proteases, nucleases, lipases, hyaluronate lyase and staphylokinase facilitate tissue invasion [5]. Secreted toxins (e.g. toxin shock syndrome toxin, enterotoxins and exfoliative toxins) elicit sepsis and/or induce specific toxinoses. Nevertheless, despite the recognition of this plethora of virulence factors, we are still far from identifying exactly which ones are necessary for infection and which ones may be targeted by new therapies [6]. S. aureus can modulate the transcriptional activity of virulence genes in response to environmental changes controlled by global regulatory elements that are of two major types; two-component regulatory systems and the SarA family of DNA binding-proteins [7]. A major two-component regulatory system is encoded by the agr locus which controls the expression of many virulence factors including proteases, MSCRAMMs and toxins in a growth phase and density-dependent manner [8]. The synthesis of many cell- wall associated proteins is stimulated during the initial stages of growth but is repressed post-exponentially. In contrast, the expression of extracellular proteins, including toxins and proteases, is activated during the post-exponential growth phase converting bacteria from adherent to invasive. Several other regulatory loci control the expression of virulence factors by modulating agr expression, including the alternative Sigma factor B (sB) [9,10]. Such a multi-leveled regulatory system enables S. aureus to express virulence factors under varying conditions, which is of clinical significance since mutants of regulatory loci have severely impaired virulence [11–13]. Although S. aureus is considered to be an extracellular, pyogenic pathogen, an emerging body of evidence indicates that intracel- lular reservoirs of S. aureus may contribute to persistence such as occurs in recurrent staphylococcal rhinosinusitis and to relapses of infection after antimicrobial therapy [2,14]. Recent in vitro studies have revealed that S. aureus can invade a variety of non- professional phagocytic cells, including keratinocytes, fibroblasts, endothelial and epithelial cells, enterocytes and osteoblasts [15– 21]. Bacterial uptake is promoted by fibronectin which acts as a bridging molecule between integrin a5b1 on the cell surface and the S. aureus fibronectin-binding proteins [16,19,22,23]. This activates the Src family of protein-tyrosine kinases and stimulates internalization of adherent bacterium by the ‘zipper mechanism’ [24,25,26]. Internalized bacteria reside in endosomal vacuoles or are diverted from the endosomal pathway to autophagosomes depending on the cell type invaded and/or the S. aureus strain [27]. Subsequently, S. aureus escapes into the cytoplasm where it eventually kills the host cell through the induction of apoptosis [15,18,20,28,29]. Although secretion of a-toxin by internalized bacteria has been shown to kill host cells [18,30] it is becoming increasingly apparent that other signals from internalized, metabolically active staphylococci are required to induce apoptosis and that this process requires multiple virulence factors [29,31– 33]. Professional phagocytes such as neutrophils, macrophages and dendritic cells are designed to actively engulf microbes and kill them. Only a few types of microbial pathogen can survive phagocytosis by neutrophils and macrophages and they do so by using a several distinct mechanisms to avoid destruction in phagolysosomes [34,35]. Surprisingly, S. aureus also appears to be resistant to bactericidal attack inside the phagocytic vacuoles of neutrophils which can contain viable intracellular bacteria when isolated from sites of infection [36–38]. Recent in vitro studies confirmed the high level of resistance of S. aureus to killing by neutrophils [39]. Furthermore neutrophils can create an environ- ment in vivo that promotes intracellular growth of S. aureus and contributes to dissemination during infection [40]. Neutrophils, however, are unlikely cells to carry bacteria from the focus of Survival in Macrophages infection into the circulation since they are short-lived [41]. Macrophages are far better suited to this purpose because they are long lived and mobile cells that take phagocytosed material to the lymphoid tissue [42,43]. If carrying live S. aureus macrophages may contribute to the ability of the organism to invade the vascular system from localized infection sites [44]. However, very little is known about the interaction of S. aureus with macrophages. S. aureus is able to survive in mice and rat macrophages [45–48]. In addition, Elliott et al. [49] have demonstrated short-term survival of S. aureus inside human alveolar macrophages. However, none of these investigations have addressed specifically which bacterial factors are important for intracellular persistence. The objective of this study was to investigate the long-term ability of S. aureus to survive uptake by human monocyte-derived macrophages (hMDMs). We found that significant numbers of S. aureus cells which had avoided killing during the first 4–5 days post-phagocytosis were suddenly able to lyse the macrophages in which no signs of apoptosis or necrosis were previously demonstrated. Furthermore these newly escaped S. aureus cells were then able to proliferate abundantly in the conditioned media. Bacterial survival and escape from phagocytes was dependent not only on a-hemolysin, but also on functional agr and sB loci, as well as on the expression of sortase A and the metalloprotease, aureolysin. Thus S. aureus survived inside macrophages in a metabolically active form until the intracellular environment become suitable for escape. Our findings suggest that the ability of S. aureus to survive phagocytosis by human macrophages may contribute to dissemination of the infection and may be detrimental to the host. RESULTS Infection of macrophages by S. aureus does not damage adherent phagocytes Previous reports have shown that hMDMs and murine macro- phages that have ingested virulent bacteria such as Streptococcus pneumoniae, Shigella flexnerii, Burkholderia pseudomallei, Yersinia ssp., Salmonella typhimurium, die by cytolytic processes that involve cell swelling, plasma membrane disintegration and karyolysis or by apoptosis [50–55]. Contrary to this we have found that the phagocytosis of opsonised S. aureus strain Newman by hMDMs in ratios bacteria to macrophages of up to 50:1 (multiplicity of infection, MOI = 50), led to a prolonged, survival of bacteria in phagocytes (see below) that did not affect the viability of the host cells. Indeed hMDMs maintained an intact plasma membrane up to 5 days post-phagocytosis as discerned by a lack of staining with propidium iodide (Fig. 1A), an impermeable fluorophore which can only enter cells with compromised plasma membranes. The infected cells were metabolically active for up to 5 days after uptake of S. aureus as indicated by a MTT cell proliferation and cytotoxicity assay which measures the reduction of tetrazolium (MTT) into an insoluble formazan product in viable cells (data not shown). Furthermore, the intracellular staphylococci apparently stimulated lipid metabolism since infected cells were stained with Bodipy 493/503, a marker of neutral lipids and lipid droplets, more vividly than control cells. This difference was visible at 1 day post-uptake (Fig. 1B). At this time point the enhanced accumu- lation of lipid inside infected cells was also apparent by TEM analysis (Fig. 1C). The integrity of the plasma membrane of hMDMs infected with different MOIs of S. aureus was shown by measuring lactate dehydrogenase (LDH) released into the culture media at 2 h and 24 h post-phagocytosis. As shown in Fig. 1D cytotoxicity (%) calculated based on the LDH activity of culture media of hMDMs PLoS ONE | www.plosone.org 2 January 2008 | Issue 1 | e1409