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.

FIG. 1.

Immunoblot analysis of PNAG expression by strains of S. aureus (listed along the left-hand side of the blot) evaluated in animal models of infection. Undiluted and 1:10 diluted boiled EDTA extracts from 5 × 10⁹ cells were applied by a vacuum manifold to the membrane which was probed with 200 ng/ml of affinity-purified goat antibody raised to a conjugate of dPNAG and diphtheria toxoid (43) followed by swine anti-goat immunoglobulin G conjugated to horseradish peroxidase.

FIG. 2.

Comparison of CFU/ml of blood 2 and 4 h postinfection using three strains of S. aureus with either a WT ica locus, the ica locus deleted and replaced with a tetracycline resistance cassette (Δica), or with the WT ica locus placed back into the chromosome (complemented [Comp] strain) of the Δica strain. Bars represent mean CFU/ml blood, error bars the standard deviations. Eight mice per group were used. The lower limit of detection is 2 CFU/ml. P values for strain 10833 represent pairwise comparisons between the Δica strain and both the WT and Comp strains determined by Tukey's multiple-comparison test. Overall ANOVA for all three strains yielded a P value of <0.001. There was no significant difference (P > 0.05) in CFU/ml blood comparing any of the WT and isologous S. aureus Comp strains.

FIG. 3.

Comparison of the method of growth of the challenge inoculum or the route of injection on the CFU/ml in blood 2 and 4 h postinfection achieved by S. aureus strain Newman either deleted for the ica locus (Δica) or with the WT ica locus placed back into the chromosome (complemented [Comp] strain). Bars represent mean CFU/ml blood, error bars the standard deviations. Eight mice per group were used. The lower limit of detection is 2 CFU/ml. P values were determined by t test.

FIG. 4.

Comparison of CFU/g kidney 5 days after i.v. infection with ∼5 × 10⁸ CFU of three strains of S. aureus with either a wild-type (WT) ica locus (⧫), the Δica strain with the WT ica locus placed back into the chromosome (complemented [Comp] strain) (▪), or the Δica strain (▴). For S. aureus strains MN8 and 10833, we also performed infections with the sarA::erm strain (ΔsarA) (•). Each point is the result from one mouse. The lower limit of detection is 10 CFU/g kidney. Kidneys in mice found dead were not analyzed for CFU/g due to postmortem effects on bacterial levels. Overall ANOVA for results with all three strains gave a P value of <0.001. The results of pairwise comparisons for each strain using Tukey's multiple-comparison test are shown. P values of <0.05 are highlighted in boldface type.

FIG. 5.

Susceptibility of WT, ica-complemented (Comp), Δica (ica−) and ΔsarA (sarA−) strains of S. aureus to opsonic killing mediated by various concentrations of infant rabbit serum as a source of complement.