Survival in Macrophages Figure 4. Macrophages stimulated with interferon-c kill engulfed S. aureus strains: Newman (A), ATCC 25923 (B), and COL (C) more efficiently than non-stimulated cells. Cells were stimulated with recombinant human IFNc overnight at concentrations equivalent to human therapeutic doses (100 U ml21), and then allowed to phagocytose three different strains of S. aureus for 2 h. Infected cultures were processed as described in the legend for Fig. 3A and live bacteria in the whole cultures (CFU) were enumerated up to 14 days postphagocytosis. Since at the longer timepoints no bacterial growth was detected (CFU = 0) these points were not presented on the graph. The data shown is representative of at least three separate experiments, performed in triplicate, using hMDM derived from different donors. Bars represent mean CFU value 6SD. *, p,0.05; **, p,0.01; ***, p,0.001. NS-not significant. doi:10.1371/journal.pone.0001409.g004 mutant was unable to survive phagocytosis, with all ingested bacteria killed within 2 days (Fig. 6C). The significance of agr and sarA was studied using isogenic mutants of S. aureus Newman. The inactivation of agr eliminated the ability of S. aureus to survive within hMDMs. By the second day of infection no viable bacteria were apparent in cell lysates, indicating that the engulfed S. aureus had been completely destroyed (Fig. 6D). Somewhat surprisingly, inactivation of sarA had no effect on intracellular survival (Fig. 6E) and the sarA mutant had a pronounced tendency to lyse the cells two days earlier than the parental strain (Fig. 6F). Taken together these results argue that overlapping, but seemingly distinct, subsets of genes are crucial for S. aureus persistence in hMDMs and other cell types, including neutrophils and non-professional phagocytes. Expression of a-toxin, MSCRAMMs, and aureolysin, but not other proteases, contributes to S. aureus persistence in macrophages SarA and agr differentially regulate the expression of virulence factors, especially surface-associated proteins (MSCRAMMs) and secreted enzymes [8]. In addition, it has previously been established that a-hemolysin is vital for the escape of S. aureus from phagosomes into the cytoplasm of infected, non-professional phagocytes [15,62]. Therefore we examined the effects of inactivation of hla (Fig. 7), the major extracellular proteases (aureolysin, the V8 protease and staphopain B), and surface adhesins (via a sortase A mutant) (Fig. 8) on the survival of S. aureus Newman phagocytosed by hMDMs. PLoS ONE | www.plosone.org 7 January 2008 | Issue 1 | e1409