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The Role of Proline Catabolism in Candida Albicans Pathogenesis

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The Role of Proline Catabolism in Candida Albicans Pathogenesis Fitz Gerald S. Silao The Role of Proline Catabolism in Candida albicans Pathogenesis The Role of Proline Catabolism in Catabolism Role of Proline The Fitz Gerald S. Silao Candida albicans Pathogenesis ISBN 978-91-7797-837-4 Department of Molecular Biosciences, The Wenner-Gren Institute Doctoral Thesis in Molecular Bioscience at Stockholm University, Sweden 2019 The Role of Proline Catabolism in Candida albicans Pathogenesis Fitz Gerald S. Silao Academic dissertation for the Degree of Doctor of Philosophy in Molecular Bioscience at Stockholm University to be publicly defended on Friday 8 November 2019 at 10.00 in Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20. 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. Keywords: Candida albicans, proline metabolism, ATP, P5C, virulence, macrophage, hyphae, filamentation, Ras1/ cAMP/PKA, mitochondria, Proline dehydrogenase, P5C dehydrogenase, reactive oxygen species, Proline-P5C cycle. Stockholm 2019 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-173362 ISBN 978-91-7797-837-4 ISBN 978-91-7797-838-1 Department of Molecular Biosciences, The Wenner-Gren Institute Stockholm University, 106 91 Stockholm THE ROLE OF PROLINE CATABOLISM IN CANDIDA ALBICANS PATHOGENESIS Fitz Gerald S. Silao The Role of Proline Catabolism in Candida albicans Pathogenesis Fitz Gerald S. Silao ©Fitz Gerald S. Silao, Stockholm University 2019 ISBN print 978-91-7797-837-4 ISBN PDF 978-91-7797-838-1 Printed in Sweden by Universitetsservice US-AB, Stockholm 2019 To Joan and Lukas List of papers Paper I. Silao FGS, Ward M, Ryman K, Wallstrom A, Brindefalk B, Udekwu K, and Ljungdahl, P. Mitochondrial proline catabolism activates Ras1/cAMP/PKA-induced filamentation in Candida albicans. PLoS Genet. 2019;15(2):e1007976. Paper II. Silao FGS, Kühbacher A, Uwamohoro N, Nogueira F, Ryman K, Ward M, Lion T, Urban C, Rupp S, and Ljungdahl P. Proline catabolism contributes to reactive oxygen species homeostasis in Candida albicans. 2019. Manuscript. Paper III. Silao FGS, Ryman K, Ward M, Hansmann N, and Ljungdahl, P. Glutamate dehydrogenase (Gdh2)-dependent alkalization is dispensable for survival and escape of Candida albicans from macrophages. 2019. Manuscript (to be submitted to PLoS Pathogens). 1 Table of Contents Table of Figures ............................................................................................................................................ 3 List of abbreviations..................................................................................................................................... 4 Summary ....................................................................................................................................................... 6 Sammanfattning ........................................................................................................................................... 8 Introduction .................................................................................................................................................10 Candida albicans: The Model Fungal Pathogen ....................................................................................10 Virulence Factors of C. albicans .................................................................................................................12 Pleomorphism in Candida albicans - Morphological Switching .........................................................13 Signaling pathways involved in C. albicans morphogenesis ...............................................................15 Ras1, The Master Hyphal Regulator ................................................................................................16 The cAMP/PKA signaling cascade ...................................................................................................17 The Cek1-MAPK Signaling Cascade ................................................................................................18 Adhesion ...................................................................................................................................................20 Secretion of hydrolytic enzymes and cytolytic toxins ..........................................................................21 Metabolic Regulation of Candida albicans Virulence .............................................................................22 Carbon assimilation ................................................................................................................................22 Extracellular glucose sensing ............................................................................................................23 Intracellular glucose repression pathway ........................................................................................24 Regulatory Control of Carbon Utilization .......................................................................................25 Nitrogen assimilation .............................................................................................................................26 Global Crosspathway Regulation.....................................................................................................26 Tuned Regulatory Pathways .............................................................................................................28 The RIM101/pH control pathway ...........................................................................................................31 PROLINE METABOLISM ..........................................................................................................................33 Proline: a unique amino acid ..................................................................................................................33 Mitochondria: the site of proline catabolism .......................................................................................34 Electron transport chain (ETC) ..........................................................................................................34 Additional Respiratory Pathways in C. albicans ............................................................................35
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