTPases to ribosomal subunits compared to the GTP-bound state both in vitro and within bacterial cells by inducing the OFF-state conformation. We propose that in this conformation, the G2/switch I loop adopts a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated growth control.

microbiology↗

bioRxiv2018

Stringent response-mediated control of rRNA processing via the ribosomal assembly GTPase Era

P-loop GTPases are widely conserved across all domains of life. These enzymes act as molecular switches, cycling between inactive GDP-bound, and active GTP-bound states. Our previous work identified the Staphylococcus aureus GTPase Era as a binding target for the stringent response alarmone (p)ppGpp. Here we show that, unlike in Escherichia coli, Era is not essential in S. aureus but is important for 30S ribosomal subunit assembly. We employ bacterial two-hybrid and split luciferase approaches to show that Era interacts with the endonuclease YbeY, a protein of unknown function YbeZ, and the DEAD-box RNA helicase CshA in E. coli and natively in S. aureus. We determine that both Era and CshA are cold shock proteins required for virulence and rRNA processing. Era and CshA also form direct interactions with the (p)ppGpp synthetase RSH, an interaction required for controlling (p)ppGpp levels in response to cold shock. Taken together, we conclude that Era acts as an intermediary protein, directing enzymes involved in rRNA maturation to their site of action, an activity which, under stress, is controlled by the stringent response.

microbiology↗