Morphology changes in phagocytosed <i>C. albicans</i> cells.

<p>Morphology changes in phagocytosed <i>C. albicans</i> cells.</p

Neutralization of the phagosome is sufficient for hyphal morphogenesis and escape from the macrophages.

<p>Cells lacking <i>STP2</i> were co-cultured at 1∶1 ratio with RAW264.7 macrophages that were pre-treated with 50 nM Bafilomycin A1. Co-cultures were imaged using time-lapse microscopy. Representative images are shown, but the experiments were performed in triplicate and morphogenesis was quantitated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003995#ppat-1003995-t001" target="_blank">Table 1</a>. White arrows point to <i>C. albicans</i> cells in yeast morphology, whereas black arrows show germinated cells.</p

Environmental alkalinization by <i>C. albicans</i> occurs under host niche-simulating conditions.

<p>Wild-type cells, <i>stp2Δ</i> mutant or complemented cells were grown in <b>A.</b> artificial saliva, pH 4.5 or <b>B.</b> vaginal simulating fluid, pH 4.2. The pH change of the growing cultures was measured at the indicated time points. The experiment was performed in triplicate.</p

<i>C. albicans</i> cells alkalinize the macrophage phagosome.

<p>FITC-stained wild type, <i>stp2Δ</i> mutant, <i>STP2</i> complemented or heat killed wild type cells were co-cultured with macrophages preloaded with Lysotracker Red. The cultures were fixed at the indicated times, imaged and phagosomal pH was calculated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003995#s4" target="_blank">Materials and Methods</a>. Statistical analysis was performed using a two-way anova; asterisks indicate p<0.05 compared to the other conditions. Data from three independent experiments is shown.</p

Cells lacking <i>STP2</i> fail to form hyphae and escape from phagocytosis.

<p>Log phase wild type cells or <i>stp2Δ</i> mutant cells were co-cultured with RAW264.7 macrophages in RPMI medium. The interaction was observed using time-lapse microscopy as noted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003995#s4" target="_blank">Materials and Methods</a>. Representative images are shown. White arrows indicate cells in yeast morphology, whereas black arrows point to germinated cells.</p

Modulation of Phagosomal pH by <i>Candida albicans</i> Promotes Hyphal Morphogenesis and Requires Stp2p, a Regulator of Amino Acid Transport

<div><p><i>Candida albicans</i>, the most important fungal pathogen of humans, has a unique interaction with macrophages in which phagocytosis induces a switch from the yeast to hyphal form, allowing it to escape by rupturing the immune cell. While a variety of factors induce this switch in vitro, including neutral pH, it is not clear what triggers morphogenesis within the macrophage where the acidic environment should inhibit this transition. In vitro, <i>C. albicans</i> grown in similar conditions in which amino acids are the primary carbon source generate large quantities of ammonia to raise the extracellular pH and induce the hyphal switch. We show here that <i>C. albicans</i> cells neutralize the macrophage phagosome and that neutral pH is a key inducer of germination in phagocytosed cells by using a mutant lacking <i>STP2</i>, a transcription factor that regulates the expression of multiple amino acid permeases, that is completely deficient in alkalinization <i>in vitro</i>. Phagocytosed <i>stp2Δ</i> mutant cells showed significant reduction in hypha formation and escaped from macrophages less readily compared to wild type cells; as a result <i>stp2Δ</i> mutant cells were killed at a higher rate and caused less damage to RAW264.7 macrophages. Stp2p-regulated import leads to alkalinization of the phagosome, since the majority of the wild type cells fail to co-localize with acidophilic dyes, whereas the <i>stp2Δ</i> mutant cells were located in acidic phagosomes. Furthermore, <i>stp2Δ</i> mutant cells were able to form hyphae and escape from neutral phagosomes, indicating that the survival defect in these cells was pH dependent. Finally, these defects are reflected in an attenuation of virulence in a mouse model of disseminated candidiasis. Altogether our results suggest that <i>C. albicans</i> utilizes amino acids to promote neutralization of the phagosomal pH, hyphal morphogenesis, and escape from macrophages.</p></div

<i>C. albicans</i> alkalinizes the macrophage phagosome.

<p>FITC-stained <i>C. albicans</i> wild type, <i>stp2Δ</i> mutant, <i>STP2</i> complemented cells or heat-killed wild type cells were co-cultured with RAW264.7 macrophages preloaded with Lysotracker Red at a 1∶1 ratio and imaged after one hour of co-culture. Representative images are shown.</p

<i>C. albicans</i> s<i>tp2Δ</i> mutant cells fail to change the environmental pH.

<p><b>A.. </b><i>C. albicans</i> strains of the indicated genotypes were grown in YNB+1% CAA, pH 4.5 at 37°C and growth was assessed by measuring OD₆₀₀ at the indicated time points. <b>B.</b> The pH of the same cultures was measured. <b>C.. </b><i>C. albicans</i> strains were grown on GM-BCP agar to promote alkalinization and ammonia release was quantified as noted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003995#s4" target="_blank">Materials and Methods</a>. Each experiment was performed in triplicate.</p

Sensing mechanism and transport systems for the utilization of different carbon sources.

<p>A) Sensing of monosaccharides via Hgt4 up-regulates the expression of sugar transporters and metabolic genes. Organic acid uptake is facilitated via Jen transporters. B) Sensing of peptides and amino acids via the Ssy1-Ptr3-Ssy5 (SPS) complex up-regulates the expression of amino acid permeases (AAPs) and oligopeptide transporters (OPTs) as well as secreted proteases and amino acid catabolic genes. Resulting ammonia from amino acid catabolism is extruded via ammonia transporters (ATOs).</p

Immunomodulatory activities of <i>C. albicans</i>.

<p>Extracellular <i>C. albicans</i> inhibits complement deposition and detoxifies ROS via secreted mediators (SODs, catalase, Pra1), while phagocytosed cells proceed along an altered trafficking pathway to end up in an ER-associated compartment characterized by the membrane calnexin (Cnx) and a loss of LAMP-1, peripheral actin, and vATPase.</p