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Candida albicans cells 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. albicans strains lacking GDH2 (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 of GDH2 expression and mitochondrial function. Consistently, inhibition of oxidative phosphorylation or mitochondrial translation by antimycin A or chloramphenicol, respectively, prevents alkalization. GDH2 expression 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 primary carbon source. Using time-lapse microscopy, we document that gdh2-/- cells survive, filament and escape from primary murine macrophages at rates indistinguishable from wildtype. In intact hosts, such as in fly and murine models of systemic candidiasis, gdh2-/- mutants are as virulent as wildtype. 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. Consistently, using the pH-sensitive dye (pHrodo), we observed no significant difference between wildtype and gdh2-/- mutants in phagosomal pH modulation. Following engulfment of fungal cells, the phagosomal compartment is rapidly acidified and hyphal growth initiates and sustained under consistently acidic conditions within phagosomes. Together, our results demonstrate that amino acid-dependent alkalization is not essential for hyphal growth, survival in macrophages and hosts. An accurate understanding of the microenvironment within macrophage phagosomes and the metabolic events underlying the survival of phagocytized C. albicans cells and their escape are critical to understanding the host-pathogen interactions that ultimately determine the pathogenic outcome., Competing Interests: The authors have declared that no competing interests exist.

(C) Fractionation of extracts from cells expressing Gdh2-GFP and Put2-HA (CFG397 or CFG404) or Gdh2-GFP and Put1-RFP (CFG398 or CFG407) (upper left panels). Subsequent Mass Spec analysis of immunoprecipitated GFP constructs (GFP-pulldowns) confirmed that the presumed Gdh2-GFP in CFG273 was in fact Put2-GFP. After this article [[1]] was published, we discovered that the results reported for Gdh2-GFP in Figs 2-4 instead represented results from a strain expressing Put2-GFP. (B) CFG273 (GDH2-GFP) or CFG219 (PUT2-GFP) cells were co-cultured in CO2-independent medium with RAW264.7 macrophages, pre-stained with Lysotracker Red (LST) at MOI of 1:1 (C:M). [Extracted from the article]