Survival in Macrophages r Figure 3. After being phagocytosed by hMDM S. aureus strain Newman decreasingly persists intracellularly for several days until a burst of growth on day 6. A. CFU of S. aureus in cell lysates and culture medium on six consecutive days post-infection. Macrophages were allowed to engulf S. aureus at a MOI of 25 for 2 h, washed, and extracellular bacteria killed by gentamycin. Macrophages were cultured in media without antibiotics. At consecutive days post-phagocytosis media was aspired and hMDMs were lysed. Both conditioned media and cell lysates were plated onto TSA for CFU enumeration. The data shown details of one representative experiment (means6SD) of the 76 we performed in triplicate. B. CFU of S. aureus and LDH activity levels (A490 nm) in the conditioned culture medium on six consecutive days post infection (left panel); and transmission (upper row) and fluorescent (propidium iodide staining, lower row) light micrographs (x40) of S. aureus-infected cells maintained for the indicated time interval post infection. C. Confocal fluorescence microscopy images of hMDMs after 1, 3, and 5 days post-infection at MOI of 25. Cells were fixed, treated with RNase and stained with acridine orange (see Experimental Procedures section). Internalized bacteria are observed as green spots. All scale bars = 10 mm. D. Confocal fluorescence microscopy images of viable bacteria in hMDMs on the 5th day post-phagocytosis. Infected hMDMs were collected, permeabilized with 0.2 % Triton X-100 and double- labeled with propidium iodide and Syto9 (LIVE/DEAD BacLight Kit). Viable S. aureus cells are stained green while red signals represent dead bacteria and the host cell’s nuclear DNA stained mainly with propidium. Scale bar = 7.5 mm. E. Specific PCR amplification of the S. aureus 16S rRNA gene (left panel) from cell lysates and the MLVF pattern (right panel) of bacteria infecting cells on consecutive days post-phagocytosis (numbers above lanes). Lane 0, sample collected immediately upon completion of phagocytosis (2h). Lane N, the MLVF pattern of S. aureus Newman before infection. doi:10.1371/journal.pone.0001409.g003 obtained from a number of different donors. Despite some minor variations in CFU on individual days we found a consistently high level of phagocytosis, a reproducible decrease in cultivable bacteria 4–5 days post-phagocytosis, and the sudden growth of S. aureus on day 5–6 which occurred simultaneously with, or proceeding, macrophages lysis. An occasional deviation from the pattern shown in Fig. 3A was the proliferation of S. aureus on day 3 or 4 (11 cases). We also observed the complete elimination of bacteria (7 cases), and in a single case intracellular S. aureus had converted into small colony variant (SCV) that persisted in hMDMs without causing lysis (data not shown). These variations can be explained by differences amongst donors. Analysis of the data in Fig. 3A indicates that only a small number of bacteria survived intracellular killing by macrophages. To confirm the presence of intracellular S. aureus within hMDMs at various times post-uptake, cells were fixed and stained with acridine orange (AO) and examined by confocal microscopy. As seen in Fig. 3C the number of intracellular bacteria decreased from day 1 to day 3, reaching the lowest level on day 5 which correlates with viable counts. We assessed whether intracellular bacteria at day 5 were viable by staining cells with the LIVE/ DEAD BacLight Kit which differentiates live and dead S. aureus by staining the latter with a red fluorophore and viable bacteria with a green fluorophore (Fig. 3D). This showed that although the majority of bacteria were alive, a significant number of dead intracellular staphylococci were present. The presence of S. aureus within hMDMs was confirmed by the amplification of 16S rRNA PCR fragment, using S. aureus-specific primers, from total DNA isolated from the lysates of infected hMDMs (Fig. 3E, left panel). Finally, multi-locus VNTR fingerprinting (MLVF) [56] confirmed that hMDMs cultures were not contaminated with other strains of S. aureus. Identical banding patterns (Fig. 3E, right panel) indicated that strain Newman was present throughout. Priming macrophages with interferon-c helps the cells eradicate intracellular S. aureus Interferon-c (IFNc) plays an essential role in stimulating cells to eliminate intracellular pathogens [57]. Therefore we checked the effect of IFNc on the course of hMDMs infection with three different S. aureus strains. As can be clearly seen in Fig. 4, from day 2 onwards there are significant differences between the CFU from the lysates of IFNc-pretreated or non-treated macrophages. The most dramatic difference was observed on day 6 for Newman (Fig. 4A) and day 5 for strains ATCC 25923 and COL (Fig. 4BC). While S. aureus escaped from non-treated cells into the media, where proliferated explosively, IFNc-stimulated cells curtailed the numbers of intracellular staphylococci, eliminating strain Newman on day 7, and strains ATCC 25923 or COL on day 6; yielding cultures that were sterile for up to 6 consecutive days (end of experiment) after the intracellular infection was cleared. These results demonstrate that as in the case of other intracellular pathogens [57], the infection of hMDMs by S. aureus can be resolved if macrophages are primed with IFNc. S. aureus persists in intracellular vacuoles The strategies employed by obligate intracellular pathogens to avoid intracellular killing by professional phagocytes can be broadly categorized as immediate escape from the phagosome into the cytoplasm or modification of the phagosome preventing it from fusing with lysosomes [58,59]. In order to investigate the intracellular localization of staphylococci we have analyzed hMDMs infected with S. aureus strain Newman by transmission electron microscopy. Macrophages engulfed the bacteria by forming phagocytic cups (Fig. 5B). At 2h post-inoculation (Fig. 5B) internalized S. aureus cells could be seen in very tight membranous compartments (71.2% of internalized bacteria), or in clearly defined vacuoles (28.8%). After only 24 h the majority of S. aureus cells (61.2%) were located in single, membrane-bound vacuoles (Fig. 5C). At 2–3 days post-phagocytosis staphylococci were found exclusively inside vacuoles (Fig. 5DEF) some of which were spacious and occupied by several bacterial cells (Fig. 5E). Significantly, on day 4 considerable numbers of bacteria were found in vacuoles whose membranes were partially or totally degraded (Fig. 5GH). Finally, it is important to underline that at any point post-infection, dividing intact bacteria were frequently observed in vacuoles. Collectively these results strongly support our contention that S. aureus can survive intracellular killing inside vesicular compartments. Intracellular survival of S. aureus depends on functional agr and sigB loci, but not on sarA The intracellular survival of S. aureus in neutrophils, as well as in non-professional phagocytes, depends on multiple virulence factors, including global regulators of gene expression [32,40]. Therefore we compared the uptake and intracellular killing/survival of S. aureus strains deficient in agr, sarA or sigB. First we examined the survival of S. aureus strain 8325-4 in which an 11bp deletion in the rsbU gene renders the sigB operon essentially non-functional [60]. Macro- phages cleared intracellular 8325-4 within two days, apparently by killing the bacteria in phagolysosome (Fig. 6A). The importance of sigB was confirmed by the finding that S. aureus strain SH1000, 8325-4 with a restored rsbU gene and a functional sigB operon [61], survived phagocytosis (Fig. 6B). Furthermore, a Newman sigB PLoS ONE | www.plosone.org 6 January 2008 | Issue 1 | e1409