Jean C. Lee

Staphylococcus aureus Capsular Polysaccharides

SUMMARY Serotype 5 and 8 capsular polysaccharides predominate among clinical isolates of Staphylococcus aureus. The results of experiments in animal models of infection have revealed that staphylococcal capsules are important in the pathogenesis of S. aureus infections. The capsule enhances staphylococcal virulence by impeding phagocytosis, resulting in bacterial persistence in the bloodstream of infected hosts. S. aureus capsules also promote abscess formation in rats. Although the capsule has been shown to modulate S. aureus adherence to endothelial surfaces in vitro, animal studies suggest that it also promotes bacterial colonization and persistence on mucosal surfaces. S. aureus capsular antigens are surface associated, limited in antigenic specificity, and highly conserved among clinical isolates. With the emergence of vancomycin-resistant S. aureus in the United States in 2002, new strategies are needed to combat staphylococcal infections. Purified serotype 5 and 8 capsular polysaccharides offer promise as target antigens for a vaccine to prevent staphylococcal infections, although the inclusion of other antigens is likely to be essential in the development of an effective S. aureus vaccine. The genetics and mechanisms of capsule biosynthesis are complex, and much work remains to enhance our understanding of capsule biosynthesis and its regulation.

Comparison of Virulence in Community-Associated Methicillin-Resistant Staphylococcus aureus Pulsotypes USA300 and USA400 in a Rat Model of Pneumonia

BACKGROUND The predominant genetic background of community-associated methicillin-resistant Staphylococcus aureus has transitioned from USA400 to USA300 in most US communities. The explanation for this shift is unclear. We hypothesized that USA300 must be more pathogenic--specifically, that USA300 would have increased virulence when compared with USA400 in an animal model. METHODS Rats were inoculated intratracheally with 1 of 6 S. aureus isolates from the USA300 and USA400 backgrounds. We assessed mortality, in vivo bacterial growth, and histopathology. We assessed the in vitro expression of capsule and of selected genes believed to be important in virulence in S. aureus, including agr, saeRS, sarA, alpha-toxin (hla), and Panton-Valentine leukocidin (pvl). RESULTS USA300 isolates were more lethal, produced more severe pneumonia, and had higher in vivo bacterial density in the lung than did USA400 isolates. In vitro expression of agr, saeRS, sarA, hla, and pvl were greater in USA300 isolates. USA300 isolates were unencapsulated, whereas 2 of 3 USA400 isolates produced capsule. CONCLUSIONS USA300 isolates were more virulent than USA400 isolates in a model of necrotizing pneumonia. The explanation for this is unclear, but it likely results from increased expression of S. aureus regulatory systems (e.g., agr, saeRS, and sarA) and the resultant upregulation of key virulence factors including alpha-toxin and PVL.

Immunization with Staphylococcus aureus Clumping Factor B, a Major Determinant in Nasal Carriage, Reduces Nasal Colonization in a Murine Model

ABSTRACT Staphylococcus aureus is responsible for a wide range of infections, including soft tissue infections and potentially fatal bacteremias. The primary niche for S. aureus in humans is the nares, and nasal carriage is a documented risk factor for staphylococcal infection. Previous studies with rodent models of nasal colonization have implicated capsule and teichoic acid as staphylococcal surface factors that promote colonization. In this study, a mouse model of nasal colonization was utilized to demonstrate that S. aureus mutants that lack clumping factor A, collagen binding protein, fibronectin binding proteins A and B, polysaccharide intercellular adhesin, or the accessory gene regulator colonized as well as wild-type strains colonized. In contrast, mutants deficient in sortase A or clumping factor B (ClfB) showed reduced nasal colonization. Mice immunized intranasally with killed S. aureus cells showed reduced nasal colonization compared with control animals. Likewise, mice that were immunized systemically or intranasally with a recombinant vaccine composed of domain A of ClfB exhibited lower levels of colonization than control animals exhibited. A ClfB monoclonal antibody (MAb) inhibited S. aureus binding to mouse cytokeratin 10. Passive immunization of mice with this MAb resulted in reduced nasal colonization compared with the colonization observed after immunization with an isotype-matched control antibody. The mouse immunization studies demonstrate that ClfB is an attractive component for inclusion in a vaccine to reduce S. aureus nasal colonization in humans, which in turn may diminish the risk of staphylococcal infection. As targets for vaccine development and antimicrobial intervention are assessed, rodent nasal colonization models may be invaluable.

The Staphylococcus aureus allelic genetic loci for serotype 5 and 8 capsule expression contain the type-specific genes flanked by common genes

The nucleotide sequences of two gene clusters, cap5 and cap8, involved in the synthesis of Staphylococcus aureus type 5 and type 8 capsular polysaccharides (CPs), respectively were determined. Each gene cluster contained 16 ORFs, which were named cap5A through cap5P for type 5 CP and cap8A through cap8P for type 8 CP. The cap5 and cap8 loci were allelic and were mapped to the SmaI-G fragment in the standard SmaI map of Staph. aureus strain NCTC 8325. The predicted gene products of cap5A through cap5G and cap5L through cap5P are essentially identical to those of cap8A through cap8G and cap8L through cap8P, respectively, with very few amino acid substitutions. Four ORFs located in the central region of each locus are type-specific. A comparison of the predicted amino acid sequences of cap5 and cap8 with sequences found in the databases allowed tentative assignment of functions to 15 of the 16 ORFs. The majority of the capsule genes are likely to be involved in amino sugar synthesis; the remainder are likely to be involved in sugar transfer, capsule chain-length regulation, polymerization and transport.

Structural rationale for the modulation of abscess formation by Staphylococcus aureus capsular polysaccharides

Staphylococcus aureus is a medically important bacterial pathogen that is a common cause of superficial and deep-seated abscesses in humans. Most S. aureus isolates produce either a serotype 5 or 8 capsular polysaccharide (CP) that has been shown to enhance bacterial virulence. We investigated the role of S. aureus CPs in modulating abscess formation in an experimental animal model of intraabdominal infection. Structural studies of CP8 revealed that it has a zwitterionic charge motif conferred by the negatively charged carboxyl group of N-acetylmannosaminuronic acid and free amino groups available on partially N-acetylated fucosamine residues. We report that purified CP5 and CP8 facilitated intraabdominal abscess formation in animals when given i.p. with a sterile cecal contents adjuvant. Chemical modifications that neutralized the positively or negatively charged groups on CP8 abrogated its ability to provoke abscesses. Rats prophylactically treated with CP8 s.c. were protected against abscess formation induced by homologous or heterologous zwitterionic polysaccharides. Likewise, treatment with CP8 protected against challenge with viable S. aureus strains PS80 (a capsule type 8 strain) or COL (a methicillin-resistant capsule type 5 strain). Purified CP8 was a potent activator of rat and human CD4+ T cells in vitro. When transferred to naïve rats, these activated T cells modulated the development of intraabdominal abscess formation. These results provide a structure/function rationale for abscess formation by S. aureus and expand the sphere of encapsulated organisms that interact directly with T cells to regulate this host response to bacterial infection.

Vaccination and passive immunisation against Staphylococcus aureus.

Staphylococcus aureus, an important bacterial pathogen in the hospital and the community, has become increasingly resistant to multiple antibiotics. Non-antimicrobial approaches to controlling S. aureus are clearly needed. Because many individuals who are susceptible to staphylococcal infections are not competent to mount an effective immune response, passive as well as active immunisation strategies have been explored. A capsular polysaccharide-based vaccine (StaphVAX) showed promise in an initial phase III trial in haemodialysis patients, but was found to be ineffective in a confirmatory trial. Likewise, a human immunoglobulin G (IgG) preparation known as INH-A21 (Veronate) with elevated levels of antibodies to the staphylococcal surface adhesins ClfA and SdrG made it into phase III testing, where it failed to show a clinical benefit in neonates. A number of novel antigens are in pre-clinical trials, including cell-wall-anchored adhesins, surface polysaccharides and exotoxoids. Given the multiple and sometimes redundant virulence factors of S. aureus that enable it to be such a crafty pathogen, if a vaccine is to prove effective it will of necessity be multicomponent, incorporating a number of surface proteins, toxoids and surface polysaccharides.

Reversion of the Glycopeptide Resistance Phenotype in Staphylococcus aureus Clinical Isolates

ABSTRACT The recent identification of glycopeptide intermediate-resistantStaphylococcus aureus (GISA) clinical isolates has provided an opportunity to assess the stability of the glycopeptide resistance phenotype by nonselective serial passage and to evaluate reversion-associated cell surface changes. Three GISA isolates from the United States (MIC of vancomycin = 8 μg/ml) and two from Japan (MICs of vancomycin = 8 and 2 μg/ml) were passaged daily on nutrient agar with or without vancomycin supplementation. After 15 days of passage on nonselective medium, vancomycin- and teicoplanin-susceptible revertants were obtained from each GISA isolate as determined by broth dilution MIC. Revertant isolates were compared with parent isolates for changes in vancomycin heteroresistance, capsule production, hemolysis phenotype, coagulase activity, and lysostaphin susceptibility. Several revertants lost the subpopulations with intermediate vancomycin resistance, whereas two revertants maintained them. Furthermore, although all of the parent GISA isolates produced capsule type 5 (CP5), all but one revertant tested no longer produced CP5. In contrast, passage on medium containing vancomycin yielded isolates that were still intermediately resistant to vancomycin, had no decrease in the MIC of teicoplanin, and produced detectable CP5. No consistent changes in the revertants in hemolysis phenotype, lysostaphin susceptibility, or coagulase activities were discerned. These data indicate that the vancomycin resistance phenotype is unstable in clinical GISA isolates. Reversion of the vancomycin resistance phenotype might explain the difficulty in isolating vancomycin-resistant clinical isolates from the blood of patients who fail vancomycin therapy and, possibly, may account for some of the difficulties in identifying GISA isolates in the clinical laboratory.

Adherence of Staphylococcus aureus to Endothelial Cells: Influence of Capsular Polysaccharide, Global Regulator agr, and Bacterial Growth Phase

ABSTRACT The adherence of Staphylococcus aureus to human endothelial cells (EC) is probably an important step in the pathogenesis of systemic staphylococcal infections. We examined the influence of type 5 capsular polysaccharide (CP5) production, the global regulator agr, and the bacterial growth phase onS. aureus adherence to EC. Whereas S. aureusNewman showed maximal adherence to EC in the logarithmic phase of growth, an isogenic agr mutant showed maximal adherence in the stationary growth phase. S. aureus adherence to EC and CP5 expression were negatively correlated: a mutation in theagr locus diminished CP5 production and led to increased adherence. Likewise, induction of CP5 expression by addition of NaCl to the growth medium resulted in reduced staphylococcal adherence to EC.S. aureus Newman cells that adhered to EC did not express CP5. A Newman cap5O mutant was acapsular and showed significantly greater adherence to EC than the parental strain did (P < 0.005). Complementation of the cap5Omutation in trans restored CP5 expression and reduced EC adherence to a level similar to that of the parental strain. The enhanced adherence shown by the cap5O mutant was similar in magnitude to that of the agr mutant or the cap5O agr double mutant. Cells of the cap5O mutant andcap5O agr double mutant harvested from stationary-phase cultures adhered significantly better than did cells harvested in the exponential growth phase. These data are consistent with the postexponential and agr-independent expression by S. aureus of at least one putative EC adhesin, whose binding domain may be masked by CP5.

Staphylococcus aureus Strains That Express Serotype 5 or Serotype 8 Capsular Polysaccharides Differ in Virulence

ABSTRACT Most isolates of Staphylococcus aureus produce a serotype 5 (CP5) or 8 (CP8) capsular polysaccharide. To investigate whether CP5 and CP8 differ in their biological properties, we created isogenic mutants of S. aureus Reynolds that expressed CP5, CP8, or no capsule. Biochemical analyses of CP5 and CP8 purified from the isogenic S. aureus strains were consistent with published structures. The degree of O acetylation of each polysaccharide was similar, but CP5 showed a greater degree of N acetylation. Mice challenged with the CP5+ strain showed a significantly higher bacteremia level than mice challenged with the CP8+ strain. Similarly, the CP5+ strain survived preferentially in the bloodstream and kidneys of infected mice challenged with a mixed inoculum containing both strains. The enhanced virulence of the CP5+ strain in vivo correlated with its greater resistance to in vitro killing in whole mouse blood. Likewise, in vitro opsonophagocytic killing assays with human neutrophils and sera revealed greater survival of the Reynolds (CP5) strain, even though the kinetics of opsonization by C3b and iC3b was similar for both the CP5+ and CP8+ strains. Electron micrographs demonstrated C3 molecules on the cell wall beneath the capsule layer for both serotype 5 and 8 strains. Purified CP5 and CP8 stimulated a modest oxidative burst in human neutrophils but failed to activate the alternative complement pathway. These results indicate that CP5 and CP8 differ in a number of biological properties, and these differences likely contribute to the relative virulence of serotype 5 and 8 S. aureus in vivo.

The Effectiveness of the Controlled Release of Gentamicin from Polyelectrolyte Multilayers in the Treatment of Staphylococcus aureus Infection in a Rabbit Bone Model

While the infection rate of orthopedic implants is low, the required treatment, which can involve six weeks of antibiotic therapy and two additional surgical operations, is life threatening and expensive, and thus motivates the development of a one-stage re-implantation procedure. Polyelectrolyte multilayers incorporating gentamicin were fabricated using the layer-by-layer deposition process for use as a device coating to address an existing bone infection in a direct implant exchange operation. The films eluted about 70% of their payload in vitro during the first three days and subsequently continued to release drug for more than four additional weeks, reaching a total average release of over 550 microg/cm(2). The coatings were demonstrated to be bactericidal against Staphylococcus aureus, and degradation products were generally nontoxic towards MC3T3-E1 murine preosteoblasts. Film-coated titanium implants were compared to uncoated implants in an in vivo S. aureus bone infection model. After a direct exchange procedure, the antimicrobial-coated devices yielded bone homogenates with a significantly lower degree of infection than uncoated devices at both day four (p < 0.004) and day seven (p < 0.03). This study has demonstrated that a self-assembled ultrathin film coating is capable of effectively treating an experimental bone infection in vivo and lays the foundation for development of a multi-therapeutic film for optimized, synergistic treatment of pain, infection, and osteomyelitis.