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

Abstract

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 aciddependent alkalization is not essential for hyphal growth, survival in macrophages and

Correction: Glutamate dehydrogenase (Gdh2)-dependent alkalization is dispensable for escape from macrophages and virulence of Candida albicans

“…Based on localization of a Gdh2-GFP reporter protein, we provide evidence that Gdh2 is localized in the cytoplasm, instead of in the mitochondria as originally reported [ 1 ]. The updated results ( Fig 2 ) indicate that Gdh2-GFP co-fractionates with the cytoplasmic protein Tdh3.…”

“…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. This error was discovered when validating a novel set of strains individually carrying GFP-tagged mitochondrial proteins.…”

Proline catabolism is key to facilitatingCandida albicanspathogenicity

“…In mammalian cells, GDH is localized in the mitochondria and performs an important anaplerotic function by directly feeding the TCA cycle with α-KG (17) . In C. albicans , however, GDH (Gdh2; C2_07900W) is cytosolic (18) . Gdh2 is the key ammonia generating enzyme when C. albicans cells utilize amino acids as sole energy source, and it is responsible for the observed alkalization of the extracellular milieu (18) .…”

Fungal resilience and host–pathogen interactions: Future perspectives and opportunities

“…55,58,60 C. albicans was thought to drive metabolic alkalinization to help maintain a neutral pH within the phagosome and escape phagocytic killing. [61][62][63] More recent data suggest that alternative mechanisms may drive alkalinization, [64][65][66] including yeast-hypha morphogenesis which can lead to rupture of the phagosome and death of the macrophage. [67][68][69] Macrophages attempt to overcome hyphal development and fungal escape by physically folding hyphae.…”