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The Role of Proline Catabolism in Candida albicans Pathogenesis
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.ORCID iD: 0000-0002-4350-8395
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2019. , p. 82
Keywords [en]
Candida albicans, proline metabolism, ATP, P5C, virulence, macrophage, hyphae, filamentation, Ras1/cAMP/PKA, mitochondria, Proline dehydrogenase, P5C dehydrogenase, reactive oxygen species, Proline-P5C cycle
National Category
Biological Sciences
Research subject
Molecular Bioscience
Identifiers
URN: urn:nbn:se:su:diva-173362ISBN: 978-91-7797-837-4 (print)ISBN: 978-91-7797-838-1 (electronic)OAI: oai:DiVA.org:su-173362DiVA, id: diva2:1353303
Public defence
2019-11-08, Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript.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

Available from: 2019-10-16 Created: 2019-09-23 Last updated: 2019-10-09Bibliographically approved
List of papers
1. Mitochondrial proline catabolism activates Ras1/cAMP/PKA-induced filamentation in Candida albicans
Open this publication in new window or tab >>Mitochondrial proline catabolism activates Ras1/cAMP/PKA-induced filamentation in Candida albicans
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2019 (English)In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 15, no 2, article id e1007976Article in journal (Refereed) Published
Abstract [en]

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. Author summary Candida albicans is an opportunistic fungal pathogen that exists as a benign member of the human microbiome. Immunosuppression, or microbial dysbiosis, can predispose an individual to infection, enabling this fungus to evade innate immune cells and initiate a spectrum of pathologies, including superficial mucocutaneous or even life-threatening invasive infections. Infectious growth is attributed to an array of virulence characteristics, a major one being the ability to switch morphologies from round yeast-like to elongated hyphal cells. Here we report that mitochondrial proline catabolism is required to induce hyphal growth of C. albicans cells in phagosomes of engulfing macrophages, which is key to evade killing by macrophages. The finding that proline catabolism, also required for the utilization of arginine and ornithine, is required to sustain the energy demands of hyphal growth underscores the central role of mitochondria in fungal virulence. In contrast to existing dogma, we show that in C. albicans, mitochondrial function is subject to glucose repression, amino acid-induced signals are strictly dependent on Ras1 and the SPS-sensor is the primary sensor of extracellular amino acids. The results provide a clear example of how C. albicans cells sense and respond to host nutrients to ensure proper nutrient uptake and survival.

National Category
Biological Sciences
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-167542 (URN)10.1371/journal.pgen.1007976 (DOI)000459970100049 ()30742618 (PubMedID)
Available from: 2019-04-15 Created: 2019-04-15 Last updated: 2019-09-23Bibliographically approved
2. Proline catabolism contributes to reactive oxygen species homeostasis in Candida albicans
Open this publication in new window or tab >>Proline catabolism contributes to reactive oxygen species homeostasis in Candida albicans
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-173428 (URN)
Available from: 2019-09-24 Created: 2019-09-24 Last updated: 2019-09-25Bibliographically approved
3. Glutamate dehydrogenase (Gdh2)-dependent alkalization is dispensable for survival and escape of Candida albicans from macrophages
Open this publication in new window or tab >>Glutamate dehydrogenase (Gdh2)-dependent alkalization is dispensable for survival and escape of Candida albicans from macrophages
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-173431 (URN)
Available from: 2019-09-24 Created: 2019-09-24 Last updated: 2019-09-25Bibliographically approved

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