Biology subjects

Silao, F. G. S.

Publications and source records attributed to Silao, F. G. S..

4 recordsLinked to original sources

bioRxiv2023

Diverse mechanisms control amino acid-dependent environmental alkalization by Candida albicans

Candida albicans has the remarkable capacity to neutralize acidic growth environments by releasing ammonia derived from the catabolism of amino acids. The molecular components and mechanisms controlling this capacity remain poorly understood. Here, we present an integrative model with the cytosolic NAD+-dependent glutamate dehydrogenase (Gdh2) as the principal component. We show that the alkalization defect of a strain lacking the SPS-sensor regulated transcription factor STP2 is due to the inability to fully derepress GDH2 and the two proline catabolic enzymes, PUT1 and PUT2. Notably, the Stp2-dependent regulation of PUT1 and PUT2 occurs independent of Put3, the proline-dependent activator. Accordingly, a stp2-/- put3-/- strain is unable to derepress the expression of these enzymes resulting in a severe alkalization defect that nearly phenocopies the abrogated alkalization of a gdh2-/- strain. In wildtype cells, alkalization is tightly dependent on mitochondrial activity and occurs as long as conditions permit respiratory growth. As alkalization proceeds, Gdh2 levels decrease and glutamate is transiently extruded from cells. Together these two processes constitute a rudimentary regulatory system enabling cells to prevent the rapid intracellular build-up of ammonia. Similar to C. albicans, Gdh2-dependent alkalization is dispensable for C. glabrata and C. auris virulence as assessed using a whole-blood infection model. Intriguingly, fungal-dependent alkalization does not influence the growth or proliferation of Lactobacillus crispatus, a potent antagonist of C. albicans that normally resides in the acidic vaginal microenvironment. Our data suggest that it is time to reconsider the idea that pH modulation driven by pathogenic fungi plays a crucial role in shaping the architecture and dynamics of (poly)microbial communities. Other factors are likely to be more critical in contributing to dysbiosis and that favor virulent growth.

cell biology↗

bioRxiv2023

Proline catabolism is key to facilitating Candida albicans pathogenicity

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 P5C, which inhibits mitochondrial respiration. The put1-/- and put2-/- mutations attenuate virulence in Drosophila and murine candidemia models. Using intravital 2-photon microscopy and label-free non-linear imaging, we visualized the initial stages of C. albicans cells colonizing 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.

microbiology↗

bioRxiv2020

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

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 -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 carbon source. Using time-lapse microscopy, we document that gdh2-/- 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 phagocytosed C. albicans cells and their escape are critical to understanding the host-pathogen interactions that ultimately determine the pathogenic outcome. Author SummaryCandida 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 -ketoglutarate, is responsible for the observed environmental alkalization when C. albicans catabolize 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 of C. albicans infections.

microbiology↗

bioRxiv2018

Mitochondrial proline catabolism activates Ras1/cAMP/PKA-induced filamentation in Candida albicans

Amino acids are among the earliest identified inducers of yeast-to-hyphal transitions in Candida albicans, an opportunistic fungal pathogen of humans. Here, we show that the morphogenic amino acids arginine, ornithine and proline are internalized and metabolized in mitochondria via a PUT1- and PUT2-dependent pathway that results in enhanced ATP production. Elevated ATP levels correlate with Ras1/cAMP/PKA pathway activation and Efg1-induced gene expression. The magnitude of amino acid-induced filamentation is linked to glucose availability; high levels of glucose repress mitochondrial function thereby dampening filamentation. Furthermore, arginine-induced morphogenesis occurs more rapidly and independently of Dur1,2-catalyzed urea degradation, indicating that mitochondrial-generated ATP, not CO2, is the primary morphogenic signal derived from arginine metabolism. The important role of the SPS-sensor of extracellular amino acids in morphogenesis is the consequence of induced amino acid permease gene expression, i.e., SPS-sensor activation enhances the capacity of cells to take up morphogenic amino acids, a requisite for their catabolism. C. albicans cells engulfed by murine macrophages filament, resulting in macrophage lysis. Phagocytosed put1-/- and put2-/- cells do not filament and do not lyse macrophages, consistent with a critical role of mitochondrial proline metabolism in virulence.

microbiology↗