Glutamate dehydrogenase (Gdh2)-dependent alkalization is dispensable for escape from macrophages and virulence ofCandida albicans

Abstract

AbstractCandida albicanscells depend on the energy derived from amino acid catabolism to induce and sustain hyphal growth inside phagosomes of engulfing macrophages. The concomitant deamination of amino acids is thought to neutralize the acidic microenvironment of phagosomes, a presumed requisite for survival and initiation of hyphal growth. Here, in contrast to an existing model, we show that mitochondrial-localized NAD+-dependent glutamate dehydrogenase (GDH2) catalyzing the deamination of glutamate to α-ketoglutarate, and not the cytosolic urea amidolyase (DUR1,2), accounts for the observed alkalization of media when amino acids are the sole sources of carbon and nitrogen.C. albicansstrains lackingGDH2(gdh2-/-) are viable and do not extrude ammonia on amino acid-based media. Environmental alkalization does not occur under conditions of high glucose (2%), a finding attributable to glucose-repression ofGDH2expression and mitochondrial function. Consistently, inhibition of oxidative phosphorylation or mitochondrial translation by antimycin A or chloramphenicol, respectively, prevents alkalization.GDH2expression and mitochondrial function are derepressed as glucose levels are lowered from 2% (∼110 mM) to 0.2% (∼11 mM), or when glycerol is used as carbon source. Using time-lapse microscopy, we document thatgdh2-/- cells survive, filament and escape from primary murine macrophages at rates indistinguishable from wildtype. Consistently,gdh2-/- strains are as virulent as wildtype in fly and murine models of systemic candidiasis. Thus, although Gdh2 has a critical role in central nitrogen metabolism, Gdh2-catalyzed deamination of glutamate is surprisingly dispensable for escape from macrophages and virulence, demonstrating that amino acid-dependent alkalization is not essential for hyphal growth, survival in macrophages and hosts. An accurate description of the microenvironment within the phagosomal compartment of macrophages and the metabolic events underlying the survival of phagocytosedC. albicanscells and their escape are critical to understanding the host-pathogen interactions that ultimately determine the pathogenic outcome.Author SummaryCandida albicansis a commensal component of the human microflora and the most common fungal pathogen. The incidence of candidiasis is low in healthy populations. Consequently, environmental factors, such as interactions with innate immune cells, play critical roles. Macrophages provide the first line of defense and rapidly internalizeC. albicanscells within specialized intracellular compartments called phagosomes. The microenvironment within phagosomes is dynamic and ill defined, but has a low pH, and contains potent hydrolytic enzymes and oxidative stressors. Despite the inhospitable conditions, phagocytizedC. albicanscells catabolize amino acids to obtain energy to survive and grow. Here, we have critically examined amino acid catabolism and ammonia extrusion inC. albicans, the latter is thought to neutralize the phagosomal pH and induce the switch of morphologies from round “yeast-like” to elongated hyphal cells that can pierce the phagosomal membrane leading to escape from macrophages. We report that Gdh2, which catalyzes the deamination of glutamate to α-ketoglutarate, is responsible for the observed environmental alkalization whenC. albicanscatabolize amino acids. Strikingly, Gdh2 is dispensable for escape from macrophages and virulent growth. These results provide new insights into host-pathogen interactions that determine the pathogenic outcome ofC. albicansinfections.