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- Silao, Fitz Gerald S., 1985-, et al.
(author)
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Amino Acid Sensing and Assimilation by the Fungal Pathogen Candida albicans in the Human Host
- 2022
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In: Pathogens. - : MDPI AG. - 2076-0817. ; 11:1
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Journal article (peer-reviewed)abstract
- Nutrient uptake is essential for cellular life and the capacity to perceive extracellular nutrients is critical for coordinating their uptake and metabolism. Commensal fungal pathogens, e.g., Candida albicans, have evolved in close association with human hosts and are well-adapted to using diverse nutrients found in discrete host niches. Human cells that cannot synthesize all amino acids require the uptake of the “essential amino acids” to remain viable. Consistently, high levels of amino acids circulate in the blood. Host proteins are rich sources of amino acids but their use depends on proteases to cleave them into smaller peptides and free amino acids. C. albicans responds to extracellular amino acids by pleiotropically enhancing their uptake and derive energy from their catabolism to power opportunistic virulent growth. Studies using Saccharomyces cerevisiae have established paradigms to understand metabolic processes in C. albicans; however, fundamental differences exist. The advent of CRISPR/Cas9-based methods facilitate genetic analysis in C. albicans, and state-of-the-art molecular biological techniques are being applied to directly examine growth requirements in vivo and in situ in infected hosts. The combination of divergent approaches can illuminate the biological roles of individual cellular components. Here we discuss recent findings regarding nutrient sensing with a focus on amino acid uptake and metabolism, processes that underlie the virulence of C. albicans.
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| 2. |
- Silao, Fitz Gerald S., 1985-, et al.
(author)
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Diverse mechanisms control amino acid-dependent environmental alkalization by Candida albicans
- 2024
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In: Molecular Microbiology. - 0950-382X .- 1365-2958. ; 121:4, s. 696-716
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Journal article (peer-reviewed)abstract
- Candida albicans has the capacity to neutralize acidic growth environments by releasing ammonia derived from the catabolism of amino acids. The molecular components underlying alkalization and its physiological significance remain poorly understood. Here, we present an integrative model with the cytosolic NAD+-dependent glutamate dehydrogenase (Gdh2) as the principal ammonia-generating component. We show that alkalization is dependent on the SPS-sensor-regulated transcription factor STP2 and the proline-responsive activator Put3. These factors function in parallel to derepress GDH2 and the two proline catabolic enzymes PUT1 and PUT2. Consistently, a double mutant lacking STP2 and PUT3 exhibits a severe alkalization defect that nearly phenocopies that of a gdh2-/- strain. Alkalization is dependent on mitochondrial activity and in wild-type cells occurs as long as the conditions permit respiratory growth. Strikingly, Gdh2 levels decrease and cells transiently extrude glutamate as the environment becomes more alkaline. Together, these processes constitute a rudimentary regulatory system that counters and limits the negative effects associated with ammonia generation. These findings align with Gdh2 being dispensable for virulence, and based on a whole human blood virulence assay, the same is true for C. glabrata and C. auris. Using a transwell co-culture system, we observed that the growth and proliferation of Lactobacillus crispatus, a common component of the acidic vaginal microenvironment and a potent antagonist of C. albicans, is unaffected by fungal-induced alkalization. Consequently, although Candida spp. can alkalinize their growth environments, other fungal-associated processes are more critical in promoting dysbiosis and virulent fungal growth.
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| 3. |
- Silao, Fitz-Gerald S., 1985-, et al.
(author)
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Proline catabolism is a key factor facilitating Candida albicans pathogenicity
- 2023
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In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 19:11 NOVEMBER
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Journal article (peer-reviewed)abstract
- 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.
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| 4. |
- Silao, Fitz Gerald S., 1985-
(author)
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The Role of Proline Catabolism in Candida albicans Pathogenesis
- 2019
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Doctoral thesis (other academic/artistic)abstract
- Candida albicans is an opportunistic fungal pathogen that has evolved in close association with human hosts. Pathogenicity is linked to an array of virulence characteristics expressed in response to environmental cues and that reflect the requirement to take up and metabolize nutrients available in the host. Metabolism generates the energy to support the bioenergetic demands of infectious growth, including the ability to reversibly switch morphologies from yeast to filamentous hyphal forms. Amino acids are among the most versatile nutrients available in the hosts as they can serve as both carbon and nitrogen sources, be transformed to key metabolic intermediates, or utilized to modulate extracellular pH via deamination forming ammonia. Of the proteinogenic amino acids, proline is unique in having a secondary amine covalently locked within an imine ring. Accumulating evidence implicates proline catabolism as being critical in the pathogenesis of many human diseases, ranging from bacterial and parasitic infections to cancer progression. This work focuses on the role of proline catabolism on C. albicans pathogenesis.Paper I describes how proline induces filamentous growth in C. albicans. Hyphal growth is induced by an increase in intracellular ATP, a positive regulator of the Ras1/cAMP/PKA pathway. Proline is a direct substrate for ATP production, its catabolism in the mitochondria by proline oxidase (Put1) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase (Put2) leads to the generation of FADH2 and NADH, respectively. Arginine and ornithine induce filamentous growth due to being catabolized to proline. Strikingly, mitochondrial proline catabolism is essential for hyphal growth and escape from macrophages.Paper II documents that proline catabolism is an important regulator of reactive oxygen species (ROS) homeostasis in C. albicans. When cells depend on proline as an energy source, the activities of the two catabolic enzymes Put1 and Put2 must operate in synchrony; perturbation of these highly regulated catabolic steps exerts deleterious effects on growth. Cells lacking PUT2 exhibit increased sensitivity to exogenous proline. This sensitivity is linked to ROS generation, likely due to the accumulation of the toxic intermediate P5C. Consistently, a put2-/- mutant is avirulent in Drosophila and in a 3D skin infection model, and hypovirulent in neutrophils and a systemic murine infection model.Paper III shows that the enzymatic step directly downstream of Put2, the deamination of glutamate to α-ketoglutarate catalyzed by glutamate dehydrogenase (Gdh2), releases the ammonia responsible for the alkalization of the extracellular environment when C. albicans cells grow in the presence of amino acids. Cells lacking GDH2 do not alkalinize the medium. Alkalization is thought to induce hyphal growth in cells engulfed by macrophages. Surprisingly, filamentous growth of gdh2-/- cells is not impaired in filament-inducing media, or importantly, in situ in the phagosome of primary murine macrophages. Thus, alkalization is not a requisite for filamentous growth within macrophages.The results demonstrate that under physiologically relevant host conditions, proline catabolism is important for C. albicans pathogenesis. Further studies are warranted to determine the applicability of this pathway as a potential target for therapeutic approaches aimed at combating this major fungal pathogen.
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