The Role of Proline Catabolism in Candida albicans Pathogenesis

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.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.</p

Amino Acid Sensing and Assimilation by the Fungal Pathogen Candida albicans in the Human Host

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 &ldquo;essential amino acids&rdquo; 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&nbsp;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

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 exhibit reduced viability, consistent with a critical role of mitochondrial proline metabolism in virulence

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 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

Calcineurin Is Required for Pseudohyphal Growth, Virulence, and Drug Resistance in <em>Candida lusitaniae</em>

<div><p><em>Candida lusitaniae</em> is an emerging fungal pathogen that infects immunocompromised patients including HIV/AIDS, cancer, and neonatal pediatric patients. Though less prevalent than other <em>Candida</em> species, <em>C. lusitaniae</em> is unique in its ability to develop resistance to amphotericin B. We investigated the role of the calcium-activated protein phosphatase calcineurin in several virulence attributes of <em>C. lusitaniae</em> including pseudohyphal growth, serum survival, and growth at 37°C. We found that calcineurin and Crz1, a <em>C. albicans</em> Crz1 homolog acting as a downstream target of calcineurin, are required for <em>C. lusitaniae</em> pseudohyphal growth, a process for which the underlying mechanism remains largely unknown in <em>C. lusitaniae</em> but hyphal growth is fundamental to <em>C. albicans</em> virulence. We demonstrate that calcineurin is required for cell wall integrity, ER stress response, optimal growth in serum, virulence in a murine systemic infection model, and antifungal drug tolerance in <em>C. lusitaniae</em>. To further examine the potential of targeting the calcineurin signaling cascade for antifungal drug development, we examined the activity of a calcineurin inhibitor FK506 in combination with caspofungin against echinocandin resistant <em>C. lusitaniae</em> clinical isolates. Broth microdilution and drug disk diffusion assays demonstrate that FK506 has synergistic fungicidal activity with caspofungin against echinocandin resistant isolates. Our findings reveal that pseudohyphal growth is controlled by the calcineurin signaling cascade, and highlight the potential use of calcineurin inhibitors and caspofungin for emerging drug-resistant <em>C. lusitaniae</em> infections.</p> </div

Transcription factor Crz1 plays a greater role than calcineurin in controlling Ca²⁺ ion homeostasis in <i>C. lusitaniae</i>.

<p>(<b>A</b>) Cells were grown overnight in YPD at 30°C, 5-fold serially diluted, and spotted onto YPD medium with or without CaCl₂ at the concentrations indicated, and incubated at 37°C for 48 h. (<b>B</b>) The growth kinetics of <i>C. lusitaniae</i> wild-type and mutant strains on YPD containing 1 M CaCl₂ at 37°C. Cells were grown overnight at 30°C, washed twice with dH₂O, diluted to 0.2 OD₆₀₀/ml in fresh liquid YPD medium, and incubated at 37°C with shaking at 250 rpm. The OD₆₀₀ of cultures was measured at 0, 3, 6, 9, 12, 15, 18, 21, 24, 48, 72, 96, and 120 h (upper panel). The lower panel shows the growth kinetics between 0 and 24 h extracted from the upper panel. The experiments were performed in triplicate, and data was plotted using Prism 5.03. Strains tested were wild-type (ATCC42720), <i>cnb1</i> mutants (YC198 and YC202), and <i>crz1</i> mutants (YC187 and YC467). (<b>C</b>) Doubling time of wild-type and calcineurin pathway mutants in 1 M CaCl₂. *<i>P</i> = 0.0002, **<i>P</i><0.0001.</p

Calcineurin is required for drug tolerance in <i>C. lusitaniae</i>.

#<p>Cells were grown overnight at 30°C and washed twice with dH₂O. Then 0.5 OD (in 500 µl) of cells was spread on RPMI 1640 media (Remel; R04067). After 20 min, the E-test strips (bioMérieux Corp.) were transferred to the surface of the media. The minimum inhibitory concentrations (MIC) were read after 24 h incubation at 35°C according to the manufacturer’s instructions.</p

Calcineurin contributes to kidney tissue colonization in a murine systemic infection model.

<p>(<b>A</b>) The fungal burden in the kidneys and spleen of immunocompetent mice was determined at day 14 after challenge with 10⁷ yeast cells via lateral tail vein injection. Strains tested were wild-type (ATCC42720), <i>cnb1</i> mutants (YC198 and YC202), and <i>crz1</i> mutants (YC187 and YC467). The <i>P</i> value (ANOVA, Dunnett’s Multiple Comparison) between wild-type and mutants is shown. (<b>B</b>) The fungal burden in the kidneys and spleen of immunocompromised mice (cyclophosphamide-treated) was determined at day 7 after challenge with 10⁷ yeast cells via lateral tail vein injection. The <i>P</i> value (ANOVA, Dunnett’s Multiple Comparison) between wild-type and mutants is shown. (<b>C</b>) Histopathological sections of kidneys dissected from immunocompromised mice infected with wild-type, <i>cnb1</i>, or <i>crz1</i> mutant strains. The mice were challenged with 10⁷ cells and sacrificed at day 7. Gomori Methenamine Silver stain was used to observe <i>C. lusitaniae</i> colonization (black dots). Bar = 50 µm.</p

Optimal growth in serum is controlled by calcineurin in <i>C. lusitaniae</i>.

<p>(<b>A</b>) Cells were grown overnight in YPD at 30°C, 5-fold serially diluted, and spotted onto agar water medium containing 10% or 50% fetal bovine serum, and incubated at 37°C for 48 h. (<b>B</b>) The growth kinetics of <i>C. lusitaniae</i> wild-type and mutant strains on 100% serum at 37°C. Cells were grown overnight at 30°C, washed twice with dH₂O, diluted to 0.2 OD₆₀₀/ml in 100% serum, and incubated at 37°C with shaking at 250 rpm. The OD₆₀₀ of cultures was measured at 0, 3, 6, 9, 24, 30, 48, 72, and 96 h (upper panel). The lower panel shows the growth kinetics between 0 and 9 h extracted from the upper panel. The experiments were performed in triplicate, and data was plotted using Prism 5.03. Strains tested were wild-type (ATCC42720), <i>cnb1</i> mutants (YC198 and YC202) and <i>crz1</i> mutants (YC187 and YC467). (<b>C</b>) Doubling time of wild-type and calcineurin pathway mutants in 100% serum. * <i>P</i><0.01, ** <i>P</i><0.0001.</p