The Candida albicans ATO Gene Family Promotes Neutralization of the Macrophage Phagolysosome

Abstract

Candida albicans is an opportunistic human fungal pathogen that causes a variety of diseases, ranging from superficial mucosal to life-threatening systemic infections, the latter particularly in patients with defects in innate immune function. C. albicans cells phagocytosed by macrophages undergo a dramatic change in their metabolism in which amino acids are a key nutrient. We have shown that amino acid catabolism allows the cell to neutralize the phagolysosome and initiate hyphal growth. We show here that members of the 10-gene ATO family, which are induced by phagocytosis or the presence of amino acids in an Stp2-dependent manner and encode putative acetate or ammonia transporters, are important effectors of this pH change in vitro and in macrophages. When grown with amino acids as the sole carbon source, the deletion of ATO5 or the expression of a dominantnegative ATO1G53D allele results in a delay in alkalinization, a defect in hyphal formation, and a reduction in the amount of ammonia released from the cell. These strains also form fewer hyphae after phagocytosis, have a reduced ability to escape macrophages, and reside in more acidic phagolysosomal compartments than wild-type cells. Furthermore, overexpression of many of the 10 ATO genes accelerates ammonia release, and an ato5⌬ ATO1 G53D double mutant strain has additive alkalinization and ammonia release defects. Taken together, these results indicate that the Ato protein family is a key mediator of the metabolic changes that allow C. albicans to overcome the macrophage innate immunity barrier. Candida albicans is an opportunistic pathogen that colonizes the skin and gastrointestinal and genitourinary tracts of most healthy individuals but also causes a range of diseases, from nonlethal mucosal infections, such as oral thrush and vaginitis, to disseminated hematogenous candidiasis, the latter in immunocompromised individuals (1-3). As the fourth most prevalent cause of hospital-acquired infection, disseminated candidiasis is very difficult to treat, prolongs hospitalization, and has a mortality rate of ϳ40% (4-6). The high mortality rates and large health care burden associated with C. albicans infection highlight the importance of understanding the physiology, virulence factors, and host-pathogen interactions of C. albicans.The healthy immune system is able to effectively prevent systemic candidiasis; however, advances in health care have increased the population of individuals surviving despite immune dysfunctions. Conditions that predispose individuals to disseminated candidiasis include hematological malignancies, genetic immune disorders, HIV/AIDS, and iatrogenic interventions, including organ transplantation, chemotherapy, and invasive procedures (3, 7). The interaction between the innate immune system and C. albicans is a primary determinant of disease progression, as those with innate immune defects are more susceptible to serious infection (8). Macrophages, along with other professional phagocytes, are key components of the innate immune respons...

Phagosomal Neutralization by the Fungal Pathogen Candida albicans Induces Macrophage Pyroptosis

“…Some of these same mutations compromise the ability of C. albicans to escape from the immune cells (8,36,37). Indeed, we have shown that strains defective in the utilization of amino acids cannot neutralize the phagosome and germinate less readily, which are reflected by the improved clearance by the macrophages (8,12,13). Thus, the ability of C. albicans to adapt these pathways to alter the extracellular environment and undergo hyphal morphogenesis in certain host niches might directly contribute to survival in vivo.…”

“…albicans cells grown on amino acids release increasing amounts of ammonia as a mechanism to neutralize the environment, a process abrogated in mutants that fail to modulate the pH (8,(11)(12)(13). Thus, we assayed ammonia release by the newly constructed strains following growth on amino acids.…”

“…While both Stp2 and Ahr1 regulate the ability of the fungus to neutralize environments, including the phagosome, they are also transcription factors with potentially pleiotropic effects. We have previously identified a number of mutations that alter alkalinization in vitro and in macrophages (8,(11)(12)(13). To establish a broader linkage between fungus-driven changes in phagosomal pH and IL-1␤ release, we measured macrophage lysis and extracellular cytokine levels from J774A.1 macrophages cocultured with some of these alkalinization-defective mutants in the presence or absence of the caspase-1 inhibitor YVAD.…”

“…When C. albicans utilizes amino acids as the sole carbon source, it generates excess ammonia that can raise the extracellular pH from pH 4 to pH 7 to 8 in a matter of hours; some key factors in this phenomenon have been identified via genetic and genomic approaches (8,11). These are enriched in functions related to amino acid uptake and catabolism and include the SPS plasma membrane amino acid sensor; its regulatory target, Stp2p; a transcription factor that controls expression of amino acid permeases; and a family of putative ammonia transporters, at least two of which (Ato1 and Ato5) are required for the neutralization of acidic environments (11)(12)(13). The stp2Δ mutant completely fails to alkalinize in vitro and does not autoinduce hyphal morphogenesis (11).…”

Candida albicans morphology: still in focus

“…However, active alkalinization of the phagosome seems to be the main mechanism, which triggers intracellular hyphal formation. This modulation of phagosomal pH requires Stp2p [59], a regulator of amino acid transport, and members of the ATO gene family for transport of ammonia into the phagosome, which in turn causes neutralization of the phagosomal pH [60]. Although it has long been thought that physical piercing of the macrophage membrane by hyphae is the only cause of macrophage cell death, two studies have shown that killing by intracellular C. albicans cells is a two-step mechanism.…”