Figure 7. An a-hemolysin mutant of S. aureus strain Newman is efficiently killed in phago(lyso)somes within two days of phagocy- tosis. Intracellular persistence of S. aureus was determined as described in the legend of Fig. 3A. Bars represent mean CFU values 6SD and are representative of at least three separate experiments, performed in triplicate, using hMDMs derived from different donors. An electron microphotograph of infected hMDMs was obtained 1 day post- phagocytosis and shows partially (white arrows) and totally degraded (black arrows) bacterial cells inside tight vacuoles. At this time point some apparently intact bacterial cells are also present (black arrowheads). Magnification: x32,500. The photograph presented is a representative of a minimum of 20 fields observed. doi:10.1371/journal.pone.0001409.g007 applied to detect apoptosis, all of which were negative, despite initial transient phosphatidyl serine exposure on the cell surface (annexin V binding) and procaspase 3 activation (Koziel et al.– manuscript in preparation). Taken together these data suggest that by escaping phagolysosomal killing, S. aureus may be able to manipulate macrophages differentially compared to the other cell types in which it triggers apoptosis. However, it must be kept in mind that although hMDMs are an excellent model for in vitro studies, they are not tissue or inflammatory macrophages, and consequently the in vivo interaction of S. aureus with these professional phagocytes is affected by a multitude of pathophys- iological factors. The kinetics of intracellular killing of bacteria, and the timing of the escape of internalized S. aureus into the conditioned medium were consistent and reproducible for a given staphylococcal strain, despite the fact that macrophages were derived from many different donors. The occasional deviations from typical patterns of intracellular survival and cell lysis (Fig. 3A) were likely the result of donor variation. By examining TEM micrographs of infected macrophages several important observations were made. First, S. aureus was engulfed by the formation of typical phagocytic cups (Fig. 5B). This efficient phagocytosis was apparently carried out by a class A and B (CD36) scavenger receptors with the assistance of serum proteins present in the culture media. These proteins act as opsonins by binding to the bacterial cell surface, facilitating interaction with specific phagocyte receptors, as well as promoting bacterial engulfment [68–70]. Second, immediately after comple- tion of phagocytosis the majority of S. aureus cells occurred in tight vacuoles, some of which were apparently later converted into spacious membranous compartments. Akin to this, two types of phagocytic vacuoles were also noticed in a study on the Survival in Macrophages phagocytosis of S. aureus by neutrophils, where it was suggested that effective bacterial killing only occurred within tight phago- somes [40]. The same would appear to be true of macrophages, and is consistent with the observation that the rapidly-eradicated S. aureus hla mutant was only found in tighter vacuoles (Figs. 5 and 7). Third, at all stages seemingly intact S. aureus cells that were often in the process of dividing persisted in vacuolar compart- ments. Finally, as the time of macrophage lysis approached we observed an increasing number of bacteria-bearing vacuoles whose membranes were at differing stages of degradation. TEM findings as well as data obtained from the confocal microscopy imaging showed many living bacterial cells in the macrophages (Fig. 3D). In comparison to well studied intracellular pathogens such as Shigella flexneri, Chlamydia spp., Mycobacterium spp., Legionella pneumophila, Listeria monocytogenes, Salmonella spp., and Brucella abortus we know little about how S. aureus can survive phagocytosis. Recently, it has been shown that the subversion of autophagy by S. aureus internalized by HeLa cells and fibroblasts can be central to intracellular endurance in non-professional phagocytes [27]. However, in the case of macrophages, we have neither observed in our TEM micrographs multilamellar structures attached to, or fused with the membranes of S. aureus vacuoles, nor double membranes enveloping the bacteria which are the main morphological criteria for autophagosomes. Conversely, S. aureus was consistently found inside well defined single-membrane vacuoles. Intracellular persistence of S. aureus in non-professional phagocytes depends on the expression of the global regulatory locus agr, and on the alternative sigma factor, sigB, but not on sarA [32]. In contrast to our findings with macrophages it has previously been demonstrated that sarA is required for the survival of staphylococci upon phagocytosis by neutrophils [40]. SarA was not only dispensable for intracellular persistence in human macrophages, but the sarA mutant survived inside phagocytes with higher numbers of viable cells and initiated proliferation at least one day prior to that of the parental strain (Fig. 6). This effect may well be attributable to the pleiotropic regulatory activity of SarA. In contrast to the sarA mutant, strains lacking SigB were efficiently killed with bacteria being totally eradicated within 48 h. The importance of SigB was emphasized when comparing 8325-4, which contains a natural mutation in the rsbU locus that renders sigB essentially non-functional, and its derivative SH1000, which has restored SigB function. Strain 8325-4 was unable to survive engulfment by macrophages whereas SH1000 was the most aggressive of all S. aureus strains tested in surviving this challenge. It is not yet possible to identify which factors controlled by SigB are vital for S. aureus survival since it is a pleiotropic regulator that modulates the expression of large numbers of genes, including those encoding virulence factors and proteins involved in the environmental stress response [71,72]. Strains defective in sigB lack the golden pigmentation that is characteristic of S. aureus, which has been shown to be essential in protecting S. aureus against reactive oxygen species [73]. It is possible that lack of pigment may be directly responsible for the efficient killing of strain 8325-4 and the Newman sigB mutant. The agr locus has previously been shown to be absolutely essential for the intracellular survival of S. aureus in a variety of different cell types by facilitating the escape of bacteria from the endosome into the cytoplasm [32,33,74]. Here we show that it is indispensable for survival in macrophages. Although at least 100 genes are differentially regulated by agr [75], it’s most significant impact on intracellular survival may be via the regulated expression of a-toxin. However, while S. aureus, with the help of PLoS ONE | www.plosone.org 10 January 2008 | Issue 1 | e1409