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The secretion of proteins that damage host tissue is well established as integral to the infectious processes of many bacterial pathogens. However, recent advances in our understanding of the activity of toxins suggest that the attributes we have assigned to them from early in vitro experimentation have misled us into thinking of them as merely destructive tools. Here, we will discuss the multifarious ways in which toxins contribute to the lifestyle of bacteria and, by considering their activity from an evolutionary perspective, demonstrate how this extends far beyond their ability to destroy host tissue.

Clinical isolates of Staphylococcus aureus can express biofilm phenotypes promoted by the major cell wall autolysin and the fibronectin-binding proteins or the icaADBC-encoded polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG). Biofilm production in methicillin-susceptible S. aureus (MSSA) strains is typically dependent on PIA/PNAG whereas methicillin-resistant isolates express an Atl/FnBP-mediated biofilm phenotype suggesting a relationship between susceptibility to β-lactam antibiotics and biofilm. By introducing the methicillin resistance gene mecA into the PNAG-producing laboratory strain 8325-4 we generated a heterogeneously resistant (HeR) strain, from which a homogeneous, high-level resistant (HoR) derivative was isolated following exposure to oxacillin. The HoR phenotype was associated with a R₆₀₂H substitution in the DHHA1 domain of GdpP, a recently identified c-di-AMP phosphodiesterase with roles in resistance/tolerance to β-lactam antibiotics and cell envelope stress. Transcription of icaADBC and PNAG production were impaired in the 8325-4 HoR derivative, which instead produced a proteinaceous biofilm that was significantly inhibited by antibodies against the mecA-encoded penicillin binding protein 2a (PBP2a). Conversely excision of the SCCmec element in the MRSA strain BH1CC resulted in oxacillin susceptibility and reduced biofilm production, both of which were complemented by mecA alone. Transcriptional activity of the accessory gene regulator locus was also repressed in the 8325-4 HoR strain, which in turn was accompanied by reduced protease production and significantly reduced virulence in a mouse model of device infection. Thus, homogeneous methicillin resistance has the potential to affect agr- and icaADBC-mediated phenotypes, including altered biofilm expression and virulence, which together are consistent with the adaptation of healthcare-associated MRSA strains to the antibiotic-rich hospital environment in which they are frequently responsible for device-related infections in immuno-compromised patients.

The incidence of Staphylococcus aureus bacteremia is on a steady incline in many parts of the world. Given the associated mortality rates have changed little in the last 10 years, this is a major health concern. One contributing problem is that antibiotics effective against the bacteria in vitro are failing to cure many patients, e.g., the use of daptomycin to treat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we present a mechanistic study that may explain why this occurs. The expression of mecA , which confers the methicillin resistance of MRSA, reduces Agr activity, and this decreases the release of phenol-soluble modulins from the bacteria. Without these, the phospholipids that can block daptomycin activity are free to do so, rendering MRSA less sensitive to both antibiotics. This study, bridging clinical and molecular biology, provides an explanation for a significant clinical problem and may inform how antibiotic combinations should be managed in future trials.

Staphylococcus aureus is an important human pathogen and a commensal of the human nose and skin. Survival and persistence during colonisation are likely major drivers of S. aureus evolution. Here we applied a genome-wide mutation enrichment approach to a genomic dataset of 3060 S. aureus colonization isolates from 791 individuals. Despite limited within-host genetic diversity, we observed an excess of protein-altering mutations in metabolic genes, in regulators of quorum-sensing ( agrA and agrC ) and in known antibiotic targets ( fusA , pbp2 , dfrA and ileS ). We demonstrated the phenotypic effect of multiple adaptive mutations in vitro, including changes in haemolytic activity, antibiotic susceptibility, and metabolite utilisation. Nitrogen metabolism showed the strongest evidence of adaptation, with the assimilatory nitrite reductase ( nasD ) and urease ( ureG ) showing the highest mutational enrichment. We identified a nasD natural mutant with enhanced growth under urea as the sole nitrogen source. Inclusion of 4090 additional isolate genomes from 731 individuals revealed eight more genes including sasA / sraP , darA/pstA , and rsbU with signals of adaptive variation that warrant further characterisation. Our study provides a comprehensive picture of the heterogeneity of S. aureus adaptive changes during colonisation, and a robust methodological approach applicable to study in host adaptive evolution in other bacterial pathogens. The authors applied a genomic and evolutionary approach to study Staphylococcus aureus in host adaptation during colonisation revealing signals of adaption in metabolic genes, in regulators of quorum-sensing and in known antibiotic targets.

The S. aureus cell envelope, comprising the cytoplasmic membrane, a thick peptidoglycan layer, and the anionic polymers lipoteichoic acid and wall teichoic acids, is fundamental for bacterial growth and division, as well as being the main interface between the pathogen and the host. It has become increasingly apparent that the synthesis and turnover of cell envelope components also affect the virulence of S. aureus . In this study, we show that MspA, an effector of S. aureus virulence, contributes to the maintenance of normal levels of lipoteichoic acid in the cell wall, with implications on cell cycle and size. These findings further our understanding of the connections between envelope synthesis and pathogenicity and suggest that MspA represents a promising target for the development of future therapeutic strategies.

Entry of Staphylococcus aureus into the bloodstream can lead to metastatic abscess formation and infective endocarditis. Crucial to the development of both these conditions is the interaction of S. aureus with endothelial cells. In vivo and in vitro studies have shown that the staphylococcal invasin FnBPA triggers bacterial invasion of endothelial cells via a process that involves fibronectin (Fn) bridging to alpha(5)beta(1) integrins. The Fn-binding region of FnBPA usually contains 11 non-identical repeats (FnBRs) with differing affinities for Fn, which facilitate the binding of multiple Fn molecules and may promote integrin clustering. We thus hypothesized that multiple repeats are necessary to trigger the invasion of endothelial cells by S. aureus. To test this we constructed variants of fnbA containing various combinations of FnBRs. In vitro assays revealed that endothelial cell invasion can be facilitated by a single high-affinity, but not low-affinity FnBR. Studies using a nisin-inducible system that controlled surface expression of FnBPA revealed that variants encoding fewer FnBRs required higher levels of surface expression to mediate invasion. High expression levels of FnBPA bearing a single low affinity FnBR bound Fn but did not invade, suggesting that FnBPA affinity for Fn is crucial for triggering internalization. In addition, multiple FnBRs increased the speed of internalization, as did higher expression levels of FnBPA, without altering the uptake mechanism. The relevance of these findings to pathogenesis was demonstrated using a murine sepsis model, which showed that multiple FnBRs were required for virulence. In conclusion, multiple FnBRs within FnBPA facilitate efficient Fn adhesion, trigger rapid bacterial uptake and are required for pathogenesis.

Bacteria responsible for the greatest global mortality colonize the human microbiota far more frequently than they cause severe infections. Whether mutation and selection among commensal bacteria are associated with infection is unknown. We investigated de novo mutation in 1163 Staphylococcus aureus genomes from 105 infected patients with nose colonization. We report that 72% of infections emerged from the nose, with infecting and nose-colonizing bacteria showing parallel adaptive differences. We found 2.8-to-3.6-fold adaptive enrichments of protein-altering variants in genes responding to rsp, which regulates surface antigens and toxin production; agr, which regulates quorum-sensing, toxin production and abscess formation; and host-derived antimicrobial peptides. Adaptive mutations in pathogenesis-associated genes were 3.1-fold enriched in infecting but not nose-colonizing bacteria. None of these signatures were observed in healthy carriers nor at the species-level, suggesting infection-associated, short-term, within-host selection pressures. Our results show that signatures of spontaneous adaptive evolution are specifically associated with infection, raising new possibilities for diagnosis and treatment.

Background Fitness costs imposed on bacteria by antibiotic resistance mechanisms are believed to hamper their dissemination. The scale of these costs is highly variable. Some, including resistance of Staphylococcus aureus to the clinically important antibiotic mupirocin, have been reported as being cost-free, which suggests that there are few barriers preventing their global spread. However, this is not supported by surveillance data in healthy communities, which indicate that this resistance mechanism is relatively unsuccessful. Results Epistasis analysis on two collections of MRSA provides an explanation for this discord, where the mupirocin resistance-conferring mutation of the ileS gene appears to affect the levels of toxins produced by S. aureus when combined with specific polymorphisms at other loci. Proteomic analysis demonstrates that the activity of the secretory apparatus of the PSM family of toxins is affected by mupirocin resistance. As an energetically costly activity, this reduction in toxicity masks the fitness costs associated with this resistance mutation, a cost that becomes apparent when toxin production becomes necessary. This hidden fitness cost provides a likely explanation for why this mupirocin-resistance mechanism is not more prevalent, given the widespread use of this antibiotic. Conclusions With dwindling pools of antibiotics available for use, information on the fitness consequences of the acquisition of resistance may need to be considered when designing antibiotic prescribing policies. However, this study suggests there are levels of depth that we do not understand, and that holistic, surveillance and functional genomics approaches are required to gain this crucial information.

The bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.

Background Large-scale genomic studies of within-host diversity in Staphylococcus aureus bacteraemia (SAB) are needed to understanding bacterial adaptation underlying persistence and thus refining the role of genomics in management of SAB. However, available comparative genomic studies of sequential SAB isolates have tended to focus on selected cases of unusually prolonged bacteraemia, where secondary antimicrobial resistance has developed. Methods To understand bacterial genetic diversity during SAB more broadly, we applied whole genome sequencing to a large collection of sequential isolates obtained from patients with persistent or relapsing bacteraemia. After excluding genetically unrelated isolates, we performed an in-depth genomic analysis of point mutations and chromosome structural variants arising within individual SAB episodes. Results We show that, while adaptation pathways are heterogenous and episode-specific, isolates from persistent bacteraemia have a distinctive molecular signature, characterised by a low mutation frequency and high proportion of non-silent mutations. Analysis of structural genomic variants revealed that these often overlooked genetic events are commonly acquired during SAB. We discovered that IS 256 insertion may represent the most effective driver of within-host microevolution in selected lineages, with up to three new insertion events per isolate even in the absence of other mutations. Genetic mechanisms resulting in significant phenotypic changes, such as increases in vancomycin resistance, development of small colony phenotypes, and decreases in cytotoxicity, included mutations in key genes ( rpoB , stp , agrA ) and an IS 256 insertion upstream of the walKR operon. Conclusions This study provides for the first time a large-scale analysis of within-host genomic changes during invasive S. aureus infection and describes specific patterns of adaptation that will be informative for both understanding S. aureus pathoadaptation and utilising genomics for management of complicated S. aureus infections.