Identification of Genes Involved in Polysaccharide-Independent Staphylococcus aureus Biofilm Formation

Staphylococcus aureus is a potent biofilm former on host tissue and medical implants, and biofilm growth is a critical virulence determinant for chronic infections. Recent studies suggest that many clinical isolates form polysaccharide-independent biofilms. However, a systematic screen for defective mutants has not been performed to identify factors important for biofilm formation in these strains. We created a library of 14,880 mariner transposon mutants in a S. aureus strain that generates a proteinaceous and extracellular DNA based biofilm matrix. The library was screened for biofilm defects and 31 transposon mutants conferred a reproducible phenotype. In the pool, 16 mutants overproduced extracellular proteases and the protease inhibitor α₂-macroglobulin restored biofilm capacity to 13 of these mutants. The other 15 mutants in the pool displayed normal protease levels and had defects in genes involved in autolysis, osmoregulation, or uncharacterized membrane proteins. Two transposon mutants of interest in the GraRS two-component system and a putative inositol monophosphatase were confirmed in a flow cell biofilm model, genetically complemented, and further verified in a community-associated methicillin-resistant S. aureus (CA-MRSA) isolate. Collectively, our screen for biofilm defective mutants identified novel loci involved in S. aureus biofilm formation and underscored the importance of extracellular protease activity and autolysis in biofilm development.     

Prevalence of Clonal Complexes and Virulence Genes among Commensal and Invasive Staphylococcus aureus Isolates in Sweden

Staphylococcus aureus encodes a remarkable number of virulence factors which may contribute to its pathogenicity and ability to cause invasive disease. The main objective of this study was to evaluate the association between S. aureus invasiveness and bacterial genotype, in terms of the presence of virulence genes and affiliation to clonal complexes. Also, the significance of different virulence genes, mainly adhesins, for the development of infective endocarditis was investigated.DNA microarray technology was used to analyze 134 S. aureus isolates, all methicillin-susceptible, derived from three groups of clinically well-characterized patients: nasal carriers (n=46), bacteremia (n=55), and bacteremia with infective endocarditis (n=33).Invasive isolates were dominant in four of the major clonal complexes: 5, 8, 15, and 25. Of the 170 virulence genes examined, those encoding accessory gene regulator group II (agr II), capsule polysaccharide serotype 5 (cap5), and adhesins such as S. aureus surface protein G (sasG) and fibronectin-binding protein B (fnbB) were found to be associated with invasive disease. The same was shown for the leukocidin genes lukD/lukE, as well as the genes encoding serine protease A and B (splA/splB), staphylococcal complement inhibitor (scn) and the staphylococcal exotoxin-like protein (setC or selX). In addition, there was a trend of higher prevalence of certain genes or gene clusters (sasG, agr II, cap5) among isolates causing infective endocarditis compared to other invasive isolates. In most cases, the presence of virulence genes was linked to clonal complex affiliation.In conclusion, certain S. aureus clonal lineages harboring specific sets of virulence genes seem to be more successful in causing invasive disease.     

The lysine-peptoid hybrid LP5 maintain activity under physiological conditions and affects virulence gene expression in Staphylococcus aureus

The antimicrobial peptide, LP5, is a lysine-peptoid hybrid, with antimicrobial activity against clinically relevant bacteria. Here, we investigated how various environmental conditions affect the antimicrobial activity of LP5 against Staphylococcus aureus (S. aureus). We found that LP5 maintained activity under host physiological conditions of NaCl, MgCl₂ and pH. However, when exposed to serum, LP5 lost activity. Furthermore, when increasing NaCl concentration and lowering pH, the peptide showed reduces activity. When investigating the tolerance mechanisms of S. aureus toward antimicrobial peptides, we found that LP5 was protease resistant. However, the dltA and vraF genes, involved in reducing the net anionic charge of the bacterial cell envelope and sensing of antimicrobial peptides, respectively, played a role in the tolerance of S. aureus against LP5. In addition, the exposure of S. aureus to sub-inhibitory concentrations of LP5 affected the expression of the major virulence factors of S. aureus, revealing a potential as anti-virulence compound. Thus, these results show how environmental factors affect the peptide efficiency and further add to the knowledge on how the peptide affects S. aureus, which is crucial information for designing new peptides for optimizing antimicrobial therapy.     

A small regulatory RNA alters Staphylococcus aureus virulence by titrating RNAIII activity

Staphylococcus aureus is an opportunistic human and animal pathogen with an arsenal of virulence factors that are tightly regulated during bacterial infection. The latter is achieved through a sophisticated network of regulatory proteins and regulatory RNAs. Here, we describe the involvement of a novel prophage-carried small regulatory S. aureus RNA, SprY, in the control of virulence genes. An MS2-affinity purification assay reveals that SprY forms a complex in vivo with RNAIII, a major regulator of S. aureus virulence genes. SprY binds to the 13th stem-loop of RNAIII, a key functional region involved in the repression of multiple mRNA targets. mRNAs encoding the repressor of toxins Rot and the extracellular complement binding protein Ecb are among the targets whose expression is increased by SprY binding to RNAIII. Moreover, SprY decreases S. aureus hemolytic activity and virulence. Our results indicate that SprY titrates RNAIII activity by targeting a specific stem loop. Thus, we demonstrate that a prophage-encoded sRNA reduces the pathogenicity of S. aureus through RNA sponge activity.     

A bacterial regulatory RNA attenuates virulence,spread and human host cell phagocytosis

Staphylococcus aureus pathogenesis is directed by regulatory proteins and RNAs. We report the case of an RNA attenuating virulence and host uptake, possibly to sustain commensalism. A S. aureus sRNA, SprC (srn_3610), reduced virulence and bacterial loads in a mouse infection model. S. aureus deleted for sprC became more virulent and increased bacterial dissemination in colonized animals. Conversely, inducing SprC expression lowered virulence and the bacterial load. Without sprC, S. aureus phagocytosis by monocytes and macrophages was higher, whereas bacteria were internalized at lower yields when SprC expression was stimulated. Without sprC, higher internalization led to a greater number of extracellular bacteria, facilitating colonization. SprC expression decreased after phagocytosis, concurring with the facilitated growth of bacteria lacking the sRNA in the presence of an oxidant. The major staphylococcal autolysin facilitates S. aureus uptake by human phagocytes. ATL proved to be negatively regulated by SprC. The SprC domains involved in pairing with atl mRNA were analyzed. The addition of ATL reduced phagocytosis of bacteria lacking sprC with no effects on wild-type bacterial uptake, implying that SprC influences phagocytosis, at least in part, by controlling ATL. Since the control of SprC on ATL was modest, other factors must contribute to atl regulation.     

Exploring extra-cellular proteins in methicillin susceptible and methicillin resistant Staphylococcus aureus by liquid chromatography–tandem mass spectrometry

Staphylococcus aureus (S. aureus) strains cause several diseases in humans from minor skin infections to severe lethal infections. To explore the virulence determinants of this important microorganism, two clinical isolates of methicillin susceptible S. aureus (MSSA) and methicillin resistant S. aureus (MRSA) were subjected to proteomic analysis of their extracellular products using liquid chromatography–tandem mass spectrometry. The numbers of proteins identified in MSSA and MRSA extracellular products were 168 and 261; respectively, from them 117 were shared, while 144 proteins were unique to MRSA. The shared proteins, having a higher protein score with increased number of peptide matches in MRSA over MSSA, reflect the relatively active secretory state of MRSA rather than biased analytical variances. Characteristic determinants for MRSA were identified; mostly found to play a role in the virulence. We conclude that MRSA produces distinct proteins considered as its virulence determinants and we found that the shared extracellular products are more abundant in MRSA than MSSA that supporting the high invasiveness of MRSA over MSSA in pathogenesis.     

Expression of virulence factors by Staphylococcus aureus grown in serum

Staphylococcus aureus produces many virulence factors, including toxins, immune-modulatory factors, and exoenzymes. Previous studies involving the analysis of virulence expression were mainly performed by in vitro experiments using bacterial medium. However, when S. aureus infects a host, the bacterial growth conditions are quite different from those in a medium, which may be related to the different expression of virulence factors in the host. In this study, we investigated the expression of virulence factors in S. aureus grown in calf serum. The expression of many virulence factors, including hemolysins, enterotoxins, proteases, and iron acquisition factors, was significantly increased compared with that in bacterial medium. In addition, the expression of RNA III, a global regulon for virulence expression, was significantly increased. This effect was partially restored by the addition of 300 μM FeCl₃ into serum, suggesting that iron depletion is associated with the increased expression of virulence factors in serum. In chemically defined medium without iron, a similar effect was observed. In a mutant with agr inactivated grown in serum, the expression of RNA III, psm, and sec4 was not increased, while other factors were still induced in the mutant, suggesting that another regulatory factor(s) is involved. In addition, we found that serum albumin is a major factor for the capture of free iron to prevent the supply of iron to bacteria grown in serum. These results indicate that S. aureus expresses virulence factors in adaptation to the host environment.     

sarA‐mediated repression of protease production plays a key role in the pathogenesis of Staphylococcus aureus USA300 isolates

Mutation of staphylococcal accessory regulator (sarA) results in increased production of extracellular proteases in Staphylococcus aureus, which has been correlated with decreased biofilm formation and decreased accumulation of extracellular toxins. We used murine models of implant‐associated biofilm infection and S. aureus bacteraemia (SAB) to compare virulence of USA300 strain LAC, its isogenic sarA mutant, and derivatives of each of these strains with mutations in all 10 of the genes encoding recognized extracellular proteases. The sarA mutant was attenuated in both models, and this was reversed by eliminating production of extracellular proteases. To examine the mechanistic basis, we identified proteins impacted by sarA in a protease‐dependent manner. We identified 253 proteins where accumulation was reduced in the sarA mutant compared with the parent strain, and was restored in the sarA/protease mutant. Additionally, in SAB, the LAC protease mutant exhibited a hypervirulent phenotype by comparison with the isogenic parent strain, demonstrating that sarA also positively regulates production of virulence factors, some of which are subject to protease‐mediated degradation. We propose a model in which attenuation of sarA mutants is defined by their inability to produce critical factors and simultaneously repress production of extracellular proteases that would otherwise limit accumulation of virulence factors.     

Characterization of Insertion Sequence ISSau2 in the Human and Livestock-Associated Staphylococcus aureus

Mobile genetic elements play important roles in evolution and diversification of bacterial genomes. ISSau2 is 1660bp in length with terminal 5’-TG and CA-3’ dinucleotides and has two overlapping reading frames orfA and orfB. It has been found in a wide range of S. aureus, such as HA-MRSA252, LGA251, MRSA S0385 and ED133. To determine distribution of ISSau2, 164 S. aureus isolates from milk samples of mastitic cows from our laboratory and all the S. aureus strains from the National Center for Biotechnology Information (NCBI) database were screened for the presence of ISSau2. Next, in order to explore a potential relationship among S. aureus ISSau2-containing strains and isolates, a relationship among 10 ISSau2-positive S. aureus isolates and 27 ISSau2-positive S. aureus strains was investigated by a phylogenetic analysis. These ISSau2 isolates and strains could be classified into four groups (A, B, C and D). The strains or isolates in Group D were all isolated from mammary glands, suggesting tissue specificity. All strains in Group B had an identical ISSau2 derivative, termed ISSau2₁₆₂₈, with 32bp deletion at the 3’ terminus. ISSau2₁₆₂₈ in strain ST398 from Group B was closely related to ISSau2 in strain LGA251 from Group D.     

Golden Pigment Production and Virulence Gene Expression Are Affected by Metabolisms in Staphylococcus aureus

The pathogenesis of staphylococcal infections is multifactorial. Golden pigment is an eponymous feature of the human pathogen Staphylococcus aureus that shields the microbe from oxidation-based clearance, an innate host immune response to infection. Here, we screened a collection of S. aureus transposon mutants for pigment production variants. A total of 15 previously unidentified genes were discovered. Notably, disrupting metabolic pathways such as the tricarboxylic acid cycle, purine biosynthesis, and oxidative phosphorylation yields mutants with enhanced pigmentation. The dramatic effect on pigment production seems to correlate with altered expression of virulence determinants. Microarray analysis further indicates that purine biosynthesis impacts the expression of ∼400 genes involved in a broad spectrum of functions including virulence. The purine biosynthesis mutant and oxidative phosphorylation mutant strains exhibit significantly attenuated virulence in a murine abscess model of infection. Inhibition of purine biosynthesis with a known small-molecule inhibitor results in altered virulence gene expression and virulence attenuation during infection. Taken together, these results suggest an intimate link between metabolic processes and virulence gene expression in S. aureus. This study also establishes the importance of purine biosynthesis and oxidative phosphorylation for in vivo survival.Staphylococcus aureus causes a variety of infections in humans, ranging from minor skin and wound infections to life-threatening diseases (31). The pathogenesis of staphylococcal infections is a multifactorial process that depends on the expression of different virulence factors controlled by multiple regulatory systems in conjunction with environmental and nutritional signals (46). The high degree of variability in the expression of virulence genes is modulated by a complex network regulated by factors such as the agr locus (RNAIII), SarA, and SigB (5, 9), which allows the bacterium to adapt to changing environmental conditions for survival and developing infection.The species epithet of S. aureus reflects its characteristic surface pigmentation (aureus, meaning “golden” in Latin) (43). The yellowish-orange (golden) pigment produced by S. aureus has been linked to virulence, owing to its antioxidant property (29, 30). The golden pigmentation of S. aureus is the product of a C₃₀ triterpenoid carotenoid biosynthesis pathway, and the carotenoid pigment biosynthesis genes are organized in an operon crtOPQMN controlled by the alternative sigma factor SigB (3, 39). Since many virulence genes are coordinately regulated in S. aureus (5, 9, 31), we hypothesized that genes affecting pigmentation may also influence the production of virulence determinants and have an impact on the pathogenesis of S. aureus.Herein, we present an analysis of S. aureus golden pigment biosynthesis and regulatory pathways at the genomic level by screening the Phoenix (ΦNΞ) library, a collection of defined transposon insertions into 1,812 open reading frames of S. aureus strain Newman (1). This study indicates an intimate link between metabolic processes and virulence gene expression. It demonstrates the importance of purine biosynthesis and oxidative phosphorylation for in vivo survival and pathogenesis of S. aureus. Our results show that targeting purine biosynthesis is a promising strategy to develop anti-S. aureus therapies.     

A Proteomic View of an Important Human Pathogen – Towards the Quantification of the Entire Staphylococcus aureus Proteome

The genome sequence is the “blue-print of life,” but proteomics provides the link to the actual physiology of living cells. Because of their low complexity bacteria are excellent model systems to identify the entire protein assembly of a living organism. Here we show that the majority of proteins expressed in growing and non-growing cells of the human pathogen Staphylococcus aureus can be identified and even quantified by a metabolic labeling proteomic approach. S. aureus has been selected as model for this proteomic study, because it poses a major risk to our health care system by combining high pathogenicity with an increasing frequency of multiple antibiotic resistance, thus requiring the development of new anti-staphylococcal therapy strategies. Since such strategies will likely have to target extracellular and surface-exposed virulence factors as well as staphylococcal survival and adaptation capabilities, we decided to combine four subproteomic fractions: cytosolic proteins, membrane-bound proteins, cell surface-associated and extracellular proteins, to comprehensively cover the entire proteome of S. aureus. This quantitative proteomics approach integrating data ranging from gene expression to subcellular localization in growing and non-growing cells is a proof of principle for whole-cell physiological proteomics that can now be extended to address physiological questions in infection-relevant settings. Importantly, with more than 1700 identified proteins (and 1450 quantified proteins) corresponding to a coverage of about three-quarters of the expressed proteins, our model study represents the most comprehensive quantification of a bacterial proteome reported to date. It thus paves the way towards a new level in understanding of cell physiology and pathophysiology of S. aureus and related pathogenic bacteria, opening new avenues for infection-related research on this crucial pathogen.     

Clindamycin suppresses virulence expression in inducible clindamycin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> strains

Clindamycin is a protein synthesis inhibitory agent that has the ability to suppress the expression of virulence factors in Staphylococcus aureus. Recent guidelines recommend the use of clindamycin for the treatment of toxin-mediated infections. Clindamycin modulates virulence expression at sub-inhibitory concentrations (sub-MICs) in clindamycin-susceptible S. aureus strains but previous report shown that this effect was supressed for constitutive clindamycin resistant strains. However, no data are currently available on the impact of clindamycin at sub-MICs on the virulence of inducible clindamycin-resistant S. aureus strains. Here, we show that sub-MICs of clindamycin decrease Panton–Valentine leucocidin, toxic-shock-staphylococcal toxin (TSST-1) and alpha-haemolysin (Hla) expression in six inducible clindamycin-resistant isolates cultivated in vitro in CCY medium. These results suggest that the clindamycin anti-toxin effect is retained for inducible clindamycin-resistant S. aureus isolates; therefore, its usage should be considered within the treatment regimen of toxin related infections for inducible clindamycin-resistant S. aureus.     

Expression of Panton-Valentine Leukocidin mRNA among Staphylococcus aureus Isolates Associates with Specific Clinical Presentations

Panton-Valentine leukocidin (PVL; gene designation lukF/S-PV) is likely an important virulence factor for Staphylococcus aureus (S. aureus), as qualitative expression of the protein correlates with severity for specific clinical presentations, including skin and soft tissue infections (SSTIs). Development of genetic approaches for risk-assessment of patients with S. aureus infections may prove clinically useful, and whether lukF/S-PV gene expression correlates with specific clinical presentations for S. aureus has been largely unexplored. In the present study, we quantified lukS-PV mRNA among 96 S. aureus isolates to determine whether expression levels correlated with specific clinical presentations in adults and children. Expression level of lukS-PV mRNA among isolates from skin and soft tissue infections (SSTIs) was significantly greater than among isolates from blood stream infection (BSIs), and expression level of lukS-PV mRNA among BSI isolates from children was significantly greater than for BSI isolates among adults. Moreover, expression level of lukS-PV mRNA among community-acquired (CA) isolates was significantly greater than for hospital-acquired (HA) isolates. These data justify additional studies to determine the potential clinical utility for lukS-PV mRNA quantification as a predictive tool for severity of S. aureus infection.     

Mapping the Pathways to Staphylococcal Pathogenesis by Comparative Secretomics

The gram-positive bacterium Staphylococcus aureus is a frequent component of the human microbial flora that can turn into a dangerous pathogen. As such, this organism is capable of infecting almost every tissue and organ system in the human body. It does so by actively exporting a variety of virulence factors to the cell surface and extracellular milieu. Upon reaching their respective destinations, these virulence factors have pivotal roles in the colonization and subversion of the human host. It is therefore of major importance to obtain a clear understanding of the protein transport pathways that are active in S. aureus. The present review aims to provide a state-of-the-art roadmap of staphylococcal secretomes, which include both protein transport pathways and the extracytoplasmic proteins of these organisms. Specifically, an overview is presented of the exported virulence factors, pathways for protein transport, signals for cellular protein retention or secretion, and the exoproteomes of different S. aureus isolates. The focus is on S. aureus, but comparisons with Staphylococcus epidermidis and other gram-positive bacteria, such as Bacillus subtilis, are included where appropriate. Importantly, the results of genomic and proteomic studies on S. aureus secretomes are integrated through a comparative “secretomics” approach, resulting in the first definition of the core and variant secretomes of this bacterium. While the core secretome seems to be largely employed for general housekeeping functions which are necessary to thrive in particular niches provided by the human host, the variant secretome seems to contain the “gadgets” that S. aureus needs to conquer these well-protected niches.     

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.     

Genome sequence of Staphylococcus aureus strain Newman and comparative analysis of staphylococcal genomes: polymorphism and evolution of two major pathogenicity islands

Strains of Staphylococcus aureus, an important human pathogen, display up to 20% variability in their genome sequence, and most sequence information is available for human clinical isolates that have not been subjected to genetic analysis of virulence attributes. S. aureus strain Newman, which was also isolated from a human infection, displays robust virulence properties in animal models of disease and has already been extensively analyzed for its molecular traits of staphylococcal pathogenesis. We report here the complete genome sequence of S. aureus Newman, which carries four integrated prophages, as well as two large pathogenicity islands. In agreement with the view that S. aureus Newman prophages contribute important properties to pathogenesis, fewer virulence factors are found outside of the prophages than for the highly virulent strain MW2. The absence of drug resistance genes reflects the general antibiotic-susceptible phenotype of S. aureus Newman. Phylogenetic analyses reveal clonal relationships between the staphylococcal strains Newman, COL, NCTC8325, and USA300 and a greater evolutionary distance to strains MRSA252, MW2, MSSA476, N315, Mu50, JH1, JH9, and RF122. However, polymorphism analysis of two large pathogenicity islands distributed among these strains shows that the two islands were acquired independently from the evolutionary pathway of the chromosomal backbones of staphylococcal genomes. Prophages and pathogenicity islands play central roles in S. aureus virulence and evolution.