Survival in Macrophages Figure 5. Apparently structurally normal, intact dividing and viable S. aureus Newman cells persist in the intracellular vacuolar compartment on four consecutive days post-phagocytosis. The photographs presented are representative of a minimum of 20 fields observed. hMDMs were allowed to engulf bacteria for 2h at a MOI of 25, and infected macrophages were fixed with Karnovsky’s fixative either immediately following phagocytosis (2 h), or on consecutive days post-phagocytosis, before being processed by standard electron microscopic techniques. At any given point post- phagocytosis dividing bacterial cells were visible (arrowheads). Magnified views of selected bacteria framed on main micrographs are shown in the bottom row labeled from A’ to H’, respectively. A. Control, non-infected hMDM has a morphological appearance typical for that of professional phagocyte, with vesicles localized around the nucleus (N) and only few lipid droplets (arrows). Magnification: x14,000. B. Immediately post- phagocytosis (2h), the bacteria were located predominantly in very tight membranous compartments (arrows). Magnification: x50,000. C. Already 1 day post-phagocytosis the majority of S. aureus cells were found in clearly defined membrane-enclosed vacuoles (arrows). A partially degraded bacterium in the vacuole is framed. Magnification: x20,000. D and E. At day 2 the cells of S. aureus can only be found in well defined vacuoles (arrows), occasionally spacious vacuoles (asterisk) containing several bacteria. Magnification: x32,500. F. On day 3 S. aureus persists in vacuoles which often reveal signs of partial membrane discontinuity (arrows). Magnification: x32,500. G and H. On day 4, the majority of bacteria were found in vacuoles with profoundly damaged or fully disintegrated membranes (arrow). Magnification: x54,000 (G) and x32,000 (H). doi:10.1371/journal.pone.0001409.g005 In comparison to the wild-type, which persisted intracellularly for 5 days before lysing macrophages, hla-deficient mutant was eradicated by day 2 (Fig. 7). TEM examination of macrophages directly after phagocytosis did not show any significant difference in the intracellular distribution of wild-type or hla mutant cells (data not shown). Significantly, however, in contrast to the parental strain, on day 1 the majority of hla mutant cells were found to be either partly or completely degraded (Fig. 7, inset) which correlates well with the lack of viable S. aureus at day 2. Of the three major extracellular proteases only aureolysin was shown to have a role in the survival of S. aureus inside macrophages (Fig. 8A). The ssp mutant, deficient in both the V8 protease (SspA) and staphopain B (SspB), had a phenotype that was indistinguish- able from the parental strain (Fig. 8C). In comparison to agr, sigB and hla mutants (see Figs. 6 and 7), the rate of intracellular killing of the aur mutant was slower, with live staphylococci consistently present in cell lysates up to day 3. Also, macrophages infected with a sortase A mutant, which is deficient in multiple MSCRAMMs, cleared bacteria more slowly (Fig. 8B) at a rate that was comparable to that of the aur mutant. Interestingly, the S. aureus protein A deficient mutant , was killed by macrophages with kinetics remarkably similar to those of the sortase A mutant (compare Fig. 8A and 8D). Significantly, the lack of MSCRAMMs did not affect the internalization of S. aureus cells since there were no statistically significant differences between the number of initially phagocytosed MSCRAMM-deficient cells versus the number of wild-type or the aur or ssp mutants. DISCUSSION In this study we detail findings that confirm previous observations that S. aureus can survive phagocytosis by macrophages [45,47,49]. Most importantly, however, our data shows for the first time that this bacterium can not only persist for several days inside macrophages, but ultimately is able to escape the intracellular confinement and proliferate rapidly in the conditioned media. Throughout the course of intracellular infection with each S. aureus strain tested (Newman, COL, and SH1000) macrophages showed no signs of necrosis until the day of lysis. In the case of strain Newman, the plasma membrane of infected macrophages maintained their integrity for up to 5 days with mitochondria remaining functional and cells actively metabolizing lipids (Fig. 1). Over the course of the infection macrophages appeared to be able PLoS ONE | www.plosone.org 8 January 2008 | Issue 1 | e1409