Imperial College London

DrAndrewEdwards

Faculty of MedicineDepartment of Infectious Disease

Senior Lecturer
 
 
 
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a.edwards Website

 
 
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5.40AFlowers buildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

40 results found

Ha KP, Clarke R, Kim G-L, Brittan J, Rowley J, Mavridou D, Parker D, Clarke T, Nobbs A, Edwards Aet al., 2020, Staphylococcal DNA repair is required for infection, mBio, ISSN: 2150-7511

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.

Journal article

Clarke R, Bruderer M, Ha KP, Edwards Aet 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.

Journal article

Lim C, Ha KP, Clarke R, Gavin L-A, Cook D, Hutton J, Sutherell C, Edwards A, Evans L, Tate E, Lanyon-Hogg Tet 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.

Journal article

Lacoma A, Edwards AM, Young BC, Dominguez J, Prat C, Laabei Met al., 2019, Cigarette smoke exposure redirects Staphylococcus aureus to a virulence profile associated with persistent infection, SCIENTIFIC REPORTS, Vol: 9, ISSN: 2045-2322

Journal article

Edwards AM, 2019, Silence is golden for Staphylococcus, NATURE MICROBIOLOGY, Vol: 4, Pages: 1073-1074, ISSN: 2058-5276

Journal article

Evans LE, Krishna A, Ma Y, Webb TE, Marshall DC, Tooke CL, Spencer J, Clarke TB, Armstrong A, Edwards Aet 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.

Journal article

Pee CJE, Pader V, Ledger EVK, Edwards AMet 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.

Journal article

Sabnis A, Klöckner A, Becce M, Hagart KLH, Evans LE, Furniss RCD, Mavridou DAI, Larrouy-Maumus GJ, Stevens MM, Edwards AMet al., 2018, Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane, Publisher: Cold Spring Harbor Laboratory

<jats:title>Summary</jats:title><jats:p>Colistin is an antibiotic of last resort for infections caused by drug-resistant Gram-negative pathogens such as <jats:italic>Pseudomonas aeruginosa</jats:italic> and <jats:italic>Escherichia coli</jats:italic>. For this reason, high rates of treatment failure and increasing resistance to this antibiotic are very concerning and attempts to resolve these issues are hampered by a poor understanding of colistin’s mode of action. Whilst it is well established that colistin binds to lipopolysaccharide in the bacterial outer membrane, it was unclear how this led to bacterial killing. Here, we show that colistin also targets lipopolysaccharide in the cytoplasmic membrane and that this interaction is essential for cytoplasmic membrane permeabilisation, cell lysis and the bactericidal activity of the antibiotic. We also found that MCR-1-mediated colistin resistance confers protection against the antibiotic via the presence of modified lipopolysaccharide within the cytoplasmic membrane, rather than the outer membrane. These findings reveal key details about the mechanism by which colistin kills bacteria, providing the foundations for the development of new approaches to enhance therapeutic outcomes.</jats:p>

Working paper

Pee CJE, Pader V, Ledger EVK, Edwards AMet al., 2018, A FASII inhibitor prevents staphylococcal evasion of daptomycin by inhibiting phospholipid decoy production, Publisher: Cold Spring Harbor Laboratory

<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 d

Working paper

Krishna A, Holden M, Peacock S, Edwards A, Wigneshweraraj Set 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.

Journal article

Sabnis A, Ledger E, Pader V, Edwards Aet al., 2018, Antibiotic interceptors: creating safe spaces for bacteria, PLoS Pathogens, Vol: 14, ISSN: 1553-7366

Journal article

Painter K, Hall A, Ha KP, Edwards Aet 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.

Journal article

Painter KL, Hall A, Ha KP, Edwards Aet 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.

Journal article

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.

Working paper

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.

Journal article

Pader V, Edwards AM, 2017, Daptomycin: new insights into an antibiotic of last resort, FUTURE MICROBIOLOGY, Vol: 12, Pages: 461-464, ISSN: 1746-0913

Journal article

Pader V, Hakim S, Painter KL, Wigneshweraraj S, Clarke TB, Edwards Aet al., 2016, Staphylococcus aureus inactivates daptomycin by releasing membrane phospholipids, Nature Microbiology, Vol: 2, ISSN: 2058-5276

Journal article

Painter KL, Strange E, Parkhill J, Bamford KB, Armstrong-James D, Edwards AMet 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

Journal article

Painter KL, Krishna A, Wigneshweraraj S, Edwards AMet 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

Journal article

Stemberk V, Jones RPO, Moroz O, Atkin KE, Edwards AM, Turkenburg JP, Leech AP, Massey RC, Potts JRet 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

Journal article

Rudkin JK, Laabei M, Edwards AM, Joo H-S, Otto M, Lennon KL, O'Gara JP, Waterfield NR, Massey RCet al., 2014, Oxacillin Alters the Toxin Expression Profile of Community-Associated Methicillin-Resistant Staphylococcus aureus, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Vol: 58, Pages: 1100-1107, ISSN: 0066-4804

Journal article

Pader V, James EH, Painter KL, Wigneshweraraj S, Edwards AMet 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

Journal article

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.

Journal article

Rudkin JK, Edwards AM, Bowden MG, Brown EL, Pozzi C, Waters EM, Chan WC, Williams P, O'Gara JP, Massey RCet 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

Journal article

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.

Journal article

Edwards AM, Bowden MG, Brown EL, Laabei M, Massey RCet 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.

Journal article

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.

Journal article

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

Journal article

Edwards AM, Potter U, Meenan NA, Potts JR, Massey RCet al., 2011, Staphylococcus aureus keratinocyte invasion is dependent upon multiple high-affinity fibronectin-binding repeats within FnBPA, PLoS.One., Vol: 6

Staphylococcus aureus is a commensal organism and a frequent cause of skin and soft tissue infections, which can progress to serious invasive disease. This bacterium uses its fibronectin binding proteins (FnBPs) to invade host cells and it has been hypothesised that this provides a protected niche from host antimicrobial defences, allows access to deeper tissues and provides a reservoir for persistent or recurring infections. FnBPs contain multiple tandem fibronectin-binding repeats (FnBRs) which bind fibronectin with varying affinity but it is unclear what selects for this configuration. Since both colonisation and skin infection are dependent upon the interaction of S. aureus with keratinocytes we hypothesised that this might select for FnBP function and thus composition of the FnBR region. Initial experiments revealed that S. aureus attachment to keratinocytes is rapid but does not require FnBRs. By contrast, invasion of keratinocytes was dependent upon the FnBR region and occurred via similar cellular processes to those described for endothelial cells. Despite this, keratinocyte invasion was relatively inefficient and appeared to include a lag phase, most likely due to very weak expression of alpha(5)beta(1) integrins. Molecular dissection of the role of the FnBR region revealed that efficient invasion of keratinocytes was dependent on the presence of at least three high-affinity (but not low-affinity) FnBRs. Over-expression of a single high-affinity or three low-affinity repeats promoted invasion but not to the same levels as S. aureus expressing an FnBPA variant containing three high-affinity repeats. In summary, invasion of keratinocytes by S. aureus requires multiple high-affinity FnBRs within FnBPA, and given the importance of the interaction between these cell types and S. aureus for both colonisation and infection, may have provided the selective pressure for the multiple binding repeats within FnBPA

Journal article

Edwards AM, Massey RC, 2011, Invasion of human cells by a bacterial pathogen, J.Vis.Exp.

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO(2) at 37 degrees C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 mul tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy

Journal article

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