Date Published: March 13, 2014
Publisher: Public Library of Science
Author(s): Slavena Vylkova, Michael C. Lorenz, Damian J. Krysan.
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.
Normally a benign commensal, Candida albicans is also the most prevalent fungal pathogen in humans. Common mucosal manifestations of candidiasis are oropharyngeal thrush and vaginitis, but C. albicans can infect virtually any body site , . The most serious infection – disseminated hematogenous candidiasis – is the fourth most common acquired hospital infection with a mortality rate of about 40% , . In healthy individuals the innate immune system maintains C. albicans as a commensal and, with the exception of vaginitis, C. albicans infections are associated with defects in innate immunity. A variety of factors such as neutropenia, chemotherapy, implanted medical devices, and several genetic disorders have been linked with increased risk for disseminated candidiasis, emphasizing the important role of the immune system, including phagocytes such as macrophages and neutrophils .
Here 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. This conclusion comes from the analysis of strains lacking Stp2p, a transcriptional regulator of amino acid permease genes that is required for amino acid-driven alkalinization in vitro . Cells lacking Stp2p are defective in hypha formation during phagocytosis, show reduced survival upon interaction with the immune cell and are defective in killing the macrophages. These cells grow at wild type rates on alternative carbon sources and show normal resistance to a variety of stress conditions encountered in the phagosome, indicating that the observed defects are not due to an enhanced sensitivity to macrophage-derived stresses. However, stp2Δ mutant cells switched to the hyphal form when within neutral phagosomes, indicating a key role for pH in inducing germination of phagocytosed cells. Alkalinization is also observed in artificial saliva and vaginal simulating fluid, indicating that there may be Candida-induced pH changes at body sites normally occupied by C. albicans, such as the oropharyngeal tract and vulvovaginal tract, underlining the effect of this phenomenon on microbial and human physiology. These defects are reflected in a modest attenuation of virulence in a mouse model of disseminated candidiasis.