Publications
71 results found
Sweeney E, Sabnis A, Edwards AM, et al., 2020, <i>Effect of host-mimicking medium and biofilm growth on the ability of colistin to kill</i> Pseudomonas aeruginosa
<jats:title>Abstract</jats:title><jats:p><jats:italic>In vivo</jats:italic> biofilms cause recalcitrant infections with extensive and unpredictable antibiotic tolerance. Here, we demonstrate increased tolerance of colistin by <jats:italic>Pseudomonas aeruginosa</jats:italic> when grown in cystic fibrosis-mimicking medium <jats:italic>versus</jats:italic> standard medium in <jats:italic>in vitro</jats:italic> biofilm assays, and drastically increased tolerance when grown in an <jats:italic>ex vivo</jats:italic> CF model versus the <jats:italic>in vitro</jats:italic> assay. We used colistin conjugated to the fluorescent dye BODIPY to assess the penetration of the antibiotic into <jats:italic>ex vivo</jats:italic> biofilms and showed that poor penetration partly explains the high doses of drug necessary to kill bacteria in these biofilms. The ability of antibiotics to penetrate the biofilm matrix is key to their clinical success, but hard to measure. Our results demonstrate both the importance of reduced entry into the matrix in <jats:italic>in vivo</jats:italic>-like biofilm, and the tractability of using a fluorescent tag and benchtop fluorimeter to assess antibiotic entry into biofilms. This method could be a relatively quick, cheap and useful addition to diagnostic and R&D pipelines, allowing the assessment of drug entry into biofilms, in <jats:italic>in vivo</jats:italic>-like conditions, prior to more detailed tests of biofilm killing.</jats:p>
Ha KP, Clarke R, Kim G-L, et al., 2020, Staphylococcal DNA repair is required for infection, Publisher: bioRxiv
To cause infection, Staphylococcus aureus must withstand damage caused by host immune defences. However, the mechanisms by which staphylococcal DNA is damaged and repaired during infection are poorly understood. Using a panel of transposon mutants, we identified the rexBA operon as important for the survival of Staphylococcus aureus in whole human blood. Mutants lacking rexB were also attenuated for virulence in murine models of both systemic and skin infections. We then demonstrated that RexAB is a member of the AddAB family of helicase/nuclease complexes responsible for initiating the repair of DNA double strand breaks. Using a fluorescent reporter system, we were able to show that neutrophils cause staphylococcal DNA double strand breaks through ROS generated by the respiratory burst, which are repaired by RexAB, leading to induction of the mutagenic SOS response. We found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted survival of these pathogens in human blood, suggesting that DNA double strand break repair is required for Gram-positive bacteria to survive in host tissues. Together, these data demonstrate that DNA is a target of host immune cells, leading to double-strand breaks, and that repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection.
Clarke R, Bruderer M, Ha KP, et al., 2019, RexAB is essential for the mutagenic repair of Staphylococcus aureus DNA damage caused by co-trimoxazole, Antimicrobial Agents and Chemotherapy, Vol: 63, ISSN: 0066-4804
Co-trimoxazole (SXT) is a combination therapeutic that consists of sulfamethoxazole and trimethoprim that is increasingly used to treat skin and soft-tissue infections caused by methicillin-resistant Staphylococcus aureus (MRSA). However, the use of SXT is limited to the treatment of low-burden, superficial S. aureus infections and its therapeutic value is compromised by the frequent emergence of resistance. As a first step towards the identification of approaches to enhance the efficacy of SXT, we examined the role of bacterial DNA repair in antibiotic susceptibility and mutagenesis. We found that mutants lacking the DNA repair complex RexAB had a modest 2-fold lower SXT MIC than wild-type strains but were killed 50-5000-fold more efficiently by the combination antibiotic at the breakpoint concentration. SXT-mediated DNA damage occurred via both thymidine limitation and the generation of reactive oxygen species, and triggered induction of the SOS response in a RexAB-dependent manner. SOS induction was associated with a 50% increase in the mutation rate, which may contribute to emergence of resistant strains during SXT therapy. In summary, this work determined that SXT caused DNA damage in S. aureus via both thymidine limitation and oxidative stress, which was repaired by the RexAB complex, leading to induction of the mutagenic SOS response. Small molecule inhibitors of RexAB could therefore have therapeutic value by increasing the efficacy of SXT and decreasing the emergence of drug-resistance during treatment of infections caused by S. aureus.
Lim C, Ha KP, Clarke R, et al., 2019, Identification of a potent small-molecule inhibitor of bacterial DNA repair that potentiates quinolone antibiotic activity in methicillin-resistant Staphylococcus aureus, Bioorganic and Medicinal Chemistry, Vol: 27, Pages: 1-7, ISSN: 0968-0896
The global emergence of antibiotic resistance is one of the most serious challenges facing modern medicine. There is an urgent need for validation of new drug targets and the development of small molecules with novel mechanisms of action. We therefore sought to inhibit bacterial DNA repair mediated by the AddAB/RecBCD protein complexes as a means to sensitize bacteria to DNA damage caused by the host immune system or quinolone antibiotics. A rational, hypothesis-driven compound optimization identified IMP-1700 as a cell-active, nanomolar potency compound. IMP-1700 sensitized multidrug-resistant Staphylococcus aureus to the fluoroquinolone antibiotic ciprofloxacin, where resistance results from a point mutation in the fluoroquinolone target, DNA gyrase. Cellular reporter assays indicated IMP-1700 inhibited the bacterial SOS-response to DNA damage, and compound-functionalized Sepharose successfully pulled-down the AddAB repair complex. This work provides validation of bacterial DNA repair as a novel therapeutic target and delivers IMP-1700 as a tool molecule and starting point for therapeutic development to address the pressing challenge of antibiotic resistance.
Lacoma A, Edwards AM, Young BC, et al., 2019, Cigarette smoke exposure redirects Staphylococcus aureus to a virulence profile associated with persistent infection, Scientific Reports, Vol: 9, Pages: 1-15, ISSN: 2045-2322
Tobacco smoking represents the leading preventable cause of death worldwide. Smoking is a recognised risk factor for several pathologies and is detrimental to host immune surveillance and defence. However, the impact of smoking on microbial residents of the nasopharyngeal cavity, in contact with cigarette smoke (CS), is lacking. Staphylococcus aureus is a major human pathogen that colonises the human nasopharynx and causes a wide range of infections. We investigated the impact of CS on specific virulence phenotypes important in S aureus pathogenesis. We observed strain-dependent differences following exposure to CS, namely growth inhibition, augmented biofilm formation, increased invasion of, and persistence within, bronchial alveolar epithelial cells. Additionally, we confirm the critical role of a functional accessory gene regulator (Agr) system in mediating increased biofilm development and host cell invasion and persistence following CS exposure. Furthermore, CS exposure resulted in reduced toxin production. Importantly, exposure of S aureus to CS accelerated the frequency of mutations and resulted in a significant increase in gentamicin-resistant small colony variant (SCV) formation. Mutational analysis revealed that CS induced SCVs emerge via the SOS response DNA mutagenic repair system. Taken together, our results suggest that CS redirects certain S aureus strains to a virulence profile associated with persistence.
Edwards AM, 2019, Silence is golden for <i>Staphylococcus</i>, NATURE MICROBIOLOGY, Vol: 4, Pages: 1073-1074, ISSN: 2058-5276
Evans LE, Krishna A, Ma Y, et al., 2019, Exploitation of antibiotic resistance as a novel drug target: development of a β-lactamase-activated antibacterial prodrug., Journal of Medicinal Chemistry, Vol: 62, Pages: 4411-4425, ISSN: 0022-2623
Expression of β-lactamase is the single most prevalent determinant of antibiotic resistance, rendering bacteria resistant to β-lactam antibiotics. In this article, we describe the development of an antibiotic pro-drug that combines ciprofloxacin with a β-lactamase-cleavable motif. The pro-drug is only bactericidal after activation by β-lactamase. Bactericidal activity comparable to ciprofloxacin is demonstrated against clinically-relevant E. coli isolates expressing diverse β-lactamases; bactericidal activity was not observed in strains without β-lactamase. These findings demonstrate that it is possible to exploit antibiotic resistance to selectively target β-lactamase-producing bacteria using our pro-drug approach, without adversely affecting bacteria that do not produce β-lactamase. This paves the way for selective targeting of drug-resistant pathogens without disrupting or selecting for resistance within the microbiota, reducing the rate of secondary infections and subsequent antibiotic use.
Pee CJE, Pader V, Ledger EVK, et al., 2019, A FASII inhibitor prevents staphylococcal evasion of daptomycin by inhibiting phospholipid decoy production, Antimicrobial Agents and Chemotherapy, Vol: 63, Pages: 1-18, ISSN: 0066-4804
Daptomycin is a treatment of last resort for serious infections caused by drug-resistant Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus. We have shown recently that S. aureus can evade daptomycin by releasing phospholipid decoys that sequester and inactivate the antibiotic, leading to treatment failure. Since phospholipid release occurs via an active process, we hypothesised that it could be inhibited, thereby increasing daptomycin efficacy. To identify opportunities for therapeutic interventions that block phospholipid release, we first determined how the host environment influenced the release of phospholipids and inactivation of daptomycin by S. aureus The addition of certain host-associated fatty acids to the growth medium enhanced phospholipid release. However, in serum, the sequestration of fatty acids by albumin restricted their availability to S. aureus sufficiently to prevent their use in the generation of released phospholipids. This finding implied that in host tissues S. aureus may be completely dependent upon endogenous phospholipid biosynthesis to generate lipids for release, providing a target for therapeutic intervention. To test this, we exposed S. aureus to AFN-1252, an inhibitor of the staphylococcal FASII fatty acid biosynthetic pathway, together with daptomycin. AFN-1252 efficiently blocked daptomycin-induced phospholipid decoy production, even in the case of isolates resistant to AFN-1252, which prevented the inactivation of daptomycin and resulted in sustained bacterial killing. In turn, daptomycin prevented the fatty acid-dependent emergence of AFN-1252-resistant isolates in vitro In summary, AFN-1252 significantly enhances daptomycin activity against S. aureusin vitro by blocking the production of phospholipid decoys, whilst daptomycin blocks the emergence of resistance to AFN-1252.
Sabnis A, Hagart KLH, Klöckner A, et al., 2018, Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane
<jats:title>Summary</jats:title><jats:p>Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance is due to modified LPS at the cytoplasmic rather than outer membrane. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane. We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the cytoplasmic membrane, which resulted in increased susceptibility to colistin <jats:italic>in vitro</jats:italic> and improved treatment efficacy <jats:italic>in vivo</jats:italic>. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.</jats:p>
Pee CJE, Pader V, Ledger EVK, et al., 2018, A FASII inhibitor prevents staphylococcal evasion of daptomycin by inhibiting phospholipid decoy production
<jats:title>Abstract</jats:title><jats:p>Daptomycin is a treatment of last resort for serious infections caused by drug-resistant Gram-positive pathogens such as methicillin-resistant<jats:italic>Staphylococcus aureus</jats:italic>. We have shown recently that<jats:italic>S. aureus</jats:italic>can evade daptomycin by releasing phospholipid decoys that sequester and inactivate the antibiotic, leading to treatment failure. Since phospholipid release occurs via an active process we hypothesised that it could be inhibited, thereby increasing daptomycin efficacy. To identify opportunities for therapeutic interventions that block phospholipid release, we first determined how the host environment influenced the release of phospholipids and inactivation of daptomycin by<jats:italic>S. aureus</jats:italic>. The addition of certain host-associated fatty acids to the growth medium enhanced phospholipid release. However, in serum, the sequestration of fatty acids by albumin restricted their availability to<jats:italic>S. aureus</jats:italic>sufficiently to prevent their use in the generation of released phospholipids. This finding implied that in host tissues<jats:italic>S. aureus</jats:italic>is likely to be completely dependent upon endogenous phospholipid biosynthesis to generate lipids for release, providing a target for therapeutic intervention. To test this, we exposed<jats:italic>S. aureus</jats:italic>to AFN-1252, an inhibitor of the staphylococcal FASII fatty acid biosynthetic pathway, together with daptomycin. AFN-1252 efficiently blocked daptomycin-induced phospholipid decoy production, even in the case of isolates resistant to AFN-1252, which prevented the inactivation of daptomycin and resulted in sustained bacterial killing. In turn, daptomycin prevented the fatty acid-dependent emergence of AFN-1252-resistant isolates. In summary, AFN-1252 significantly enhances daptomycin
Krishna A, Holden M, Peacock S, et al., 2018, Naturally occurring polymorphisms in the virulence regulator Rsp modulate Staphylococcus aureus survival in blood and antibiotic susceptibility, Microbiology, Vol: 164, Pages: 1189-1195, ISSN: 1350-0872
Nasal colonization by the pathogen Staphylococcus aureus is a risk factor for subsequent infection. Loss of function mutations in the gene encoding the virulence regulator Rsp are associated with the transition of S. aureus from a colonizing isolate to one that causes bacteraemia. Here, we report the identification of several novel activity-altering mutations in rsp detected in clinical isolates, including for the first time, mutations that enhance agr operon activity. We assessed how these mutations affected infection-relevant phenotypes and found loss and enhancement of function mutations to have contrasting effects on S. aureus survival in blood and antibiotic susceptibility. These findings add to the growing body of evidence that suggests S. aureus ‘trades off’ virulence for the acquisition of traits that benefit survival in the host, and indicates that infection severity and treatment options can be significantly affected by mutations in the virulence regulator rsp.
Sabnis A, Ledger E, Pader V, et al., 2018, Antibiotic interceptors: creating safe spaces for bacteria, PLoS Pathogens, Vol: 14, ISSN: 1553-7366
Painter K, Hall A, Ha KP, et al., 2017, The electron transport chain sensitises Staphylococcus aureus and Enterococcus faecalis to the oxidative burst, Infection and Immunity, ISSN: 0019-9567
Small colony variants (SCVs) of Staphylococcus aureus typically lack a functional electron transport chain and cannot produce virulence factors such as leukocidins, hemolysins or the anti-oxidant staphyloxanthin. Despite this, SCVs are associated with persistent infections of the bloodstream, bones and prosthetic devices. The survival of SCVs in the host has been ascribed to intracellular residency, biofilm formation and resistance to antibiotics. However, the ability of SCVs to resist host defences is largely uncharacterised. To address this, we measured survival of wild-type and SCV S. aureus in whole human blood, which contains high numbers of neutrophils, the key defense against staphylococcal infection. Despite the loss of leukcocidin production and staphyloxanthin biosynthesis, SCVs defective for heme or menquinone biosynthesis were significantly more resistant to the oxidative burst than wild-type bacteria in a whole human blood model. Supplementation of the culture medium of the heme-auxotrophic SCV with heme, but not iron, restored growth, hemolysin and staphyloxanthin production, and sensitivity to the oxidative burst. Since Enterococcus faecalis is a natural heme auxotroph and cause of bloodstream infection, we explored whether restoration of the electron transport chain in this organism also affected survival in blood. Incubation of E. faecalis with heme increased growth and restored catalase activity, but resulted in decreased survival in human blood via increased sensitivity to the oxidative burst. Therefore, the lack of functional electron transport chains in SCV S. aureus and wild-type E. faecalis results in reduced growth rate but provides resistance to a key immune defence mechanism.
Painter KL, Hall A, Ha KP, et al., 2017, The electron transport chain sensitisesStaphylococcus aureus and Enterococcus faecalis to the oxidative burst, Infection and Immunity, Vol: 85, ISSN: 0019-9567
Small colony variants (SCVs) of Staphylococcus aureus typically lack a functional electron transport chain and cannot produce virulence factors such as leukocidins, hemolysins or the anti-oxidant staphyloxanthin. Despite this, SCVs are associated with persistent infections of the bloodstream, bones and prosthetic devices. The survival of SCVs in the host has been ascribed to intracellular residency, biofilm formation and resistance to antibiotics. However, the ability of SCVs to resist host defences is largely uncharacterised. To address this, we measured survival of wild-type and SCV S. aureus in whole human blood, which contains high numbers of neutrophils, the key defense against staphylococcal infection. Despite the loss of leukcocidin production and staphyloxanthin biosynthesis, SCVs defective for heme or menquinone biosynthesis were significantly more resistant to the oxidative burst than wild-type bacteria in human blood or the presence of purified neutrophils. Supplementation of the culture medium of the heme-auxotrophic SCV with heme, but not iron, restored growth, hemolysin and staphyloxanthin production, and sensitivity to the oxidative burst. Since Enterococcus faecalis is a natural heme auxotroph and cause of bloodstream infection, we explored whether restoration of the electron transport chain in this organism also affected survival in blood. Incubation of E. faecalis with heme increased growth and restored catalase activity, but resulted in decreased survival in human blood via increased sensitivity to the oxidative burst. Therefore, the lack of functional electron transport chains in SCV S. aureus and wild-type E. faecalis results in reduced growth rate but provides resistance to a key immune defence mechanism.
Ledger E, Pader V, Edwards A, 2017, Enterococcus faecalis and pathogenic streptococci inactivate daptomycin by releasing phospholipids, Publisher: Microbiology Society
Daptomycin is a lipopeptide antibiotic with activity against Gram-positive bacteria. We have shown previously that Staphylococcus aureus can survive daptomycin exposure by releasing membrane phospholipids that inactivate the antibiotic. To determine whether other pathogens possess this defence mechanism, phospholipid release and daptomycin activity were measured after incubation of Staphylococcus epidermidis, Group A or B streptococci, Streptococcus gordonii or Enterococcus faecalis with the antibiotic. All bacteria released phospholipid in response to daptomycin, which resulted in at least partial inactivation of the antibiotic. However, E. faecalis showed the highest levels of lipid release and daptomycin inactivation. As shown previously for S. aureus, phospholipid release by E. faecalis was inhibited by the lipid biosynthesis inhibitor platensimycin. In conclusion, several pathogenic Gram-positive bacteria, including E. faecalis, inactivate daptomycin by releasing phospholipids, which may contribute to the failure of daptomycin to resolve infections caused by these pathogens.
Ledger EVK, Pader V, Edwards A, 2017, Enterococcus faecalis and pathogenic streptococci inactivate daptomycin by releasing phospholipids, Microbiology, Vol: 163, Pages: 1502-1508, ISSN: 1350-0872
Daptomycin is a lipopeptide antibiotic with activity against Gram-positive bacteria. We showed previously that Staphylococcusaureus can survive daptomycin exposure by releasing membrane phospholipids that inactivate the antibiotic. To determinewhether other pathogens possess this defence mechanism, phospholipid release and daptomycin activity were measuredafter incubation of Staphylococcus epidermidis, group A or B streptococci, Streptococcus gordonii or Enterococcus faecalis withthe antibiotic. All bacteria released phospholipids in response to daptomycin, which resulted in at least partial inactivation ofthe antibiotic. However, E. faecalis showed the highest levels of lipid release and daptomycin inactivation. As shownpreviously for S. aureus, phospholipid release by E. faecalis was inhibited by the lipid biosynthesis inhibitor platensimycin. Inconclusion, several pathogenic Gram-positive bacteria, including E. faecalis, inactivate daptomycin by releasing phospholipids,which may contribute to the failure of daptomycin to resolve infections caused by these pathogens.
Pader V, Edwards AM, 2017, Daptomycin: new insights into an antibiotic of last resort, FUTURE MICROBIOLOGY, Vol: 12, Pages: 461-464, ISSN: 1746-0913
Pader V, Hakim S, Painter KL, et al., 2016, Staphylococcus aureus inactivates daptomycin by releasing membrane phospholipids, Nature Microbiology, Vol: 2, Pages: 1-8, ISSN: 2058-5276
Daptomycin is a bactericidal antibiotic of last resort for serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA)1,2. Although resistance is rare, treatment failure can occur in more than 20% of cases3,4 and so there is a pressing need to identify and mitigate factors that contribute to poor therapeutic outcomes. Here, we show that loss of the Agr quorum-sensing system, which frequently occurs in clinical isolates, enhances S. aureus survival during daptomycin treatment. Wild-type S. aureus was killed rapidly by daptomycin, but Agr-defective mutants survived antibiotic exposure by releasing membrane phospholipids, which bound and inactivated the antibiotic. Although wild-type bacteria also released phospholipid in response to daptomycin, Agr-triggered secretion of small cytolytic toxins, known as phenol soluble modulins, prevented antibiotic inactivation. Phospholipid shedding by S. aureus occurred via an active process and was inhibited by the β-lactam antibiotic oxacillin, which slowed inactivation of daptomycin and enhanced bacterial killing. In conclusion, S. aureus possesses a transient defence mechanism that protects against daptomycin, which can be compromised by Agr-triggered toxin production or an existing therapeutic antibiotic.
Painter KL, Strange E, Parkhill J, et al., 2015, Staphylococcus aureus adapts to oxidative stress by producing H2O2-resistant small colony variants via the SOS response, Infection and Immunity, ISSN: 1098-5522
Painter KL, Krishna A, Wigneshweraraj S, et al., 2014, What role does the quorum-sensing accessory gene regulator system play during Staphylococcus aureus bacteremia?, Trends in Microbiology, Vol: In Press, ISSN: 0966-842X
Stemberk V, Jones RPO, Moroz O, et al., 2014, Evidence for steric regulation of fibrinogen binding to staphylococcus aureus Fibronectin-binding Protein A ( FnBPA), Journal of Biological Chemistry, Vol: 289, Pages: 12842-12851, ISSN: 0021-9258
The adjacent fibrinogen (Fg)- and fibronectin (Fn)-binding sites on Fn-binding protein A (FnBPA), a cell surface protein from Staphylococcus aureus, are implicated in the initiation and persistence of infection. FnBPA contains a single Fg-binding site (that also binds elastin) and multiple Fn-binding sites. Here, we solved the structure of the N2N3 domains containing the Fg-binding site of FnBPA in the apo form and in complex with a Fg peptide. The Fg binding mechanism is similar to that of homologous bacterial proteins but without the requirement for “latch” strand residues. We show that the Fg-binding sites and the most N-terminal Fn-binding sites are nonoverlapping but in close proximity. Although Fg and a subdomain of Fn can form a ternary complex on an FnBPA protein construct containing a Fg-binding site and single Fn-binding site, binding of intact Fn appears to inhibit Fg binding, suggesting steric regulation. Given the concentrations of Fn and Fg in the plasma, this mechanism might result in targeting of S. aureus to fibrin-rich thrombi or elastin-rich tissues.
Rudkin JK, Laabei M, Edwards AM, et al., 2014, Oxacillin Alters the Toxin Expression Profile of Community-Associated Methicillin-Resistant <i>Staphylococcus aureus</i>, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Vol: 58, Pages: 1100-1107, ISSN: 0066-4804
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Pader V, James EH, Painter KL, et al., 2014, The Agr quorum-sensing system regulates fibronectin-binding but not hemolysis in the absence of a functional electron transport chain., Infection and Immunity
James EH, Edwards AM, Wigneshweraraj S, 2013, Transcriptional downregulation of agr expression in Staphylococcus aureus during growth in human serum can be overcome by constitutively active mutant forms of the sensor kinase AgrC, FEMS Microbiology Letters, Vol: 349, Pages: 153-162, ISSN: 0378-1097
The temporal and cell density-dependent regulation of expression of virtually all the Staphylococcus aureus virulon is under the control of the agr (accessory gene regulatory) operon. The expression of the agr operon is subject to transcriptional regulation by the AgrA/C two-component response regulator/sensor kinase pair. During bacteraemia, a frequent syndrome caused by methicillin-resistant S. aureus (MRSA), the transcriptional downregulation of agr expression has been attributed to the sequestration of the quorum-signalling molecule auto-inducing peptide (AIP) by the human serum component apolipoprotein B as part of an innate immune response to infection. However, it is not known whether transcriptional downregulation of agr expression during growth in human serum is additionally subjected to regulation by transcription regulatory proteins that either directly or indirectly affect transcription from the agr operon promoters. Here, using chromosomal fluorescence reporters of agr expression in S. aureus, we show that the transcriptional downregulation of agr expression in human serum can be overcome using constitutive active mutant forms of AgrC. Therefore, it seems that the sequestration of the AIP is likely to be the only mechanism by which the host innate immune response limits agr expression at the transcriptional level to maintain the host–pathogen balance towards a noninvasive outcome.
Edwards AM, Bowden MG, Brown EL, et al., 2012, Staphylococcus aureus extracellular adherence protein triggers TNF alpha release, promoting attachment to endothelial cells via protein A, PLoS One, Vol: 7, Pages: 1-8, ISSN: 1932-6203
Staphylococcus aureus is a leading cause of bacteraemia, which frequently results in complications such as infective endocarditis, osteomyelitis and exit from the bloodstream to cause metastatic abscesses. Interaction with endothelial cells is critical to these complications and several bacterial proteins have been shown to be involved. The S. aureus extracellular adhesion protein (Eap) has many functions, it binds several host glyco-proteins and has both pro- and anti-inflammatory activity. Unfortunately its role in vivo has not been robustly tested to date, due to difficulties in complementing its activity in mutant strains. We previously found Eap to have pro-inflammatory activity, and here show that purified native Eap triggered TNFα release in whole human blood in a dose-dependent manner. This level of TNFα increased adhesion of S. aureus to endothelial cells 4-fold via a mechanism involving protein A on the bacterial surface and gC1qR/p33 on the endothelial cell surface. The contribution this and other Eap activities play in disease severity during bacteraemia was tested by constructing an isogenic set of strains in which the eap gene was inactivated and complemented by inserting an intact copy elsewhere on the bacterial chromosome. Using a murine bacteraemia model we found that Eap expressing strains cause a more severe infection, demonstrating its role in invasive disease.
Rudkin JK, Edwards AM, Bowden MG, et al., 2012, Methicillin resistance reduces the virulence of healthcare-associated methicillin-resistant staphylococcus aureus by interfering with the agr quorum sensing system, Journal of Infectious Diseases, Vol: 205, Pages: 798-806, ISSN: 0022-1899
The difficulty in successfully treating infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has led to them being referred to as highly virulent or pathogenic. In our study of one of the major healthcare-associated MRSA (HA-MRSA) clones, we show that expression of the gene responsible for conferring methicillin resistance (mecA) is also directly responsible for reducing the ability of HA-MRSA to secrete cytolytic toxins. We show that resistance to methicillin induces changes in the cell wall, which affects the bacteria's agr quorum sensing system. This leads to reduced toxin expression and, as a consequence, reduced virulence in a murine model of sepsis. This diminished capacity to cause infection may explain the inability of HA-MRSA to move into the community and help us understand the recent emergence of community-associated MRSA (CA-MRSA). CA-MRSA typically express less penicillin-binding protein 2a (encoded by mecA), allowing them to maintain full virulence and succeed in the community environment.
Edwards AM, 2012, Phenotype-switching is a natural consequence of Staphylococcus aureus replication, Journal of Bacteriology
The pathogen Staphylococcus aureus undergoes phenotype-switching in vivo from its normal colony phenotype (NCP) to a slow-growing, antibiotic-resistant small colony variant (SCV) phenotype, which is associated with persistence in host cells and tissues. However, it is not clear whether phenotype-switching is the result of a constitutive process that is selected for under certain conditions, or is triggered by particular environmental stimuli. Examination of cultures of diverse S. aureus strains in the absence of selective pressure consistently revealed a small gentamicin-resistant SCV sub-population, which emerged during exponential-phase NCP growth and increased in number until NCP stationary phase. Treatment of replicating bacteria with the antibiotic gentamicin, which inhibited NCP but not SCV replication, resulted in an initial decrease in SCV numbers, demonstrating that SCVs arise as a consequence of NCP replication. However, SCV population expansion in the presence of gentamicin was re-established by selection of phenotype-stable SCVs and subsequent SCV replication. In the absence of selective pressure however, phenotype-switching was bi-directional and occurred at a high frequency during NCP replication, resulting in SCV turnover. In summary, these data demonstrate that S. aureus phenotype-switching occurs via a constitutive mechanism that generates a dynamic, antibiotic-resistant sub-population of bacteria that can revert to the parental phenotype. The emergence of SCVs can therefore be considered a normal part of the S. aureus life-cycle and provides an insurance policy against exposure to antibiotics that would otherwise eliminate the entire population.
Edwards AM, Bowden MG, Brown EL, et al., 2012, Staphylococcus aureus Extracellular Adherence Protein triggers TNFα release, promoting attachment to endothelial cells via protein A., Plos One, Vol: (8)
Staphylococcus aureus is a leading cause of bacteraemia, which frequently results in complications such as infective endocarditis, osteomyelitis and exit from the bloodstream to cause metastatic abscesses. Interaction with endothelial cells is critical to these complications and several bacterial proteins have been shown to be involved. The S. aureus extracellular adhesion protein (Eap) has many functions, it binds several host glyco-proteins and has both pro- and anti-inflammatory activity. Unfortunately its role in vivo has not been robustly tested to date, due to difficulties in complementing its activity in mutant strains. We previously found Eap to have pro-inflammatory activity, and here show that purified native Eap triggered TNFα release in whole human blood in a dose-dependent manner. This level of TNFα increased adhesion of S. aureus to endothelial cells 4-fold via a mechanism involving protein A on the bacterial surface and gC1qR/p33 on the endothelial cell surface. The contribution this and other Eap activities play in disease severity during bacteraemia was tested by constructing an isogenic set of strains in which the eap gene was inactivated and complemented by inserting an intact copy elsewhere on the bacterial chromosome. Using a murine bacteraemia model we found that Eap expressing strains cause a more severe infection, demonstrating its role in invasive disease.
Edwards AM, Massey RC, Clarke SR, 2012, Molecular mechanisms of Staphylococcus aureus nasopharyngeal colonization, Molecular Oral Microbiology, Vol: (1), Pages: 1-10
Staphylococcus aureus is responsible for a wide range of different infections ranging in severity from mild to fatal. However, it primarily exists as a commensal organism in a number of different anatomical sites including the nasopharynx. Although colonization itself is a harmless state, colonized individuals are at risk of endogenous infection when S. aureus enters otherwise sterile sites via wounds or indwelling medical devices. As such, studies of colonization may identify important targets for vaccines or other prophylactic approaches. Colonization is a dynamic process; S. aureus must attach to host surfaces, overcome immune components and compete with other commensal microbes. This occurs via a number of surface-attached and secreted proteins and other factors such as wall teichoic acid. In addition, colonizing S. aureus must constantly replicate to maintain its niche and exclude other strains. These myriad interactions provide a strong selective pressure for the maintenance or enhancement of mechanisms of adhesion, invasion and immune evasion. The evolutionary implications of this may explain why S. aureus is such a capable pathogen because many of the proteins involved in colonization have also been identified as virulence factors. This review describes the diverse molecular mechanisms used by S. aureus to colonize the host and discusses how the pressures that have selected for these may have led to its virulence.
Edwards AM, Massey RC, 2011, How does Staphylococcus aureus escape the bloodstream?, Trends Microbiol., Vol: 19, Pages: 184-190
Staphylococcus aureus is a major cause of bacteraemia, which frequently leads to infective endocarditis, osteomyelitis, septic arthritis and metastatic abscess formation. The development of these secondary infections is due to bacterial dissemination from the blood into surrounding tissues and is associated with significantly increased morbidity and mortality. Despite the importance of S. aureus extravasation in disease progression, there is relatively little understanding of the molecular mechanisms by which this pathogen crosses the endothelial barrier and establishes new sites of infection. Recent work has identified a number of putative routes by which S. aureus can escape the bloodstream. In this article we review these new developments and set them in the context of strategies used by other established pathogens to traverse cellular barriers
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