Abstract
Candida albicans, 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, C. albicans 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 C. albicans cells neutralize the macrophage phagosome and that neutral pH is a key inducer of germination in phagocytosed cells by using a mutant lacking STP2, a transcription factor that regulates the expression of multiple amino acid permeases, that is completely deficient in alkalinization in vitro. Phagocytosed stp2Δ mutant cells showed significant reduction in hypha formation and escaped from macrophages less readily compared to wild type cells; as a result stp2Δ 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 stp2Δ mutant cells were located in acidic phagosomes. Furthermore, stp2Δ 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 C. albicans utilizes amino acids to promote neutralization of the phagosomal pH, hyphal morphogenesis, and escape from macrophages.
Highlights
A benign commensal, Candida albicans is the most prevalent fungal pathogen in humans
We show that phagocytosed stp2D mutant cells do not underdo hyphal morphogenesis within macrophages, suggesting that amino acid uptake is necessary for this process
We show that C. albicans can neutralize the macrophage phagosome to induce hyphal morphogenesis and escape from the immune cell and we propose the first mechanism for this phenomenon: extrusion of ammonia as a byproduct of amino acid catabolism within the host cell
Summary
Candida albicans is the most prevalent fungal pathogen in humans. Bacterial pathogens such as Pseudomonas aeruginosa and Yersinia enterolitica inhibit phagocytosis through direct inhibition or by altering cell surface structure [6,7]. Other pathogens, such as Anaplasma phagocytophila, interfere with the endocytic process or with the activity of macrophage-derived antimicrobial factors [8,9]. A variety of factors can trigger morphogenesis in vitro, including neutral pH, serum, elevated CO2 concentration, physiological temperature, and N-acetyl glucosamine [15] While several of these inducing factors (37uC, elevated CO2) act on phagocytosed cells, an acidic phagosome should inhibit germination and it has been unclear what stimulates this morphological transition.
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