Staphylococcal Factors Research Articles

Staphylococcus aureus delta toxin modulates both extracellular membrane vesicle biogenesis and amyloid formation.

Extracellular membrane vesicles (MVs) produced by Staphylococcus aureus in planktonic cultures encapsulate a diverse cargo of bacterial proteins, nucleic acids, and glycopolymers that are protected from destruction by external factors. δ-toxin, a member of the phenol soluble modulin family, was shown to be critical for MV biogenesis. Amyloid fibrils co-purified with MVs generated by virulent, community-acquired S. aureus strains, and fibril formation was dependent on expression of the S. aureus δ-toxin gene (hld). Mass spectrometry data confirmed that the amyloid fibrils were comprised of δ-toxin. Although S. aureus MVs were produced in vivo in a localized murine infection model, amyloid fibrils were not observed in the in vivo setting. Our findings provide critical insights into staphylococcal factors involved in MV biogenesis and amyloid formation.

A conserved arginine residue in a staphylococcal anti-sigma factor is required to preserve its kinase activity, structure, and stability

RsbW, σB, and RsbV, encoded by Staphylococcus aureus and related bacteria, act as an anti-sigma factor, an sigma factor, and an anti-anti-sigma factor, respectively. The interaction between RsbW and σB blocks the transcription initiation activity of the latter protein. RsbW also functions as a serine kinase and phosphorylates RsbV in the presence of ATP. Our modeling study indicates that the RsbW-RsbV complex is stabilized by twenty-four intermolecular non-covalent bonds. Of the bond-forming RsbW residues, Arg 23, and Glu 49 are conserved residues. To understand the roles of Arg 23 in RsbW, rRsbW[R23A], a recombinant S. aureus RsbW (rRsbW) harboring Arg to Ala change at position 23, was investigated using various probes. The results reveal that rRsbW[R23A], like rRsbW, exists as the dimers in the aqueous solution. However, rRsbW[R23A], unlike rRsbW, neither interacted with a chimeric RsbV (rRsbV) nor formed the phosphorylated rRsbV in the presence of ATP. Furthermore, the tertiary structure and hydrophobic surface area of rRsbW[R23A] matched little with those of rRsbW. Conversely, both rRsbW[R23A] and rRsbW showed interaction with a recombinant σB (rσB). rRsbW and rRsbW[R23A] were also unfolded via the formation of at least one intermediate in the presence of urea. However, the thermodynamic stability of rRsbW significantly differed from that of rRsbW[R23A]. Our molecular dynamics (MD) simulation study also reveals the substantial change of structure, dimension, and stability of RsbW due to the above mutation. The ways side chain of critical Arg 23 contributes to maintaining the tertiary structure, and stability of RsbW was elaborately discussed. Communicated by Ramaswamy H. Sarma

Mechanisms of Pyocyanin Toxicity and Genetic Determinants of Resistance in Staphylococcus aureus.

Pseudomonas aeruginosa and Staphylococcus aureus are commonly isolated from polymicrobial infections, such as wound infections and chronic respiratory infections of persons with cystic fibrosis. Despite their coisolation, P. aeruginosa produces substances toxic to S. aureus, including pyocyanin, a blue-pigmented molecule that functions in P. aeruginosa virulence. Pyocyanin inhibits S. aureus respiration, forcing it to derive energy from fermentation and adopt a small-colony variant (SCV) phenotype. The mechanisms by which S. aureus sustains infection in the presence of pyocyanin are not clear. We sought to clarify the mechanisms of pyocyanin toxicity in S. aureus as well as identify the staphylococcal factors involved in its resistance to pyocyanin toxicity. Nonrespiring S. aureus SCVs are inhibited by pyocyanin through pyocyanin-dependent reactive oxygen species (ROS) production, indicating that pyocyanin toxicity is mediated through respiratory inhibition and ROS generation. Selection on pyocyanin yielded a menadione auxotrophic SCV capable of growth on high concentrations of pyocyanin. Genome sequencing of this isolate identified mutations in four genes, including saeS, menD, NWMN_0006, and qsrR QsrR is a quinone-sensing repressor of quinone detoxification genes. Inactivation of qsrR resulted in significant pyocyanin resistance, and additional pyocyanin resistance was achieved through combined inactivation of qsrR and menadione biosynthesis. Pyocyanin-resistant S. aureus has an enhanced capability to inactivate pyocyanin, suggesting QsrR-regulated gene products may degrade pyocyanin to alleviate toxicity. These findings demonstrate pyocyanin-mediated ROS generation as an additional mechanism of pyocyanin toxicity and define QsrR as a key mediator of pyocyanin resistance in S. aureus IMPORTANCE Many bacterial infections occur in the presence of other microbes, where interactions between different microbes and the host impact disease. In patients with cystic fibrosis, chronic lung infection with multiple microbes results in the most severe disease manifestations. Staphylococcus aureus and Pseudomonas aeruginosa are prevalent cystic fibrosis pathogens, and infection with both is associated with worse outcomes. These organisms have evolved mechanisms of competing with one another. For example, P. aeruginosa produces pyocyanin, which inhibits S. aureus growth. Our research has identified how pyocyanin inhibits S. aureus growth and how S. aureus can adapt to survive in the presence of pyocyanin. Understanding how S. aureus sustains infection in the presence of P. aeruginosa may identify means of disrupting these microbial communities.

Increased Susceptibility of Humanized NSG Mice to Panton-Valentine Leukocidin and Staphylococcus aureus Skin Infection.

Staphylococcus aureus is a leading cause of skin and soft-tissue infections worldwide. Mice are the most commonly used animals for modeling human staphylococcal infections. However a supra-physiologic S. aureus inoculum is required to establish gross murine skin pathology. Moreover, many staphylococcal factors, including Panton-Valentine leukocidin (PVL) elaborated by community-associated methicillin-resistant S. aureus (CA-MRSA), exhibit selective human tropism and cannot be adequately studied in mice. To overcome these deficiencies, we investigated S. aureus infection in non-obese diabetic (NOD)/severe combined immune deficiency (SCID)/IL2rγnull (NSG) mice engrafted with human CD34+ umbilical cord blood cells. These “humanized” NSG mice require one to two log lower inoculum to induce consistent skin lesions compared with control mice, and exhibit larger cutaneous lesions upon infection with PVL+ versus isogenic PVL- S. aureus. Neutrophils appear important for PVL pathology as adoptive transfer of human neutrophils alone to NSG mice was sufficient to induce dermonecrosis following challenge with PVL+ S. aureus but not PVL- S. aureus. PMX53, a human C5aR inhibitor, blocked PVL-induced cellular cytotoxicity in vitro and reduced the size difference of lesions induced by the PVL+ and PVL- S. aureus, but PMX53 also reduced recruitment of neutrophils and exacerbated the infection. Overall, our findings establish humanized mice as an important translational tool for the study of S. aureus infection and provide strong evidence that PVL is a human virulence factor.

Molecular basis of virulence in Staphylococcus aureus mastitis.

Background S. aureus is one of the main pathogens involved in ruminant mastitis worldwide. The severity of staphylococcal infection is highly variable, ranging from subclinical to gangrenous mastitis. This work represents an in-depth characterization of S. aureus mastitis isolates to identify bacterial factors involved in severity of mastitis infection.Methodology/Principal FindingsWe employed genomic, transcriptomic and proteomic approaches to comprehensively compare two clonally related S. aureus strains that reproducibly induce severe (strain O11) and milder (strain O46) mastitis in ewes. Variation in the content of mobile genetic elements, iron acquisition and metabolism, transcriptional regulation and exoprotein production was observed. In particular, O11 produced relatively high levels of exoproteins, including toxins and proteases known to be important in virulence. A characteristic we observed in other S. aureus strains isolated from clinical mastitis cases.Conclusions/SignificanceOur data are consistent with a dose-dependant role of some staphylococcal factors in the hypervirulence of strains isolated from severe mastitis. Mobile genetic elements, transcriptional regulators, exoproteins and iron acquisition pathways constitute good targets for further research to define the underlying mechanisms of mastitis severity.

Advances in in Vitro and in Vivo Models for Studying the Staphylococcal Factors Involved in Implant Infections

Implant infections due to staphylococci are one of the greatest threats facing patients receiving implant devices. For many years researchers have sought to understand the mechanisms involved in the adherence of the bacterium to the implanted device and the formation of the unique structure, the biofilm, which protects the indwelling bacteria from the host defence and renders them resistant to antibiotic treatment. A major goal has been to develop in vitro and in vivo models that adequately reflect the real-life situation. From the simple microtiter plate assay and scanning electron microscopy, tools for studying adherence and biofilm formation have since evolved to include specialised equipment for studying adherence, flow cell systems, real-time analysis of biofilm formation using reporter gene assays both in vitro and in vivo, and a wide variety of animal models. In this article, we discuss advances in the last few years in selected in vitro and in vivo models as well as future developments in the study of adherence and biofilm formation by the staphylococci.

Staphylococcus aureus Infection of Epidermal Keratinocytes Promotes Expression of Innate Antimicrobial Peptides

Keratinocytes upregulate expression of endogenous antimicrobial peptides in response to inflammatory stimuli. We show that both viable and heat-inactivated Staphylococcus aureus and lipoteichoic acid differentially alter expression of these peptides upon contact with human keratinocytes. The findings indicate a diversity of staphylococcal factors involved in upregulation of antimicrobial peptide expression in cutaneous epithelia.

Staphylococcus aureus interactions with the endothelium

The intravascular manifestation of Staphylococcus aureus infection is often associated with a severe, and sometimes catastrophic disease. Many host factors contribute to endothelial tropism of S.aureus including subendothelial matrix proteins, endothelial cell receptors, and platelets that are engaged together with S. aureus cell wall adhesins such as the fibronectin binding proteins. Recently, the role of secreted staphylococcal factors that were initially identified by virtue of their binding function with host proteins and ligands, has been reappraised in this regard. Among these, bacterial proteins without significant homology among each other, coagulase (Coa), the extracellular fibrinogen binding protein (Efb), the extracellular matrix binding protein (Emp), or the extracellular adhesive protein (Eap), are the most prominent ones to be associated with endovascular disease. Newly discovered interactions with host components may account for profound effects on immunmodulation and wound healing which are summarized in this short review and which ascribe an important role of these molecules in acute and chronic endo- and extravascular staphylococcal disease. Further research in the complex functional role of these "secretable expanded repertoire adhesive molecules" (SERAM) may not only help to increase our understanding in the pathogenesis of S. aureus infection but can specify novel targets for preventive or therapeutic strategies.

Development and characterization of a Staphylococcus aureus nasal colonization model in mice.

Staphylococcus aureus nasal carriage is a risk factor for infection in humans, particularly in the hospital environment. Attenuation of carriage has proven effective in reducing the prevalence of infection in some high-risk groups. To study staphylococcal factors that influence nasal colonization, a mouse model of S. aureus nasal colonization was developed. Mice were inoculated intranasally with S. aureus Reynolds, and nasal carriage was evaluated by quantitating cultures of the nasal tissues from mice sacrificed at various time points after inoculation. The majority of mice inoculated with 10(8) CFU of S. aureus maintained nasal carriage for at least 20 days. Nasal colonization rates were similar for inbred (BALB/c and C57BL/6) and outbred (ICR) mice. Colonization was not affected by mouse passage of strain Reynolds. Lower inoculum doses (<10(7) CFU) resulted in reduced colonization after 7 days. However, mice given streptomycin in their drinking water developed long-term carriage of S. aureus, and they were colonized with inocula as low as 10(5) CFU. Nasal colonization was also established with two other S. aureus strains (one strain each of human and murine origins). S. aureus recovered from the nares of experimentally colonized mice expressed high levels of capsule, and the ability of a capsule-defective mutant to persist in the nares was reduced in comparison to that of the parent strain. This nasal colonization model should prove useful for studies of factors that mediate S. aureus colonization and for assessment of targets for antimicrobial intervention or vaccine development.

Immunosuppressor control as a modality of cancer treatment: effect of plasma adsorption with Staphylococcus aureus protein A.

In tumor-bearing hosts both cellular and humoral tumor-growth-enhancing factors are present. They cause immunosuppression and facilitate the growth of tumors. Very early during tumor growth these factors are either elicited by the tumor cells or induced by the host immunocytes. Among these immunosuppressive agents, circulating immune complexes appear to play a predominant role. They also activate suppressor cell activity. Plasma adsorption of CIC and IgG by protein A of Staphylococcus aureus has been reported to cause tumor regression. Plasma adsorption with protein A-collodion charcoal, protein A-silica, or protein A-Sepharose also induced tumorilytic reactions. Even direct infusion of protein A induced tumor regressions in rat mammary tumors. Recent studies showing tumor regressions following S. aureus Wood 46 plasma adsorption or infusion of normal plasma adsorbed over S. aureus indicate that specific blocking factor removal by plasma adsorption may not be the mechanism for causing tumor destruction. Results indicate that S. aureus plasma adsorption leaches a number of staphylococcal agents. Thus, it appears that staphylococcal agents, protein A, enterotoxin, and other factors are responsible for the induction of reactions leading to tumor destruction. A unified mechanism explaining the results obtained with plasma adsorption using protein A of S. aureus, or S. aureus Wood, or direct protein A infusion, was presented.

Mechanisms of resistance of staphylococci grown in plasma to polymorph bactericidins.

The mechanisms whereby staphylococcal strains grown in plasma assume increased resistance to polymorph bactericidins were investigated. Observations reported here showed that cultural conditions could determine the path of conversion to resistance. Staphylococcal strains and mutants lacking either free coagulase or clumping factor or both all showed enhanced resistance after 10 h incubation in plasma proteins, thus giving no clear indication that these factors were involved in the interactions. In fact, prolonged incubation in bovine serum albumin (22 h) and ordinary broth medium (24 h) also resulted in increased resistance. A distinction between staphylococcal factors interacting specifically with plasma proteins and such non-specific conversions was obtained in two different ways. Stripping of a hypothetical surface protein by treatment with trypsin or 2 M potassium bromide rendered plasma- but not 24 h-broth organisms susceptible, indicating protein coating of plasma-grown organisms. Also free coagulase-positive strains and mutants incubated in plasma for 30 min were converted while those lacking both or possessing clumping factor alone were not. It therefore appears that one of the mechanisms of acquiring resistance involves a rapid interaction between staphylococcal-free coagulase and fibrinogen, resulting in the deposition of fibrin or fibrin derivatives on the bacterial surface.

The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage.

Toxic shock syndrome (TSS) is a complex of generalized symptoms caused by a local staphylococcal infection, and a circulating toxin is thought to be involved. Indeed, nearly 100% of TSS isolates produce an exoprotein, TSSE, that is thought to have an aetiological role on the basis of positive animal tests (refs 1,2 and F. Quimby, personal communication) and human serological data. Although the precise role of TSSE in TSS remains unclear (E. Kass, personal communication), no other staphylococcal factor has been implicated. Our preliminary studies of the genetics of TSSE production failed to demonstrate plasmid or phage involvement or linkage with known chromosomal genes (ref. 4 and B.N.K. et al., unpublished data); however, Schutzer et al. have found that most TSS strains harbour prophages with common plating characteristics and suggest that the toxin(s) involved in TSS are transmitted by lysogenic conversion. We show here that TSSE is not demonstrably transferred by lysogeny; moreover, we have cloned the gene and found that the cloned product is serologically and biologically indistinguishable from the native protein, and that the TSSE determinant is associated with a larger DNA segment that is absent or rearranged in TSSE- strains.

Infections caused by staphylococci.

Staphylococci are not highly virulent bacteria. They require a predisposing factor in the host to initiate an infectious process. Primary infections are rare. Typical staphylococcal infections develop on the basis of a locus minoris resistentiae with the subsequent participation of multiple cellular and extracellular staphylococcal factors. A hypothetical view of the development of staphylococcal infection is briefly presented.

A simple method for the study in vivo of bacterial growth and accompanying host response

Summary Chambers were constructed from lengths of plastic syringe barrels, having both ends sealed with membrane filters of 0·22 μm pore diameter. When implanted intraperitoneally in mice, the chambers were used in the study of in vivo growth of staphylococci, and the host response. This system permits a dynamic interchange of diffusible host and staphylococcal factors, simulating, to a certain extent, the natural state of affairs during staphylococcal infection. The bacteria, although in close proximity to phagocytes, are protected from engulfment, and these in vivo -grown bacteria can be recovered for subsequent examination in vitro . The current study noted that growth of Staphylococcus aureus in chambers was slower in vivo than in vitro . In vivo the stationary phase was more prolonged and growth attained a lesser density than it did in vitro . An inflammatory response was observed in both control and test chamber implantations. Test chambers, however, became thickly encapsulated with granulation tissue and fibrin deposits, entrapping numerous leucocytes.

Inhibition of leukocyte migration by a staphylococcal factor.

Cell wall mucopeptide isolated from virulent strains of Staphylococcus aureus has previously been found to potentiate subcutaneous staphylococcal lesions in mice. This cell wall fraction was found to inhibit the migration of polymorphonuclear leukocytes toward a chemotactic stimulus, as tested by the micropore filter chamber technique. A close correlation was shown to exist between in vivo "mouse virulence" of staphylococcal strains and the in vitro inhibition of leukocyte migration by the cell wall factor.

Serological typing of staphylococci by means of fluorescent antibodies. I. Development of specific reagents for seven serological factors.

Cohen, Jay O. (Communicable Disease Center, Atlanta, Ga.) and Per Oeding. Serological typing of staphylococci by means of fluorescent antibodies. I. Development of specific reagents for seven serological factors. J. Bacteriol. 84:735-741. 1962-Fluorescent antibody reagents for identifying seven antigenic factors of staphylococci have been prepared. The fluorescent staining reactions of these reagents were compared to the agglutination reactions with diagnostic cultures of coagulase-positive staphylococci. Correlation between the two serological tests was almost complete with factors a, b, i, and k. The c fluorescent antibody reagent had a somewhat broader spectrum of activity than the corresponding agglutination serum, whereas the m fluorescent antibody reagent stained fewer strains than were agglutinated in m serum. The fluorescent antibody reagent for h factor stained strains possessing h(1) factor but not strains possessing only h(2) factor. Fluorescent antibody reagents for specific staphylococcal factors did not stain strains of group A streptococci.

Muller's phenomenon.

MULLER'S phenomenon, which may be defined as the appearance of small circular areas or plaques of clearing around the growth of a Staphylococcus pyogenes on a serum-containing chromo-protein agar substrate, has been recently reviewed by Elek1, who suggests that this proteolytic effect is due either to a non-pathogenic virus or to a hitherto unrecognized enzyme precursor present in sera and activated by a staphylococcal factor.

Staphylococcal Muller phenomenon: relationship to the plasminogen-plasmin system.

Quie, Paul G. (University of Minnesota, Minneapolis) and Lewis W. Wannamaker. Staphylococcal Muller phenomenon: relationship to the plasminogen-plasmin system. J. Bacteriol. 82:770-783. 1961.-The staphylococcal factor that produces particulate proteolysis (Muller phenomenon) in whole blood or hemoglobinserum agar plates has been obtained in supernatants from broth cultures. This has facilitated study of the phenomenon in a sterile system. The bacterial factor has been partially separated from other extracellular products by starch zone electrophoresis. Staphylococcal Muller factor and staphylokinase are similar in heat stability, in electrophoretic mobility, and in activation of a serum proteolytic system. A relationship of the Muller phenomenon to the plasminogen-plasmin system is also suggested by the observation that purified plasminogen may be substituted for serum in the sterile system, and by the finding that inhibitors of the plasminogen-plasmin system also inhibit the Muller phenomenon. Although growing colonies of streptococci fail to exhibit the Muller phenomenon, particulate proteolysis can be demonstrated with streptokinase, using a double-diffusion technique. A similar phenomenon can be produced with urine, suggesting that urokinase may substitute for the bacterial activators in the Muller phenomenon. Although evidence from other sources suggests that the serum Muller factor is particulate, the serum factor appears to be freely diffusible in agar and is not readily sedimented in the ultracentrifuge.

Studies on the proteoclast (Muller's phenomenon).

1. When coagulase-positive staphylococci are grown on nutrient agar containing fresh mammalian serum and haemoglobin, discrete areas of clearing appear in the medium near to the growth.2. These ‘plaques’ are due to proteolysis, the haemoglobin acting as a chromoprotein indicator substrate. For convenience the effect is referred to as ‘proteoclast’.3. The effect is due to the interaction of a diffusible product of the staphylococci with constituents of normal sera. The staphylococcal factor has been obtained in a bacterium-free filtrate and its interaction with serum studied quantitatively. It resists boiling.4. The number of plaques produced under defined conditions showed a linear correlation with the amount of serum used, but not with the amount of filtrate.5. By means of serial filtration through Gradocol membranes it was shown that one factor in normal serum required for the proteoclast effect is particulate, having a size of the order 20–70 m μ diameter.6. It was found that a third factor present in fresh sera was also required.7. Serum was rendered inactive by conditions of heating and ultra-violet irradiation similar to those required to sterilize vegetative bacteria and viruses.8. It is suggested that the proteoclast effect is due either to a non-pathogenic virus, or to a hitherto unrecognized particulate enzyme precursor present in sera and activated by the staphylococcal factor.

Effect of Penicillin on Growth and Toxin Production of Enterotoxigenic Staphylococci.

SummaryThe natural resistance of 15 enterotoxic strains of staphylococci to penicillin was found to range from 0.05 to 500 units per ml. Production of staphylococcal hemolysins, lethal factor and dermonecrotoxin, but not of enterotoxin, was inhibited by growing selected naturally and artificially resistant strains in Amigen liquid medium containing sublethal concentrations of penicillin. Inhibition was not observed in similar experiments using semisolid Amigen and veal infusion media.