Poly- N -Acetylglucosamine Production in Staphylococcus aureus Is Essential for Virulence in Murine Models of Systemic Infection

Abstract

The contribution of the Staphylococcus aureus surface polysaccharide poly-N-acetylglucosamine (PNAG) to virulence was evaluated in three mouse models of systemic infection: bacteremia, renal abscess formation, and lethality following high-dose intraperitoneal (i.p.) infection. Deletion of the intercellular adhesin (ica) locus that encodes the biosynthetic enzymes for PNAG production in S. aureus strains Mn8, Newman, and NCTC 10833 resulted in mutant strains with significantly reduced abilities to maintain bacterial levels in blood following intravenous or i.p. injection, to spread systemically to the kidneys following i.p. injection, or to induce a moribund/lethal state following i.p. infection. In the bacteremia model, neither growth phase nor growth medium used to prepare the S. aureus inoculum affected the conclusion that PNAG production was needed for full virulence. As the SarA regulatory protein has been shown to affect ica transcription, PNAG synthesis, and biofilm formation, we also evaluated S. aureus strains Mn8 and 10833 deleted for the sarA gene in the renal infection model. A decrease in PNAG production was seen in sarA mutants using immunoblots of cell surface extracts but was insufficient to reduce the virulence of sarA-deleted strains in this model. S. aureus strains deleted for the ica genes were much more susceptible to antibody-independent opsonic killing involving human peripheral blood leukocytes and rabbit complement. Thus, PNAG confers on S. aureus resistance to killing mediated by these innate host immune mediators. Overall, PNAG production by S. aureus appears to be a critical virulence factor as assessed in murine models of systemic infection.Staphylococcus aureus is well-known for its ability to elaborate a broad range of virulence factors that are thought to be key to this organism's abilities to colonize, infect, and eventually cause disease in a variety of host tissues. Among these factors are bacterial proteins that bind to host extracellular matrix proteins that are referred to as MSCRAMMs (16,65,75); capsular polysaccharides (CP) such as CP5 and CP8 (54); extracellular toxins, hemolysins, and superantigens (36,53,62); protein A (20); mediators of antibiotic resistance (26, 67); proteases and lipases (11, 22); formation of biofilms (17, 59); and iron acquisition (45,66,70). Regulatory proteins also impact the production of virulence factors, with prominent systems including the products of the accessory gene regulator (agr) locus (39), the staphylococcal accessory regulator (sar) locus (6), proteins encoded by two-component regulatory systems (4, 58), and transcriptional factors and their regulators, such as sigB and rsbU (29).While acknowledging the multifactorial nature of S. aureus pathogenesis, typical virulence studies in animals for new virulence factors usually evaluate changes in tissue levels of mutant strains compared with parental controls or some change in lethality (3,7,54,58). Such studies can be useful for determining the impact of a virulence factor on the organism...

Combining biofilm matrix measurements with biomass and viability assays in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms

“…24 The S. aureus Newman, S. epidermidis RP62A and the E. coli XL 1 Blue strains are producers of PNAG and the WGA assay is well applicable to them. [25][26][27] The S. epidermidis 12228 is negative for the ica locus and it is often referred to as a non-biofilm or weak biofilm producer. 27,28 In the absence of PNAG, the WGA probe is probably unable to bind to the matrix of the biofilms formed by the S. epidermidis 12228 and when using this strain, highly diverting signals, including negative Z 0 -values were in fact registered, thus impeding the application of the WGA assay.…”

Protection against Escherichia coli infection by antibody to the Staphylococcus aureus poly- N -acetylglucosamine surface polysaccharide

“…To evaluate the expression of PNAG, we used several strains of staphylococci, including PNAG-positive S. aureus MN8 (15) and S. epidermidis M187 (39) and PNAGnegative S. aureus MN8⌬ica (8). E. coli strains used included CFT073, a urinary tract isolate previously shown to contain the pga locus (18), and 30 clinical isolates of E. coli collected over different periods of time and also geographically distant.…”

“…In both staphylococci and E. coli, the PNAG polymer promotes biofilm formation and is cell surface-associated (5,6,18). In staphylococci, PNAG has been found to be a virulence factor (8,(31)(32)(33) and target for protective Ab (14)(15)(16). In this work, we report that a high percentage of E. coli UTI and neonatal bacteremia isolates contain the icahomologous pga genetic locus and synthesize PNAG that is immunologically cross-reactive with PNAG isolated from S. aureus, and the production of PNAG by E. coli allows opsonically active antibodies raised to the S. aureus dPNAG antigen to mediate in vitro killing of these organisms.…”

“…To evaluate the in vivo protective efficacy of Ab to PNAG, we used an i.p. infection model in mice, as described previously (8,14). Briefly, groups of eight mice (FVB, female, 6-8 weeks of age) were immunized i.p.…”

Physiology and Pathology of Multidrug-Resistant Bacteria: Antibodies- and Vaccines-Based Pathogen-Specific Targeting

“…PNAG, which is synthesized by enzymes encoded in intercellular adhesin (ica) locus, contributes to bioilm formation, colonization in host tissue, and immune evasion [51,52]. Recent work showed that deacetylation of PNAG (dPNAG) by surface protein, IcaB, is a critical step for PNAG association to cell wall and plays key roles in colonization and resistance to host immune defense [53].…”