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...