Evolutionary Trade-Offs Underlie the Multi-faceted Virulence of Staphylococcus aureus

Bacterial virulence is a multifaceted trait where the interactions between pathogen and host factors affect the severity and outcome of the infection. Toxin secretion is central to the biology of many bacterial pathogens and is widely accepted as playing a crucial role in disease pathology. To understand the relationship between toxicity and bacterial virulence in greater depth, we studied two sequenced collections of the major human pathogen Staphylococcus aureus and found an unexpected inverse correlation between bacterial toxicity and disease severity. By applying a functional genomics approach, we identified several novel toxicity-affecting loci responsible for the wide range in toxic phenotypes observed within these collections. To understand the apparent higher propensity of low toxicity isolates to cause bacteraemia, we performed several functional assays, and our findings suggest that within-host fitness differences between high- and low-toxicity isolates in human serum is a contributing factor. As invasive infections, such as bacteraemia, limit the opportunities for onward transmission, highly toxic strains could gain an additional between-host fitness advantage, potentially contributing to the maintenance of toxicity at the population level. Our results clearly demonstrate how evolutionary trade-offs between toxicity, relative fitness, and transmissibility are critical for understanding the multifaceted nature of bacterial virulence.     

Neutrophil Crawling in Capillaries; A Novel Immune Response to Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA), particularly the USA300 strain, is a highly virulent pathogen responsible for an increasing number of skin and soft tissue infections globally. Furthermore, MRSA-induced soft tissue infections can rapidly progress into life-threatening conditions, such as sepsis and necrotizing fasciitis. The importance of neutrophils in these devastating soft tissue infections remains ambiguous, partly because of our incomplete understanding of their behaviour. Spinning disk confocal microscopy was used to visualize the behaviour of GR1-labelled neutrophils in subcutaneous tissue in response to GFP-expressing MRSA attached to a foreign particle (agarose bead). We observed significant directional neutrophil recruitment towards the S. aureus agarose bead but not a control agarose bead. A significant increase in neutrophil crawling within the capillaries surrounding the infectious nidus was noted, with impaired capillary perfusion in these vessels and increased parenchymal cell death. No neutrophils were able to emigrate from capillaries. The crawling within these capillaries was mediated by the β₂ and α₄ integrins and blocking these integrins 2 hours post infection eliminated neutrophil crawling, improved capillary perfusion, reduced cell death and reduced lesion size. Blocking prior to infection increased pathology. Neutrophil crawling within capillaries during MRSA soft tissue infections, while potentially contributing to walling off or preventing early dissemination of the pathogen, resulted in impaired perfusion and increased tissue injury with time.     

Identification of a Novel Staphylococcus aureus Two-Component Leukotoxin Using Cell Surface Proteomics

Staphylococcus aureus is a prominent human pathogen and leading cause of bacterial infection in hospitals and the community. Community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 are highly virulent and, unlike hospital strains, often cause disease in otherwise healthy individuals. The enhanced virulence of CA-MRSA is based in part on increased ability to produce high levels of secreted molecules that facilitate evasion of the innate immune response. Although progress has been made, the factors that contribute to CA-MRSA virulence are incompletely defined. We analyzed the cell surface proteome (surfome) of USA300 strain LAC to better understand extracellular factors that contribute to the enhanced virulence phenotype. A total of 113 identified proteins were associated with the surface of USA300 during the late-exponential phase of growth in vitro. Protein A was the most abundant surface molecule of USA300, as indicated by combined Mascot score following analysis of peptides by tandem mass spectrometry. Unexpectedly, we identified a previously uncharacterized two-component leukotoxin–herein named LukS-H and LukF-G (LukGH)-as two of the most abundant surface-associated proteins of USA300. Rabbit antibody specific for LukG indicated it was also freely secreted by USA300 into culture media. We used wild-type and isogenic lukGH deletion strains of USA300 in combination with human PMN pore formation and lysis assays to identify this molecule as a leukotoxin. Moreover, LukGH synergized with PVL to enhance lysis of human PMNs in vitro, and contributed to lysis of PMNs after phagocytosis. We conclude LukGH is a novel two-component leukotoxin with cytolytic activity toward neutrophils, and thus potentially contributes to S. aureus virulence.     

Phase Variation of Poly-N-Acetylglucosamine Expression in Staphylococcus aureus

Polysaccharide intercellular adhesin (PIA), also known as poly-N-acetyl-β-(1–6)-glucosamine (PIA/PNAG) is an important component of Staphylococcus aureus biofilms and also contributes to resistance to phagocytosis. The proteins IcaA, IcaD, IcaB, and IcaC are encoded within the intercellular adhesin (ica) operon and synthesize PIA/PNAG. We discovered a mechanism of phase variation in PIA/PNAG expression that appears to involve slipped-strand mispairing. The process is reversible and RecA-independent, and involves the expansion and contraction of a simple tetranucleotide tandem repeat within icaC. Inactivation of IcaC results in a PIA/PNAG-negative phenotype. A PIA/PNAG-hyperproducing strain gained a fitness advantage in vitro following the icaC mutation and loss of PIA/PNAG production. The mutation was also detected in two clinical isolates, suggesting that under certain conditions, loss of PIA/PNAG production may be advantageous during infection. There was also a survival advantage for an icaC-negative strain harboring intact icaADB genes relative to an isogenic icaADBC deletion mutant. Together, these results suggest that inactivation of icaC is a mode of phase variation for PIA/PNAG expression, that high-level production of PIA/PNAG carries a fitness cost, and that icaADB may contribute to bacterial fitness, by an unknown mechanism, in the absence of an intact icaC gene and PIA/PNAG production.     

Membrane Damage Elicits an Immunomodulatory Program in Staphylococcus aureus

The Staphylococcus aureus HrtAB system is a hemin-regulated ABC transporter composed of an ATPase (HrtA) and a permease (HrtB) that protect S. aureus against hemin toxicity. S. aureus strains lacking hrtA exhibit liver-specific hyper-virulence and upon hemin exposure over-express and secrete immunomodulatory factors that interfere with neutrophil recruitment to the site of infection. It has been proposed that heme accumulation in strains lacking hrtAB is the signal which triggers S. aureus to elaborate this anti-neutrophil response. However, we report here that S. aureus strains expressing catalytically inactive HrtA do not elaborate the same secreted protein profile. This result indicates that the physical absence of HrtA is responsible for the increased expression of immunomodulatory factors, whereas deficiencies in the ATPase activity of HrtA do not contribute to this process. Furthermore, HrtB expression in strains lacking hrtA decreases membrane integrity consistent with dysregulated permease function. Based on these findings, we propose a model whereby hemin-mediated over-expression of HrtB in the absence of HrtA damages the staphylococcal membrane through pore formation. In turn, S. aureus senses this membrane damage, triggering the increased expression of immunomodulatory factors. In support of this model, wildtype S. aureus treated with anti-staphylococcal channel-forming peptides produce a secreted protein profile that mimics the effect of treating ΔhrtA with hemin. These results suggest that S. aureus senses membrane damage and elaborates a gene expression program that protects the organism from the innate immune response of the host.     

Biochemical and Structural Characterization of SplD Protease from Staphylococcus aureus

Staphylococcus aureus is a dangerous human pathogen. A number of the proteins secreted by this bacterium are implicated in its virulence, but many of the components of its secretome are poorly characterized. Strains of S. aureus can produce up to six homologous extracellular serine proteases grouped in a single spl operon. Although the SplA, SplB, and SplC proteases have been thoroughly characterized, the properties of the other three enzymes have not yet been investigated. Here, we describe the biochemical and structural characteristics of the SplD protease. The active enzyme was produced in an Escherichia coli recombinant system and purified to homogeneity. P1 substrate specificity was determined using a combinatorial library of synthetic peptide substrates showing exclusive preference for threonine, serine, leucine, isoleucine, alanine, and valine. To further determine the specificity of SplD, we used high-throughput synthetic peptide and cell surface protein display methods. The results not only confirmed SplD preference for a P1 residue, but also provided insight into the specificity of individual primed- and non-primed substrate-binding subsites. The analyses revealed a surprisingly narrow specificity of the protease, which recognized five consecutive residues (P4-P3-P2-P1-P1’) with a consensus motif of R-(Y/W)-(P/L)-(T/L/I/V)↓S. To understand the molecular basis of the strict substrate specificity, we crystallized the enzyme in two different conditions, and refined the structures at resolutions of 1.56 Å and 2.1 Å. Molecular modeling and mutagenesis studies allowed us to define a consensus model of substrate binding, and illustrated the molecular mechanism of protease specificity.     

Novel Inhibitors of Staphylococcus aureus Virulence Gene Expression and Biofilm Formation

Staphylococcus aureus is a major human pathogen and one of the more prominent pathogens causing biofilm related infections in clinic. Antibiotic resistance in S. aureus such as methicillin resistance is approaching an epidemic level. Antibiotic resistance is widespread among major human pathogens and poses a serious problem for public health. Conventional antibiotics are either bacteriostatic or bacteriocidal, leading to strong selection for antibiotic resistant pathogens. An alternative approach of inhibiting pathogen virulence without inhibiting bacterial growth may minimize the selection pressure for resistance. In previous studies, we identified a chemical series of low molecular weight compounds capable of inhibiting group A streptococcus virulence following this alternative anti-microbial approach. In the current study, we demonstrated that two analogs of this class of novel anti-virulence compounds also inhibited virulence gene expression of S. aureus and exhibited an inhibitory effect on S. aureus biofilm formation. This class of anti-virulence compounds could be a starting point for development of novel anti-microbial agents against S. aureus.     

Glycerol Uptake Is Important for L-Form Formation and Persistence in Staphylococcus aureus

S. aureus is a significant human pathogen and has previously been shown to form cell wall deficient forms or L-forms in vitro and in vivo during infection. Despite many previous studies on S. aureus L-forms, the mechanisms of L-form formation in this organism remain unknown. Here we established the L-form model in S. aureus and constructed a transposon mutant library to identify genes involved in L-form formation. Screening of the library for mutants defective in L-form formation identified glpF involved in glycerol uptake being important for L-form formation in S. aureus. Consistent with this observation, glpF was found to be highly expressed in L-form S. aureus but hardly expressed in normal walled form. In addition, glpF mutant was found to be defective in antibiotic persistence. The defect in L-form formation and antibiotic persistence of the glpF mutant could be complemented by the wild type glpF gene. These findings provide new insight into the mechanisms of L-form formation and persistence in S. aureus and may have implications for development of new drugs targeting persisters for improved treatment.     

The multifaceted resources and microevolution of the successful human and animal pathogen methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important bacterial pathogens based on its incidence and the severity of its associated infections. In addition, severe MRSA infections can occur in hospitalised patients or healthy individuals from the community. Studies have shown the infiltration of MRSA isolates of community origin into hospitals and variants of hospital-associated MRSA have caused infections in the community. These rapid epidemiological changes represent a challenge for the molecular characterisation of such bacteria as a hospital or community-acquired pathogen. To efficiently control the spread of MRSA, it is important to promptly detect the mecA gene, which is the determinant of methicillin resistance, using a polymerase chain reaction-based test or other rapidly and accurate methods that detect the mecA product penicillin-binding protein (PBP)2a or PBP2’. The recent emergence of MRSA isolates that harbour a mecA allotype, i.e., the mecC gene, infecting animals and humans has raised an additional and significant issue regarding MRSA laboratory detection. Antimicrobial drugs for MRSA therapy are becoming depleted and vancomycin is still the main choice in many cases. In this review, we present an overview of MRSA infections in community and healthcare settings with focus on recent changes in the global epidemiology, with special reference to the MRSA picture in Brazil.     

Hfq Is a Global Regulator That Controls the Pathogenicity of Staphylococcus aureus

The Hfq protein is reported to be an RNA chaperone, which is involved in the stress response and the virulence of several pathogens. In E. coli, Hfq can mediate the interaction between some sRNAs and their target mRNAs. But it is controversial whether Hfq plays an important role in S. aureus. In this study, we found that the deletion of hfq gene in S. aureus 8325-4 can increase the surface carotenoid pigments. The hfq mutant was more resistant to oxidative stress but the pathogenicity of the mutant was reduced. We reveal that the Hfq protein can be detected only in some S. aureus strains. Using microarray and qRT-PCR, we identified 116 genes in the hfq mutant which had differential expression from the wild type, most of which are related to the phenotype and virulence of S. aureus. Among the 116 genes, 49 mRNAs can specifically bind Hfq protein, which indicates that Hfq also acts as an RNA binding protein in S. aureus. Our data suggest that Hfq protein of S. aureus is a multifunctional regulator involved in stress and virulence.     

A Timescale for Evolution,Population Expansion,and Spatial Spread of an Emerging Clone of Methicillin-Resistant Staphylococcus aureus

Due to the lack of fossil evidence, the timescales of bacterial evolution are largely unknown. The speed with which genetic change accumulates in populations of pathogenic bacteria, however, is a key parameter that is crucial for understanding the emergence of traits such as increased virulence or antibiotic resistance, together with the forces driving pathogen spread. Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of hospital-acquired infections. We have investigated an MRSA strain (ST225) that is highly prevalent in hospitals in Central Europe. By using mutation discovery at 269 genetic loci (118,804 basepairs) within an international isolate collection, we ascertained extremely low diversity among European ST225 isolates, indicating that a recent population bottleneck had preceded the expansion of this clone. In contrast, US isolates were more divergent, suggesting they represent the ancestral population. While diversity was low, however, our results demonstrate that the short-term evolutionary rate in this natural population of MRSA resulted in the accumulation of measurable DNA sequence variation within two decades, which we could exploit to reconstruct its recent demographic history and the spatiotemporal dynamics of spread. By applying Bayesian coalescent methods on DNA sequences serially sampled through time, we estimated that ST225 had diverged since approximately 1990 (1987 to 1994), and that expansion of the European clade began in 1995 (1991 to 1999), several years before the new clone was recognized. Demographic analysis based on DNA sequence variation indicated a sharp increase of bacterial population size from 2001 to 2004, which is concordant with the reported prevalence of this strain in several European countries. A detailed ancestry-based reconstruction of the spatiotemporal dispersal dynamics suggested a pattern of frequent transmission of the ST225 clone among hospitals within Central Europe. In addition, comparative genomics indicated complex bacteriophage dynamics.     

MreC and MreD Proteins Are Not Required for Growth of Staphylococcus aureus

The transmembrane proteins MreC and MreD are present in a wide variety of bacteria and are thought to be involved in cell shape determination. Together with the actin homologue MreB and other morphological elements, they play an essential role in the synthesis of the lateral cell wall in rod-shaped bacteria. In ovococcus, which lack MreB homologues, mreCD are also essential and have been implicated in peripheral cell wall synthesis. In this work we addressed the possible roles of MreC and MreD in the spherical pathogen Staphylococcus aureus. We show that MreC and MreD are not essential for cell viability and do not seem to affect cell morphology, cell volume or cell cycle control. MreC and MreD localize preferentially to the division septa, but do not appear to influence peptidoglycan composition, nor the susceptibility to different antibiotics and to oxidative and osmotic stress agents. Our results suggest that the function of MreCD in S. aureus is not critical for cell division and cell shape determination.     

Structural Characterization of a Gcn5-Related N-Acetyltransferase from Staphylococcus aureus

The Gcn5-related N-acetyltransferases (GNATs) are ubiquitously expressed in nature and perform a diverse range of cellular functions through the acetylation of small molecules and protein substrates. Using activated acetyl coenzyme A as a common acetyl donor, GNATs catalyse the transfer of an acetyl group to acceptor molecules including aminoglycoside antibiotics, glucosamine-6-phosphate, histones, serotonin and spermidine. There is often only very limited sequence conservation between members of the GNAT superfamily, in part, reflecting their capacity to bind a diverse array of substrates. In contrast, the secondary and tertiary structures are highly conserved, but then at the quaternary level there is further diversity, with GNATs shown to exist in monomeric, dimeric, or tetrameric states. Here we describe the X-ray crystallographic structure of a GNAT enzyme from Staphyloccocus aureus with only low sequence identity to previously solved GNAT proteins. It contains many of the classical GNAT motifs, but lacks other hallmarks of the GNAT fold including the classic β-bulge splayed at the β-sheet interface. The protein is likely to be a dimer in solution based on analysis of the asymmetric unit within the crystal structure, homology with related GNAT family members, and size exclusion chromatography. The study provides the first high resolution structure of this enzyme, providing a strong platform for substrate and cofactor modelling, and structural/functional comparisons within this diverse enzyme superfamily.     

Mutation in the C-Di-AMP Cyclase dacA Affects Fitness and Resistance of Methicillin Resistant Staphylococcus aureus

Faster growing and more virulent strains of methicillin resistant Staphylococcus aureus (MRSA) are increasingly displacing highly resistant MRSA. Elevated fitness in these MRSA is often accompanied by decreased and heterogeneous levels of methicillin resistance; however, the mechanisms for this phenomenon are not yet fully understood. Whole genome sequencing was used to investigate the genetic basis of this apparent correlation, in an isogenic MRSA strain pair that differed in methicillin resistance levels and fitness, with respect to growth rate. Sequencing revealed only one single nucleotide polymorphism (SNP) in the diadenylate cyclase gene dacA in the faster growing but less resistant strain. Diadenylate cyclases were recently discovered to synthesize the new second messenger cyclic diadenosine monophosphate (c-di-AMP). Introduction of this mutation into the highly resistant but slower growing strain reduced resistance and increased its growth rate, suggesting a direct connection between the dacA mutation and the phenotypic differences of these strains. Quantification of cellular c-di-AMP revealed that the dacA mutation decreased c-di-AMP levels resulting in reduced autolysis, increased salt tolerance and a reduction in the basal expression of the cell wall stress stimulon. These results indicate that c-di-AMP affects cell envelope-related signalling in S. aureus. The influence of c-di-AMP on growth rate and methicillin resistance in MRSA indicate that altering c-di-AMP levels could be a mechanism by which MRSA strains can increase their fitness levels by reducing their methicillin resistance levels.     

Synergistic Action of Gentamicin and Bacteriophage in a Continuous Culture Population of Staphylococcus aureus

With the increasing frequency of antibiotic resistance and the decreasing frequency of new antibiotics entering the market, interest has returned to developing bacteriophage as a therapeutic agent. Acceptance of phage therapy, however, is limited by the unknown pharmacodynamics of a replicating agent, as well as the potential for the evolution of resistant bacteria. One way to overcome some of these limitations is to incorporate phage and antibiotics into a dual therapy regimen; however, this increases the complexity of the pharmacodynamics. The aim of this study is to develop an experimental system to evaluate the pharmacodynamics of dual phage-drug therapy. A continuous culture system for Staphylococcus aureus is used to simulate the pharmacokinetics of periodic antibiotic dosing alone and in combination with lytic phage. A computer model representation of the system allows further evaluation of the conditions governing the observed pharmacodynamics. The results of this experimental/modeling approach suggest that dual therapy can be more efficacious than single therapies, particularly if there is an overlap in the physiological pathways targeted by the individual agents. In this case, treatment with gentamicin induces a population of cells with a strong aggregation phenotype. These aggregators also have an increased ability to form biofilm, which is a well-known, non-genetic mechanism of drug resistance. However, the aggregators are also more susceptible than the parental strain to the action of the phage. Thus, dual treatment with gentamicin and phage resulted in lower final cell densities than either treatment alone. Unlike in the phage-only treatment, phage-resistant isolates were not detected in the dual treatment.