Survival in Macrophages Figure 8. Metalloprotease (Aur), protein A, and MSCRAMs, but not the V8 protease (SspA) and staphopain B (SspB), contribute to S. aureus intracellular persistence in infected hMDMs. Intracellular persistence of S. aureus was determined as described in the legend of Fig. 3A. Bars represent mean CFU value 6SD and are representative of at least three separate experiments, performed in triplicate, using hMDMs derived from different donors. A. Newman aur mutant B. Newman srtA mutant C. Newman ssp operon mutant D. Newman spA mutant doi:10.1371/journal.pone.0001409.g008 a-toxin, escapes endosomes within hours of engulfment by non- professional phagocytes, it persists in membranous compartments in macrophages for up to 5 days. This suggests that hla expression by S. aureus inside macrophages may be regulated differentially compared to other cell types where significant amounts of toxin are produced immediately following invasion facilitating the prompt escape of bacteria into the cytoplasm and eventually triggering cell death by activation of caspase-dependent or independent apoptosis [30,76–78]. a-toxin may work at two different stages by first facilitating disintegration of the vacuolar membranes that confine S. aureus and second by lysing the plasma membrane and facilitating escape from the cytoplasm into the conditioned medium. The effective eradication of the S. aureus hla mutant before any visible damage to the phagosomal membrane argues that lysing both vacuolar and cytoplasmic membranes is a major role of this toxin. The metalloprotease aureolysin, the V8 serine protease (SspA) and staphopain B (SspB), are each produced in vivo following phagocytosis by human neutrophils [77] but only the first contributed to the survival of S. aureus in macrophages. Nevertheless, it cannot be excluded that SspA and SspB have an effect on S. aureus survival because they are activated in a cascade-like manner, initiated by aureolysin [79]. In the absence of the metalloprotease, SspA and SspB occur as proteolytically-inactive zymogens. Possibly the proteases retard endosome maturation by cleaving one or more host protein. Alternatively, they may target proteins regulating the antibacterial response of macrophages. Both these possibilities are the focus of ongoing research in our laboratories. Finally, the anchoring of MSCRAMMs to the cell wall also contributes to S. aureus survival and/or escape from intracellular confinement as the sortase A-deficient mutant of strain Newman was efficiently killed by macrophages. The observation that the S. aureus protein A deficient strain was attenuated in intracellular persistence to the same degree as the srtA mutant, suggests the importance of protein A in surviving confrontations with macrophages. This is in agreement with the known importance of protein A for S. aureus virulence [80]. Additional surface protein(s) may also contribute to survival, but the fact that Newman is defective in its net fibronectin binding capacity due to point mutations in the two major fibronectin-binding genes (fnbA and fnbB) [81], argues against the importance of these adhesins in the process of intracellular persistence of S. aureus in macrophages. PLoS ONE | www.plosone.org 11 January 2008 | Issue 1 | e1409