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Candida albicans, the primary etiology of human mycoses, is well-adapted to catabolize proline to obtain energy to initiate morphological switching (yeast to hyphal) and for growth. We report that put1-/- and put2-/- strains, carrying defective Proline UTilization genes, display remarkable proline sensitivity with put2-/- mutants being hypersensitive due to the accumulation of the toxic intermediate pyrroline-5-carboxylate (P5C), which inhibits mitochondrial respiration. The put1-/- and put2-/- mutations attenuate virulence in Drosophila and murine candidemia models and decrease survival in human neutrophils and whole blood. Using intravital 2-photon microscopy and label-free non-linear imaging, we visualized the initial stages of C. albicans cells infecting a kidney in real-time, directly deep in the tissue of a living mouse, and observed morphological switching of wildtype but not of put2-/- cells. Multiple members of the Candida species complex, including C. auris, are capable of using proline as a sole energy source. Our results indicate that a tailored proline metabolic network tuned to the mammalian host environment is a key feature of opportunistic fungal pathogens. Author summary: Candida albicans is listed as one of four "critical priority" fungal pathogens by the World Health Organization (WHO). Intriguingly, C. albicans is a natural commensal organism thriving in symbiosis with other components of the human microflora. Given the opportunistic character of C. albicans, it is important to understand how nutrient availability within host microenvironments contribute to the transition from commensal to pathogenic growth. Here, we report that proline is catabolized by C. albicans as an important energy source, a characteristic that is conserved among other pathogenic Candida species, including the multidrug resistant C. glabrata and C. auris. Using different infection models and applying a state-of-the-art intravital imaging technique to visualize C. albicans cells infecting kidneys in a living mouse, we observed that strains unable to utilize proline exhibit significantly reduced virulence and filamentous hyphal growth. Genetic dissection of the Proline UTilization (PUT) pathway and deciphering the control mechanisms governing proline use led to the discovery that proline is toxic to cells unable to catabolize this amino acid. Our findings provide novel insights implicating proline metabolism as a key determinant of pathogenic fungal growth. [ABSTRACT FROM AUTHOR]

Summary: Fungal infections have emerged as a major global threat to human health because of the increasing incidence and mortality rates every year. The emergence of drug resistance and limited arsenal of antifungal agents further aggravates the current situation resulting in a growing challenge in medical mycology. Here, we identified that ponatinib, an FDA‐approved antitumour drug, significantly enhanced the activity of the azole fluconazole, the most widely used antifungal drug. Further detailed investigation of ponatinib revealed that its combination with fluconazole displayed broad‐spectrum synergistic interactions against a variety of human fungal pathogens such as Candida albicans, Saccharomyces cerevisiae and Cryptococcus neoformans. Mechanistic insights into the mode of action unravelled that ponatinib reduced the efflux of fluconazole via Pdr5 and suppressed the expression of the proton pump, Pma1. Taken together, our study identifies ponatinib as a novel antifungal that enhances drug activity of fluconazole against diverse fungal pathogens. [ABSTRACT FROM AUTHOR]

(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]

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. Author summary: Candida albicans is 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 internalize C. albicans cells 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, phagocytized C. albicans cells catabolize amino acids to obtain energy to survive and grow. Here, we have critically examined amino acid catabolism and ammonia extrusion in C. 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 when C. albicans catabolize amino acids in vitro. However, the phagosomes formed as macrophages engulf wildtype or gdh2-/- cells rapidly become acidified, indicating that Gdh2 has no apparent role in modulating phagosomal pH. Strikingly, and similar to wildtype cells, gdh2-/- cells initiate and sustain hyphal growth enabling them to escape from macrophages. Also, Gdh2 is dispensable for virulent growth in systemic models of infection. These results provide new insights into host-pathogen interactions that determine the pathogenic outcome of C. albicans infections. [ABSTRACT FROM AUTHOR]