level: the percentage of phagocytes engaged in phagocytosis of yeasts (either bound on the phagocyte membrane or internalized), and the survival of that same population of infected phagocytes, compared to their survival in absence of yeasts. To differentiate the CFW-labeled yeasts that were free or simply bound to the macrophage membrane from those ingested, we used the trypan blue to quench the CFW fluorescence of extra-macrophagic yeasts, leaving the internalized yeasts fluorescent. From a strict analytical point of view, our model overcomes a bias in the interpretation of the data frequently generated when the percentage of phagocytosing macrophages and the percentage of internalized fungal biomass are analyzed separately. For example, our data showed that the percentage of phagocytosing macrophages was lower for C. glabrata, which could be interpreted as a lower recognition efficiency, but the percentage of internalized fungal biomass was actually higher for C. glabrata than for the other Candida species. Also, a lower percentage of internalized fungal cells does not necessarily reflect a lower internalized fungal biomass, as extra-macrophagic fungal cells may divide over time, and increase total fungal biomass. Thus, our model takes into account not only the percentage of phagocytosing macrophages, along with the macrophage survival, but also the percentage of total fungal biomass internalized. When we compared the three Candida species, we observed differences in the proportion of macrophages engaged in phagocytosis, in the survival rate of macrophages and in the efficiency of the macrophages to internalize the fungal biomass. We established that macrophages were more efficient to phagocyte C. glabrata, as indicated by the lowest number of macrophages engaged in phagocytosis, the lowest macrophage cell death, and the highest proportion of the fungal biomass internalized. C. albicans was the more aggressive species as it triggered the highest number of macrophages engaged in phagocytosis, the highest macrophage cell death and the lowest proportion of internalized fungal biomass. We found that C. lusitaniae was more efficiently taken up than C. albicans, and in smaller amounts than C. glabrata. The differences in the aggressiveness of the Candida species toward the phagocytes highlighted in our work correlate with already published studies, especially those showing that C. glabrata was considerably less virulent than C. albicans using a mouse model [24], and in vitro competition experiments showing that J774 macrophages display strong preferences for phagocytosis of C. glabrata compared to C. albicans [25]. The differences in the interaction of the three Candida species with the macrophages may be in part attributed to a different cell wall composition, whose role in the recognition by the phagocytes has been widely described [8,15,26,27,28,29,30]. It has been reported that the cell wall of C. glabrata harbors 50% more mannose and three times less chitin than C. albicans [31]. As the mannans are exposed on the external face of the cell wall, they are likely to be more easily recognized by the receptors (Mannose Receptor, TLR4, Dectin-2, Galectine-3) of the phagocytes, whose activation initiates phagocytosis [29]. Furthermore, the cell wall composition can vary depending on the developmental state of the fungal cells [17]. Candida albicans shows a differential expression of cell wall proteins in yeast and hyphae [32,33,34], including particular glycosyltransferases that modify the glycan components, and therefore the PAMPs (Pathogen-Associated Molecular Patterns) exposed on the cell surface. Also, hyphae of C. albicans do not expose beta-glucans like yeast cells do in bud scars, and thus do not activate the Dectin-1 receptor, which is involved in phagocytosis and immune response processes [35]. Beta-glucans of C. lusitaniae may be similarly masked in the small chains of cells, Quantification of Candida - Phagocytes Interaction thus explaining why under that morphology, it is less recognized by the macrophages. Our experiments showed that the growth phase of the fungal cells is important for the host-pathogen interaction. When yeast cells were taken from the exponential phase, less C. lusitaniae and C. glabrata biomass was engulfed by the macrophages and they escaped more easily from macrophage than when taken from the stationary phase. We speculated that some cell wall components may be differentially exposed such as beta-1,2 mannosides, recognized by galectin 3 of macrophages, which are in higher amount in the cell wall of yeast from the stationary phase than from the exponential phase [36]. The experiments conducted with inactivated yeast cells allowed us to investigate the interaction independently from the morpho- logical changes and from the metabolic activity of the yeast cells. Heat treatment suppresses metabolic activity but artificially increases b-glucan exposure, altering the outer layer of mannans, whereas UV treatment was shown to maintain an intact cell wall [20]. We found that macrophages were more efficient to internalize UV-killed than heat-killed cells, emphasizing a pre- dominant role of the cell wall (and likely of the mannans) in the recognition by the macrophages. Interestingly, UV-killed yeasts were more efficiently internalized by the macrophages than live cells, despite a similar amount of macrophages engaged in phagocytosis. We speculate that live fungal cells produce a signal molecule that negatively interferes with macrophage recognition. Macrophage cell death was slightly higher when macrophages were infected by living C. glabrata and C. lusitaniae cells than when infected with dead yeasts: thus we speculated that the fungal metabolic activity of these two species did not significantly contribute to the observed macrophage killing. Up to 20% of the macrophages died in the presence of the inactivated yeast cells, irrespective of the species used. This suggests that the phagocytosis of inert yeast cells triggered macrophage killing via a macrophage- dependent mechanism. It has already been reported that the uptake of yeast polysaccharides led to a macrophage cell death of 10–20% after 6 or 24 hours of incubation [37]. In contrast, the ability of C. albicans to kill macrophages was greatly reduced after the inactivating treatment. Our data confirm already published studies showing that the metabolic activity of C. albicans, and the capacity to produce hyphae, largely contributed to macrophage killing. In our work, the three Candida species mostly survived to macrophage phagocytosis, according to three different mechan- isms. As previously shown [38–39], our data confirmed that C. albicans survived to phagolysis and rapidly produced hyphae from within the macrophages and thus escaped, killing the host cell and multiplying outside. In agreement with other work [40], we found that C. glabrata mostly survived phagolysis and divided within the macrophages, which eventually burst and released the yeast cells. Beside its ability to form pseudo-hyphae, we found that C. lusitaniae quickly formed small chains of cells, less efficiently recognized by the macrophages as confirmed by video-microscopy. Once phagocytosed, a proportion of the C. lusitaniae cells were able to survive, multiply within the macrophages and escape. Thus the present data support the hypothesis that there may be a correlation between the morphology of the Candida specie and the strategy used to escape from the macrophages. Some mechanisms important for the survival and escape of yeasts from phagocytes have recently been described. For C. albicans, beside the known yeast-to-hyphae transition [41], the contribution of other mechanisms allowing survival and resistance to phagocyte killing was reported: the inhibition of the phagosome maturation [38], the production of CO2 from arginine to induce PLoS ONE | www.plosone.org 12 March 2012 | Volume 7 | Issue 3 | e32621