Fitz Gerald S. Silao The Role of Proline Catabolism in Candida albicans Pathogenesis
The Role of Proline Catabolism in Fitz Gerald S. Silao Candida albicans Candida 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 Mitochondria and ROS production ...... 36 Mitochondria as an antifungal drug target? ...... 38 Proline Metabolism: Catabolism and Biosynthesis ...... 38 Proline-P5C Cycling ...... 41 Proline catabolism in C. albicans...... 42 C. albicans interactions with innate immune cells ...... 43 Pathogen Recognition ...... 43 Phagocytic cell types ...... 44 The Phagosome: The kill zone ...... 46 Phagosome evasion mechanism ...... 47 Genetic Modification of C. albicans ...... 50 Aims of the thesis ...... 52 Results summary ...... 53 Synthesis ...... 57 Future direction ...... 62 Acknowledgement ...... 81
2 Table of Figures
FIG. 1. CLINICAL MANIFESTATION OF CANDIDA INFECTIONS...... 10 FIG. 2. THE CANDIDA SPECIES COMPLEX...... 11 FIG. 3. SEXUAL AND PARASEXUAL CYCLES OF S. CEREVISIAE AND C. ALBICANS ...... 11 FIG. 4. VIRULENCE FACTORS OF C. ALBICANS...... 12 FIG. 5. INFECTION MODELS TO STUDY THE VIRULENCE AND PATHOGENICITY MECHANISMS OF C. ALBICANS. ..13 FIG. 6. PLEOMORPHISM IN C. ALBICANS...... 14 FIG. 7. MORPHOLOGICAL SWITCHING IN C. ALBICANS...... 15 FIG. 8. C. ALBICANS INVASION OF EPITHELIAL CELLS...... 15 FIG. 9. SIGNALING PATHWAYS MEDIATING YEAST TO HYPHA TRANSITION IN C. ALBICANS...... 16 FIG. 10. RAS1 MODULE...... 17 FIG. 11. ADENYLYL CYCLASE (CYR1) AS A SENSOR AND SIGNAL INTEGRATOR...... 18 FIG. 12. SCHEMATIC OF THE CEK1-MAPK PATHWAY...... 19 FIG. 13. SCHEMATIC OF GLUCOSE SENSING BY HGT4...... 24 FIG. 14. REWIRING OF UBIQUITYLATION TARGETS ALLOW C. ALBICANS TO SIMULTANEOUSLY UTILIZE PREFERRED (GLUCOSE) AND ALTERNATIVE CARBON SOURCES...... 26 FIG. 15. PROLINE UTILIZATION IS INSENSITIVE TO NCR...... 27 FIG. 16. EXTRACELLULAR AMINO ACID SENSING IN C. ALBICANS IS GOVERNED BY THE SPS SENSING PATHWAY...... 29 FIG. 17. GPR1-GPA2 PATHWAY FOR AMINO ACID SENSING...... 30 FIG. 18. MEP2-MEDIATED FILAMENTATION IN C. ALBICANS DURING NITROGEN LIMITATION...... 31 FIG. 19. PROLINE STRUCTURE AND BIOLOGICAL FUNCTIONS...... 33 FIG. 20. MITOCHONDRIAL STRUCTURE...... 34 FIG. 21. SCHEMATIC PRESENTATION OF OXPHOS...... 35 FIG. 22. ALTERNATIVE RESPIRATION IN C. ALBICANS...... 36 .− FIG. 23. SUPEROXIDE (O2 ) PRODUCTION IN THE MITOCHONDRIA...... 37 FIG. 24. PHYLOGENETIC TREE ILLUSTRATING THE DIVERSITY AND STRUCTURES OF PROLINE CATABOLIC ENZYMES IN BACTERIA AND EUKARYOTES...... 39 FIG. 25. OVERVIEW OF PROLINE METABOLISM...... 40 FIG. 26. PROLINE CATABOLISM IN C. ALBICANS...... 42 FIG. 27. PATHOGEN RECOGNITION BY INNATE IMMUNE CELLS...... 44 FIG. 28. PHAGOSOME MATURATION...... 47 FIG. 29. EVASION MECHANISM OF C. ALBICANS FROM MACROPHAGE...... 48 FIG. 30. PHAGOSOME STRETCHING ...... 49 FIG. 31. CRISPR/CAS9 MUTAGENESIS...... 51 FIG. 32. COLLAGEN INVASION ASSAY...... 54 FIG. 33. PUT2-/- IS SENSITIVE TO EXCESS PROLINE...... 55 FIG. 34. GDH2 CATALYZES ENVIRONMENTAL ALKALIZATION BUT IS DISPENSABLE FOR HYPHAL ESCAPE FROM MACROPHAGE...... 56 FIG. 35. PROLINE CATABOLISM IS ACTIVATED SHORTLY AFTER ENGULFMENT BY MACROPHAGES ...... 58 FIG. 36. MODEL FOR PROLINE ACQUISITION OF C. ALBICANS IN THE HOST...... 59 FIG. 37. MODEL OF PROLINE-P5C CYCLING RESULTING TO ROS FORMATION IN C. ALBICANS ...... 61 FIG. 38. QUANTIFICATION OF PROLINE-INDUCED EXPRESSION OF PUT2 USING THE PUT2-GFP/ RFP REPORTER STRAIN...... 62 FIG. 39. MODEL FOR MITOCHONDRIAL IMPORT OF PROLINE AS MEDIATED BY THE PUTATIVE TRANSPORTER, FLX1...... 63
3 List of abbreviations
ADP Adenosine diphosphate AMP Adenosine monophosphate ANT Adenine nucleotide translocator AOX Alternative oxidase APC Antigen presenting cells ATP Adenosine triphosphate cAMP cyclic adenosine 3’,5’– monophosphate CLD C-type lectin domain CLR C-type lectin receptors DC Dendritic cells eIF2 Eukaryotic Initiation Factor 2 ER Endoplasmic reticulum ETC Electron transport chain FADH2 Flavin adenine dinucleotide GAAC General amino acid control GAP GTPase activating protein G-CSF Granulocyte colony-stimulating factor GDP Guanosine 5’- diphosphate GDH Glutamate dehydrogenase GEF Guanine Exchange Factor GFP Green fluorescent protein GK γ-glutamyl kinase GM- CSF Granulocyte colony-stimulating factor GPCR G-protein coupled receptor GPI Glycosylphosphatidylinositol GPR γ-glutamyl phosphate reductase GSA Glutamic-γ-semialdehyde GTP Guanosine 5’- triphosphate HSG Hyphal-specific genes IL Interleukin IMM Inner mitochondrial membrane LRR Leucine rich repeat MAPK Mitogen-activated protein kinase MAPKK Mitogen-activated protein kinase kinase MAPKKK Mitogen-activated protein kinase kinase kinase MB Methylene blue MHC Major histocompatibility complex MM Methylsulfuron methyl MR Mannose receptor MTS Mitochondrial targeting sequence MW Molecular Weight NADH Nicotinamide adenine dinucleotide NCR Nitrogen catabolite repression NLR NOD-like receptor NET Neutrophil extracellular traps NOX NADPH oxidase OAT ornithine-δ-aminotransferase OMM Outer mitochondrial membrane OXPHOS Oxidative phosphorylation
4 PAK p21-activated kinase PAMP Pathogen associated molecular pattern PAR Parallel electron transport chain PAS Periodic acid-Schiff PCR Polymerase Chain Reaction PKA Protein Kinase A PKB Protein Kinase B PMN Polymorphonuclear leukocytes PMF Proton motive force PRODH Proline dehydrogenase PRR Pathogen recognition receptor P5C Δ1-pyrroline-5-carboxylate P5CR Δ1-pyrroline-5-carboxylate reductase P5CS Δ1-pyrroline-5-carboxylate synthase REDOX Reduction/oxidation RHE Reconstituted human epithelial ROS Reactive oxygen species SOD Superoxide dismutase SPS Ssy1-Ptr3-Ssy5 TGF Transforming growth factor TLR Toll-like receptors TNF Tumour necrosis factor TOR Target of rapamycin TCA Tricarboxylic acid cycle VDAC Voltage-dependent anion channel
5 Summary
Candida albicans is an opportunistic fungal pathogen that is intimately linked to its human hosts as a benign member of their microbiome. The capacity of C. albicans to establish persistent infections, either superficial, or invasive in immune compromised individuals, is attributed to discrete sets of virulence determinants that enable fungal cells to overcome host defenses, environmental stresses, and importantly, to assimilate nutrients to support the metabolic demands of infectious growth. Amino acids are among the most versatile nutrients available in hosts, they can be assimilated as both nitrogen and carbon precursors, transformed to key metabolic intermediates, or utilized to modulate extracellular pH via ammonia formation. Of the proteinogenic amino acids, proline is the only one with a secondary amine covalently locked within an imine ring. Interestingly, proline has long been known as one of the most potent inducers of yeast-to-hyphal transitions, a key virulence factor in C. albicans. Previous to the studies described here, only limited information existed regarding the signaling cascades that respond to the presence of proline, and correspondingly and specifically derepress the expression of hyphae-associated genes. Furthermore, most assumptions regarding proline catabolism, which occurs in mitochondria, were based on studying the yeast Saccharomyces cerevisiae, an organism, which in contrast to C. albicans, possesses mitochondria lacking the energy conserving respiratory Complex I (NADH dehydrogenase). Consequently, a thorough examination of proline catabolism in C. albicans was warranted, the premise being that important differences may exist and that may be specifically linked to the evolution of C. albicans within mammalian hosts. The overall aim of this thesis has been to provide a precise mechanistic understanding of how proline catabolism is regulated in C. albicans and how it affects the capacity of this opportunistic fungal pathogen to grow in a virulent manner.
Paper I describes studies aimed at elucidating how proline induces filamentous growth in C. albicans. Using a combination of genetic and biochemical approaches, proline was found to be catabolized in the mitochondria via the concerted action of proline oxidase (Put1) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase (Put2). These reactions lead to elevated ATP production, a positive regulator of Ras1/cAMP/PKA pathway. The increased level of
ATP is generated by the oxidation of the reduced electron carriers FADH2 and NADH formed in the reactions catalyzed by Put1 and Put2, respectively. Proline utilization in C. albicans is not subject to nitrogen catabolite repression (NCR), enabling it to utilize proline as a preferred nitrogen source. Interestingly, the same catabolic route is essential for the induction of hyphal growth by arginine and ornithine. These results reveal clear differences to S. cerevisiae, which lacks an energy conserving NADH dehydrogenase and has proline catabolic genes under strict NCR control. The capacity of proline to stimulate morphogenesis in C. albicans was found to be independent of it being sensed in the extracellular environment by the plasma membrane- localized SPS-sensor. Strikingly, proline catabolism was found to be essential to induce and support the energy demands for hyphal growth in macrophages, clearly demonstrating a vital role of mitochondria in virulence.
6 Paper II provides detailed information regarding proline catabolism and the associated consequences of the generation of reactive oxygen species (ROS) in C. albicans. Cells lacking PUT1 or PUT2 have significantly reduced levels of ATP even under conditions of nutrient abundance. In striking similarity to several cancer forms, we show that proline catabolism supports 3D growth of C. albicans in solid substrates; put1-/- and put2-/- cells are unable to efficiently invade collagen matrices. Also, mutant put2-/- cells generate excessive mitochondrial superoxide in the presence of exogenous proline. Excessive ROS is presumably due to the inability to oxidize P5C, which leads to the accumulation of this growth inhibitory substance in the mitochondria. Consistent with proline catabolism contributing to regulating the expression of important virulence properties, cells lacking PUT2 are avirulent in Drosophila in a 3D skin infection model, and have significantly reduced virulence in a systemic murine infection model and reduced survival in the presence of neutrophils.
Paper III documents that the proline catabolic pathway is essential for the ammonia- dependent alkalization of the of external environment when C. albicans cells are grown with amino acids as sole nitrogen and carbon sources. This finding led us to test whether the catabolic step directly downstream of Put2, the deamination of glutamate to α-ketoglutarate, catalyzed by glutamate dehydrogenase (Gdh2) is the primary source of the alkalinizing ammonia. The results were clear, cells lacking GDH2 are unable to alkalinize the medium. Consistent with Gdh2 localizing to the mitochondria, alkalization was found to be exquisitely sensitive to glucose. Surprisingly, filamentous growth of gdh2-/- cells is not impaired in filament-inducing media, or importantly, in situ in the phagosome of primary murine macrophages. Using time-lapse microscopy, we observed that gdh2-/- cells survive, filament and escape from macrophages at rates indistinguishable from wildtype. These results indicate that alkalization does not provide the inducing signal for filamentous growth, nor is it essential for survival of phagocytosed C. albicans. The data are contrary to existing dogma.
Together, the results presented in this thesis demonstrate that proline is an important energy source and regulator of ROS in C. albicans. Further studies are warranted to determine the applicability of this pathway as a potential target for therapeutic approaches aimed at combating this important fungal pathogen.
7 Sammanfattning
Den vanligaste svampinfektionen hos människor orsakas av Candida albicans som normalt är en del av vår mikroflora. Förmågan att etablera ihållande infektioner, antingen ytliga eller invasiva hos immunkomprimerade individer, tillskrivs virulensfaktorer. Dessa virulensfaktorer gör det möjligt för C. albicans att effektivt ta upp näringsämnen från sin omgivning och föröka sig samt övervinna värdens immunförsvar. Aminosyror är bland de mest användbara näringsämnen som finns i människan, de kan assimileras både som kväve- och kol källor, omvandlas till viktiga metaboliska mellanprodukter eller användas för att modulera extracellulärt pH.
Det har länge varit känt att prolin är en av de mest potenta inducerarna av virulent tillväxt i C. albicans. Före de studier som beskrivs i denna avhandling fanns endast begränsad information om signaleringskaskaderna som svarar på förekomsten av prolin och som specifikt inducerar cellerna till att bilda hyfer, små tunna tråder av celler, som angriper värden på ett mer aggressivt sätt och som även möjliggör överlevnad vid angrepp av fagocytiska celler. Dessutom var de flesta antaganden beträffande prolinkatabolism, som förekommer i mitokondrier, baserade på studier i jästen Saccharomyces cerevisiae, en organism, som i motsats till C. albicans, har mitokondrier som saknar komplex I (NADH-dehydrogenas). Det övergripande syftet med denna avhandling har varit att tillhandahålla en exakt mekanistisk förståelse för hur prolinkatabolism regleras i C. albicans och hur det påverkar kapaciteten för denna opportunistiska svamp att växa virulent.
Papper I belyser hur prolin inducerar tillväxten av hyfer hos C. albicans. Med användning av en kombination av genetiska och biokemiska metoder har vi kartlagt hur prolin kataboliseras i mitokondrierna genom en samordnad verkan av prolinoxidas (Put1) och Δ1-pyrrolin-5- karboxylat (P5C) dehydrogenas (Put2). Dessa reaktioner leder till förhöjd ATP-produktion, som i sin tur är en positiv regulator för Ras1/cAMP/PKA-signalvägen. Den ökade nivån av ATP genereras genom oxidation av FADH2 och NADH som bildas i reaktionerna. Prolinanvändning i C. albicans är inte utsatt för kvävereglering (NCR), vilket gör att prolin är en prioriterad kvävekälla. Dessa resultat avslöjar tydliga skillnader mot S. cerevisiae, som saknar ett energikonserverande NADH-dehydrogenas och har prolin-kataboliska gener och som är under strikt NCR-kontroll. Ett viktigt resultat är att nedbrytning av prolin krävs för att inducera och stödja energikraven och därmed möjliggör tillväxten av hyfer i makrofager som i sin tur leder till att makrofagerna dör. Studierna visar tydligt att mitokondrier har en vital roll i Candida infektioners virulens.
I papper II ges detaljerad information om prolinkatabolism och de tillhörande konsekvenserna av generering av reaktiva syreföreningar (ROS) hos C. albicans. Celler som saknar PUT1 eller PUT2 har signifikant minskade nivåer av ATP. I likhet med flera cancerformer visar vi här att prolinkatabolism stödjer 3D-tillväxten av C. albicans och det visar vi genom att put1-/- och put2-/- celler inte kan invadera kollagenmatriser effektivt. Dessutom i närvaro av prolin genererar put2-/- celler höga nivåer av skadlig ROS. Dessa syreradikaler uppstår antagligen genom den oförmåga cellen har att oxidera Δ1-pyrrolin-5-karboxylat (P5C), vilket då leder till en ackumulering av denna tillväxtinhiberande substans i mitokondrierna. C. albicans stammar som saknar PUT2 är avirulenta i Drosophila och i 3D- vävnadsmodeller, samt har signifikant minskat virulens i en systemisk murininfektionsmodell och minskad överlevnad i närvaro av neutrofiler.
8
Papper III beskriver hur prolinmetabolism ger C. albicans kapaciteten att alkalisera den extracellulära miljön när celler odlas med aminosyror som enda kväve- och kol källor. Denna upptäckt fick oss att undersöka om det kataboliska steget direkt nedströms om Put2, deamineringen av glutamat till α-ketoglutarat, katalyserat av glutamatdehydrogenas (Gdh2) är källan till den alkaliserande ammoniaken. Resultaten var tydliga, celler som saknar GDH2 kan inte alkalisera mediet. Uttrycket av GDH2 är känslig för glukos som överenskommer med att enzymet lokaliseras till mitokondrier. Överraskande var att tillväxten av gdh2-/- celler inte bara överlever, utan även bildar filament och till och med dödar makrofager lika väl som vildtyp celler. Dessa resultat indikerar att alkalisering inte ger den inducerande signal för filamentöst tillväxt och är inte heller nödvändig för överlevnad av fagocytoserade C. albicans. Våra upptäckt är inte förenligt med befintlig dogm.
Kunskapen från de resultat som beskrivs här belyser hur tillgången på näringsämnet prolin och metabolismen av denna, bidrar till att svampceller kan undvika immunsystemet och etablera skadliga infektioner. Den kunskapen behövs för att på sikt generera nya terapeutiska behandlingar, eg. antibiotika, för att bekämpa jästsvampinfektioner i den växande population av immunokomprimerade individer.
9
Introduction
Candida albicans: The Model Fungal Pathogen
Candida albicans is an opportunistic human fungal pathogen that normally thrives as a benign member of the skin and mucosal microbiota of the majority of the healthy population. It is the most frequently isolated Candida species and is considered to be the most important cause of both superficial mucocutaneous and life-threatening invasive mycoses worldwide (Fig. 1), and the most frequent cause of fungal infection-related deaths . Candida represents the fourth leading cause of hospital-acquired infections in the US and accounts for about 8 to 10% of all nosocomial blood stream infections (1, 2). The overall mortality of systemic Candida infections can be as high as 40% (3). An increasing trend of fungal infections is attributed to the widespread use of broad-spectrum antimycotic prophylaxis and immunosuppressive drugs like corticosteroids, increase in the number of HIV and AIDS patients, and wider use of medical procedures like chemotherapy and organ transplantation that considerably alter the immune system (2-4). a b c d
Fig. 1. Clinical manifestation of Candida infections. a) Oral candidiasis (5); b) Esophageal candidiasis; c) C. albicans skin infection in patient with multiple sclerosis (6); d) Nail candidiasis. Images (b) and (d) were taken from https://www.merckmanuals.com.
C. albicans belongs to the phylum Ascomycota, which includes a great variety of human pathogenic fungi (Fig. 2a). Among the first eukaryotic pathogen completely sequenced, C. albicans is a diploid organism with a haploid genome size of around 16 MB distributed to 8 chromosomes (Chromosomes R and 1-7) (7, 8). The genome is highly polymorphic and exhibits a considerable natural heterozygosity. Though thought to be an obligate diploid, C. albicans can exist in discrete ploidy states, ranging from haploid to tetraploid states, which can exhibit distinct cellular properties (9, 10). Interestingly, C. albicans and all the other CTG-clade species follow the non-canonical codon usage: CUG (CTG) is translated as serine instead of leucine (Fig. 2a), which is one of the major challenges that has hampered genetic studies of Candida species. In the clinical setting, C. albicans can be visually distinguished from other common Candida species by using CHROMagar medium where it appears bluish-green (Fig. 2b)(11). Unlike the closely related Saccharomyces cerevisiae, C. albicans has no known sexual cycle leading to meiosis (Fig. 3a/3b). As a naturally occurring diploid (2N) organism, C. albicans can reproduce asexually or parasexually (10, 12). Parasexual cycle in C. albicans involves the Mating Type-Like Locus (MTL) that is similar to S. cerevisiae MAT locus. The MTL locus is naturally heterozygous (MTLa/MTLα)(10, 12). However, under certain conditions such as growth on sorbose, one of the MTL alleles is eliminated due to the loss of one chromosome 5,
10 resulting in an aneuploid diploid cell that possesses only a MTLa or MTLα allele (13). Also, an epigenetic change can facilitate the phenotypic switching from the white (a/α) to the opaque phenotype (a/a or α/α). Opaque cells are mating competent and can mate forming a tetraploid (a/a/α/α), which subsequently revert to the diploid state, and sometimes to aneuploid progeny, through concerted chromosome loss (Fig. 3b) (13).
a b
Fig. 2. The Candida species complex. a) Phylogeny of sequenced Candida and Saccharomyces clade species. Highlighted is the CTG clade that translates CUG codon to Ser instead of Leu. Ref. (14); b) Different Candida species on CHROMagar medium. Ref. (11)
Fig. 3. Sexual and parasexual cycles of S. cerevisiae and C. albicans (a, b, respectively). Ref. (13)
11
Virulence Factors of C. albicans
As a human commensal, C. albicans can asymptomatically colonize virtually all anatomical sites in the body, including the skin, the oral and oropharyngeal cavities, the gastrointestinal and urogenital tracts, each serving as discrete niches where this opportunistic pathogen can thrive (3, 15). Each individual niche represents an entirely distinct microenvironment in terms of nutritional composition, competing microbiota, local environmental stress, and presence and rigor of innate immune defenses (16). Clearly, to survive, C. albicans requires the capacity to adapt to diverse environments to ensure successful colonization, which can subsequently lead to infectious growth (16). This remarkable ability to adapt to disparate microenvironments was earlier attributed to a large set of virulence factors, including the expression of adhesins facilitating adhesion to host cells, biofilm formation, secretion of hydrolytic enzymes and cytolytic peptide (i.e., Candidalysin), morphological switching between the yeast and filamentous forms, and phenotypic switching (Fig. 4). Recently, other fitness attributes, including tolerance to host-imposed stress, nutrient acquisition systems, and the presence of robust and flexible metabolic machinery, are now considered as contributing factors to C. albicans pathogenicity (16-18). Metabolic flexibility will be covered in greater details in the next chapter.
Fig. 4. Virulence factors of C. albicans. Image was re-drawn from ref. (18)
Experimental infection models have become an invaluable tool to examine the pathogenicity mechanisms of C. albicans. To date, there are several infection models that have been developed to study C. albicans pathogenesis ranging from low complexity cell lines to a more complex rodent models, each having their strengths and weaknesses. Though larger animals such as pigs, rabbits, and even non-human primates have been used to examine C. albicans virulence in the past, the use of mice (Fig. 5a) as a small vertebrate model has become a popular choice among researchers. The use of mice is primarily driven by economic and
12 ethical reasons, ease of handling, relative similarity in terms of anatomy and immunological response as humans, and the availability of genetically modified mouse strains (reviewed in (19, 20)). Furthermore, the use of mice allows for the development of localized (mucosal) or invasive infection models making it a very powerful tool for the investigation of host-pathogen interaction and antifungal drug development (reviewed in (19, 20)). More recently, the use of small invertebrate mini-hosts, such as the fruitfly Drosophila melanogaster (Fig. 5b)(21-23), the wax moth larvae of Galleria mellonella (Fig. 5c)(24, 25), and the nematode Caenorhabditis elegans (Fig. 5d) (26-30), have been developed to study host-fungal interaction and examine drug responses. These models have become more popular among labs due to their low cost, simple handling, high reproducibility, genetic tractability, amenability to high throughput assays, and rapid turnover of results. Though very useful, the use of invertebrates as minihosts to study host-pathogen interaction is limited to examination of innate immunity. The use of the Zebrafish as a vertebrate minihost (Fig. 5e) gives the added advantage as it can be used to study both innate and adaptive immunity (31-34). However, a major limitation of using these minihosts is that they require culturing conditions at temperatures below the physiologic human temperature of 37°C, which can potentially affect many fungal virulence traits dependent on temperature. In addition to these minihosts, human in vitro models based on reconstituted human epithelia (RHE) have been developed to analyse targeted fungal response in specific niche such as in the skin, vaginal, oral, or gut mucosa, though some work using RHE models have yielded conflicting results with respect to the role of secreted aspartyl proteases in mucosal invasion ((35-37), see commentary (38)).
Fig. 5. Infection models to study the virulence and pathogenicity mechanisms of C. albicans. Images from take from: a) (https://www.jax.org/strain/000664); b) (260); c) (262); d) C. elegans (39); e) (261).
Pleomorphism in Candida albicans - Morphological Switching
C. albicans can exist in different cellular morphologies inside the human body, and hence it is known as a pleomorphic organism (Fig. 6). Important are the three major morphologies: yeast, pseudohyphae, and true hyphae, where the latter two are classically known as filamentous morphologies (40, 41). Morphological transitions, primarily from the yeast to filamentous forms, is the focus of this thesis and will be discussed in more details later. Yeast-like cells can exist in various morphotypes. In addition to the well-known white phenotype, yeast cells can become opaque, which are elongated cells characterized by ‘pimple’-containing structures on
13 their cell surface. More recently, the GUT and Grey yeast morphotypes have been identified and have been implicated in both commensalism and virulence (9). In addition, in certain conditions like starvation, C. albicans can form a large distinct cellular structure at the terminal cells of mycelia known as the chlamydospore, the function of which is not fully understood (9, 42).
Fig. 6. Pleomorphism in C. albicans.
In response to a large repertoire of environmental cues, C. albicans can reversibly switch between the yeast and the filamentous (hyphae and pseudohyphae) forms (Fig. 7a), an attribute known to be critical to its virulence. Yeast forms are round cells that asymmetrically divide into mother and daughter cells by axial or bipolar budding. Pseudohyphae and hyphae are two distinct morphologies that can easily be confused with each other, as pseudohyphae can sometimes superficially resemble true hyphae; both filamentous forms exhibit similar long elongated structures. In reality, however, these two forms are quite distinct. Hyphae, for example, initiate as germ tubes at a bud site, and assume polarized growth with elongated cells with completely parallel sides that lack constrictions at the sites of septation. Whereas pseudohyphae are much wider and become clearly constricted at sites of septation and every subsequent septal junction (Fig. 7b) (40). Pseudohyphae appear to be an intermediate state between yeast and true hypha, but the mechanism of pseudohyphae formation more closely resembles that of the budding yeast. Pseudohyphae are sometimes viewed as chains of elongated cells with delayed isotropic growth after bud emergence. Though also implicated in virulence, the precise nature of pseudohyphae in C. albicans pathogenesis is rather unclear. There is no consensus with respect to the specific conditions needed to selectively induce and maintain pseudohyphal growth although artificial lowering of the expression of UME6, a transcriptional regulator involved in filamentous growth, yielded pseudohyphae in hyphae- inducing condition (43). Frequently, pseudohyphae appear as a mere subpopulation of cells in conditions known to trigger true hyphal induction (9).
In clinical cases of deep-seated infection, C. albicans produces characteristic invasive lesions in affected tissues arising from invading filamentous (hyphae and pseudohyphae) cells. This capacity to produce filamentous hyphal cells allows them to breach epithelial barriers and then disseminate via the bloodstream (Fig. 8). From the bloodstream, cells can infect almost all organs, but appear to preferentially invade certain organs depending upon the route of infection. When the capacity to switch is genetically disabled, C. albicans fail to mount an infection in a number of in vitro and in vivo infection models. Cumulated results
14 consistently support the concept that morphogenesis is an essential virulence attribute (40, 44- 46). However, a recent report may challenge this dogma; a large-scale genetic study provided evidence that virulence is not necessarily coupled to the ability to undergo morphogenic switching (47). The environmental signals known to stimulate morphogenesis in C. albicans reflect the diversity of microenvironments within the host (48). Among these are nutrients such as amino acids that are abundantly present in human host and that are primarily used as nitrogen sources for growth.
a b
Fig. 7. Morphological switching in C. albicans. a) C. albicans can exist in three distinct morphological forms: the yeast, the pseudohyphae, and the true hyphae; b) Similar to the baker’s yeast Saccharomyces cerevisiae, C. albicans can grow as an ovoid yeast cell and as a pseudohyphae, which are elongated yeasts formed by asymmetrical budding. Unlike S. cerevisiae, C. albicans can form true hyphae, which are similar to hyphae observed in filamentous fungi that grows by continuous polarized growth and involves significant changes in cell cycle processes. These forms can be distinguished according to the position of the septin ring. Ref. (49)
Fig. 8. C. albicans invasion of epithelial cells. The image on the right showing hyphae invading host cell tissue is from ref. (49)
Signaling pathways involved in C. albicans morphogenesis
Among the virulence factors identified in C. albicans, filamentous growth to hyphae (and also pseudohyphae) is probably the most studied. Numerous environmental cues (e.g., serum,
Lee’s medium, CO2, nutrient starvation, N-acetylglycosamine (GlcNac), and certain amino
15 acids) triggering filamentous growth in C. albicans have been identified. These environmental stimuli act by activating specific signaling cascades that eventually lead to derepression of hyphal specific genes (HSG) (Fig. 9). Positive up-regulation, or repression, of HSG in C. albicans is controlled by a set of transcription factors including Efg1, Cph1, Cph2, Tec1, Flo8, Czf1, Rim101 and Ndt80 (40). Negative regulation of filamentous growth is through the transcriptional repressor Tup1 via its partner DNA-binding proteins, Rfg1 and Nrg1. The known roles of these transcription factors and the HSG they regulate are summarized in excellent reviews by Sudberry (40) and Noble (9). The focus of this thesis will be restricted to the well-characterized mitogen-activated protein kinase (MAPK) and cyclic AMP/protein kinase A (cAMP/PKA) pathways, which act on Cph1 and Efg1, the effector transcription factors, respectively, and that depend on the GTPase Ras1 as a signal transducer (9, 40). Interestingly, these pathways are not only limited to expression of virulence genes, but also to expression of genes involved in normal cellular physiological functions.
Fig. 9. Signaling pathways mediating yeast to hypha transition in C. albicans. Ref. (9).
Ras1, The Master Hyphal Regulator
Ras proteins are small GTPases participating in numerous cellular signaling cascades in eukaryotes. Ras1 activates the adenylyl cyclase (Cyr1) resulting in an increase in intracellular cAMP concentration (50). Elevated cAMP levels are required for the activation of PKA that in
16 turn phosphorylates and activates the Efg1 transcription factor (See cAMP/PKA discussion in the following section) (50). Ras1 signaling also feeds through the MAPK pathway that activates the Cph1 transcription factor (50). Ras1 is activated when bound to GTP and is inactive when the GTP is hydrolyzed to GDP, a cycling event that is catalyzed by Cdc25 and Ira2, the guanine nucleotide exchange factor (GEF) and the GTPase activating protein (GAP), respectively (Fig. 10) (50, 51). Ras proteins are tethered to the plasma membrane via C-terminal farnesylation and palmitolyation, which is thought to be critical to its activity, and mutants devoid of these modifications are unable to undergo filamentous growth (50, 52). In addition to localization, proteolytic processing of Ras1 is also important to filamentous growth. Membrane-associated, full length Ras1 is expressed at relatively higher levels in hyphal cells, and a shorter, soluble Ras1 species arising from the proteolytic cleavage of membrane-tethered Ras1 is found in yeast form cells (53). Interestingly, the deletion of the putative cleavage site in Ras1 resulted in cells that more rapidly switched from yeast to hyphal forms, and also delayed hypha-to-yeast transitions (53). Earlier it was thought that C. albicans possesses only a single non-essential RAS gene (RAS1), however it is now known that a second Ras gene (RAS2) exists. Interestingly, Ras2 has sequence motifs that deviate with other Ras GTPases (54), and does not appear to contribute substantially to virulence of C. albicans. The deletion of RAS2 alone manifests only minor defects in filamentation. Moreover, the deletion of RAS2 in a ras1 background does not affect cell viability, but enhances the filamentous growth defect already observed in ras1 strains (54). More recent reports have shown that Ras and Cyr1 are not only restricted to the plasma membrane but also localize to the nuclear, ER and mitochondrial compartment. The distribution between plasma membrane and internal membranes is apparently dependent on the carbon source and the activity and localization of other components of the cAMP/PKA pathway, for example Gpr1-Gpa2 or PKA catalytic subunits) (50) (see below).
Fig. 10. Ras1 module. Ras1 cycling between the active (GTP-bound) and inactive (GDP-bound) forms as catalyzed respectively by Cdc25 (GEF) and Ira2 (GAP).
The cAMP/PKA signaling cascade
As the only adenylyl cyclase in C. albicans, Cyr1, has been shown to act as a signaling hub that is capable of integrating diverse signals. Cyr1 possesses multiple domains that detect distinct signaling cues (Fig. 11a) (55). Thus, the stimulatory signal from Ras1 is not the only cue that activates Cyr1. The active GTP-bound form of Ras1 activates Cyr1 via a physical interaction to the Ras associated (RA) domain of Cyr1. Cry1, together with the Cyr1-associated proteins Srv2 (Cap1) and G-actin, forms a tripartite protein complex that catalyzes the synthesis of 3’-5’-
17 cyclic adenosine monophosphate (cAMP) from ATP (50). The molecular mechanisms that determine the metabolic activation of Ras1 are not well-established. Recent work has shown that mitochondrial activity and Cyr1 function are important factors regulating Ras1 activity, and that Ras1 activity appears to be affected by a positive feedback loop dependent on intracellular ATP levels (56). According to the proposed model, an increased level of ATP interferes with the function of Ira2, the Ras1 GAP. Accordingly, this leads to an accumulation of active GTP-Ras1, which facilitates and triggers cAMP synthesis by enhanced binding and activation of Cyr1. cAMP binds to Bcy1, the regulatory subunit of protein kinase A complex, resulting in the dissociation and activation of the catalytic subunits, Tpk1 and Tpk2 (50). Tpk1 or Tpk2 activates Efg1 by phosphorylation, which then activates the expression of HSG in the nucleus. Intracellular cAMP level is negatively regulated by a phosphodiesterases, Pde1 and Pde2 that catalyze the degradation of cAMP to AMP. Pde1 is a low-affinity phosphodiesterase, whereas, Pde2 is a high-affinity phosphodiesterase (50). The Ras/cAMP/PKA pathway is summarized in Fig. 11b. In C. albicans, Efg1 activates HSG including the hyphae-specific cyclin (HGC1), hyphae-specific cell wall proteins (HYR1, HWP1 and HWP2), adhesins (ALS3), and hyphae-secreted aspartyl proteases (SAP4) (40).
Fig. 11. Adenylyl cyclase (Cyr1) as a sensor and signal integrator. a) Cyr1 contains multiple domains acting as signal sensor for a diverse set of signaling cues: RA – Ras associated domain; LRR – Leucine Rich Repeat; PP2C – protein phosphatase 2C; CYCc – Cyclase catalytic domain; CBD – Cap1 (Srv2) binding domain. Adapted and modified from: (55); b). Schematic diagram of Ras1/cAMP/PKA pathway.
The Cek1-MAPK Signaling Cascade
In eukaryotic cells, the mitogen-activated protein kinases (MAPK) signaling cascades are widespread, and have been shown to provide cells with the means to integrate environmental signals to control transcriptional responses. In C. albicans, the MAPK pathway is implicated in a number of physiological responses such as growth, cell wall repair, opaque cell mating, stress response, and invasive/filamentous growth. Currently, there are four MAPK signaling
18 cascades known to operate in C. albicans: 1) Cek1- and 2) Cek2- MAPK, 3) Mkc1-MAPK, and 4) HOG-MAPK (57). Among these MAPK cascades, the Cek1-MAPK is the most relevant to filamentous growth, although the Mkc1 has also been implicated in controlling filamentous growth (58). The MAPK signaling cascades are characterized by a conserved module of three distinct kinases: 1) MAPKKK (MAP kinase kinase kinase), 2) MAPKK (MAP kinase kinase), 3) (MAP kinase) (57) (Table 1, Fig. 12).
Table 1. Specific kinases involved in each MAPK signaling cascades
Kinase Cek1-MAPK Cek2-MAPK Mkc1-MAPK HOG-MAPK
MAPKKK STE11 STE11 BCK1 SSK2
MAPKK HST7 HST7 MKK1 PBS2
MAPK CEK1 CEK2 MKC1 HOG1
Fig. 12. Schematic of the Cek1-MAPK pathway.
In response to inducing signals, a series of phosphorylation events occur commencing with the phosphorylation of MAPKKK, which in turn phosphorylates the MAPKK, which then phosphorylates the MAPK (57). The phosphorylated MAPK activates the target transcription factor by phosphorylation to elicit the appropriate physiological response. In general, each of the MAPK signaling cascades has its own specific kinases except for the Cek1- and Cek2- MAPK cascades that share the same MAPKKK and MAPKK (57). The C. albicans Cek1-MAPK cascade is not only involved in filamentous growth, but also in biogenesis and repair of cell
19 wall components, and opaque cell mating (57). The specific MAPKKK, MAPKK, and MAPK kinases participating in Cek1-MAPK cascade are Ste11, Hst7, and Cek1, respectively. The Cek1 target transcription factor is Cph1, which is homologous to S. cerevisiae Ste12 that regulates filamentous (pseudohyphal) growth and mating in S. cerevisiae (58). The Cek1-MAPK signaling cascade can be activated through the Rho-like GTPase Cdc42 that establishes and maintains cellular polarity both in S. cerevisiae and C. albicans (59, 60). Cdc42 is activated by the GEF Cdc24 allowing high–affinity binding of Cdc42-GTP to the Cdc42-binding domain of p21- activated kinase (PAK) Cst20 and Cla4 (51, 61). How GTP-bound Ras1 can activate Cdc42 is not fully understood. Although the Cek1-MAPK signaling cascade functions in the induction of filamentous growth, it is not entirely required for filamentous growth, strains lacking components of the Cek1-MAPK cascade (Cst20, Hst7p, or Cek1) and its target transcription factor, Cph1, still respond to hyphal induction by serum (58, 62). Contrary to cph1Δ/Δ however, a cek1Δ/Δ knockout strain is avirulent in a murine systemic infection model, suggesting that Cek1 has other important functions not restricted to filamentous growth (58). The Cek-MAPK pathway is negatively regulated by Cpp1, a MAPK phosphatase belonging to the VH1 family of phosphatases, that acts on Cek1. Cpp1 is a repressor of hypha-specific gene (SAP 4-6 and HYR1) expression and deletion of CPP1 results in the constitutive derepression of these genes, which results in a hyperfilamentous phenotype even under non-inducing conditions (63).
Adhesion
Adherence to host tissues is often considered as an essential early step in the colonization of different niches. The capacity to adhere to host cells is primarily attributed to cell surface glycoproteins encoded by the ALS (agglutinin-like sequence) genes acting as multi-functional adhesive molecules. The ALS gene family consists of eight genes, ALS1 – ALS7 and ALS9, distributed on three different chromosomes: ALS3 (Chr R), ALS6 and ALS7 (Chr 3), and ALS1, ALS2, ALS4, and ALS5 (Chr 6) (64). The Als proteins are characterized by a conserved N- terminal domain, a central domain-containing tandem repeats of 108-bp units, and a highly variable C-terminal domain that is rich in serine and threonine (64). Some of the Als proteins (e.g., Als1 and Als3) are known downstream effectors of filamentous growth and facilitate adherence and flocculation. Overexpression of ALS1, the first of the ALS genes associated to filamentous growth in C. albicans, causes extensive flocculation and aggregation of cells, which is reminiscent of FLO11 overexpression in S. cerevisiae (65, 66). Interestingly, the importance of Als protein in epithelial adhesion is questioned due to opposing and conflicting results (67). For example, deletion of ALS2 and ALS3 resulted to reduced adhesion in vitro whereas that of ALS5, ALS6, or ALS7 showed the opposite (67). More work is definitely needed to clarify the role of these hypha-associated proteins in promoting virulence of C. albicans.
Another well-characterized adhesin in C. albicans is Hwp1, a hyphae-specific cell surface protein that is covalently linked to cell wall β-glucan by GPI. Hwp1 mediates tight adherence to oral epithelial cells by a mechanism that requires the participation of a specific
20 host cell surface protein. The N-terminal domain of Hwp1 serves as a substrate for mammalian transglutaminases that cross-links this protein to the protein present in the host cell surface (68). Null mutation in HWP1 (hwp1Δ/Δ) impaired the capacity of C. albicans to adhere to oral epithelial cells and displayed reduced virulence in murine systemic infection model (69).
Secretion of hydrolytic enzymes and cytolytic toxins
C. albicans secretes hydrolytic enzymes that facilitate degradation of host cell components for nutrient acquisition and also for defense, which is a common virulence mechanism among pathogens. Among these hydrolytic enzymes are proteinases encoded by the SAP gene family composed of 10-member genes (SAP1-10) and that account for the entire proteolytic activity of C. albicans (35, 70). The SAP1-10 gene products are characterized by sequence motifs typical for all aspartyl proteinase, including two aspartate residues at the catalytic site, and cysteine residues implicated in maintenance of enzyme 3D-structure (35, 70). Each of the SAP genes encodes for a pre-proenzyme that are processed in the secretory pathway, first by signal peptidase and then a Kex2-like proteinase, resulting in mature proteins with molecular weights ranging from 35 to 50 kDa (35, 70). Mature Sap1-8 proteins are secreted, whereas, the remaining two, Sap9 and Sap10, are retained in the membrane as glycophosphotidylinositol (GPI) anchored-proteins. Expression of SAPs are differentially regulated and influenced by the disease stage, and specific to the different morphological forms. For example, in vitro studies have shown that SAP1-3 expression is restricted to the yeast form, whereas SAP4-6 expression is confined to hyphal forms. In addition, Sap proteins are characterized by their discrete and yet overlapping pH optima (Sap1-3, pH 3-5, Sap4-6 at pH 5-7), and substrate specificities that permit degradation of a diverse set of proteins, either generating nutrients (peptides) for growth or for inactivating protective host defense responses. Thus, Saps provide C. albicans a competitive advantage in terms of colonization of particular host niches. Among the Saps, Sap2 is the most flexible in terms of substrate specificity, and the levels of preferred nitrogen sources in the environment tightly regulate its expression (discussed in the SPS section below). Another group of important hydrolases are phospholipases that play an important role in host cell membrane damage and invasion. Currently, there are four phospholipases identified in C. albicans: phospholipases A, B, C, and D. Increased production of phospholipases is often linked to virulence in animal models. Phospholipases are found concentrated at the hyphal tips and do not appear to increase adherence of C. albicans to host cell but rather increase the penetration of C. albicans by damaging the lipid components of the membrane, permitting invasion to other sites (71).
Recently, a novel secretory cytolytic peptide called ‘candidalysin’ was discovered from C. albicans that induces damage to epithelial membranes causing activation of the epithelial immune response and killing of macrophages via NLRP3 inflammasome activation (72-74). The secreted peptide fragment is derived from the Kex2-dependent proteolytic processing of the hyphal specific protein Ece1. Only the third peptide fragment of Ece1 (Ece1-III) is shown to induce damage.
21
Metabolic Regulation of Candida albicans Virulence
C. albicans is part of our normal microbiota. They can colonize virtually all anatomical sites in the body, which is a predisposing event for successful invasion. The opportunistic character of C. albicans suggests that it possesses efficient uptake systems for extracting nutrients from the host. The human host itself can be considered as an excellent nutrient source for C. albicans as each niche within the body offers a sizeable repertoire of readily assimilable nutrients, including sugars, amino acids, small peptides, organic acids, and simple nitrogen-containing compounds such as urea and ammonia (16, 75). However, the levels of these nutrients can vary considerably during colonization and disease progression. Notably, both the host cells living within specific niches, and other members of the endogenous microbiota, are adapted to use and depend on the same set of nutrients for growth. Essentially, fungal cells must compete with host cells for nutrients. When the availability of nutrients in these microenvironments becomes limited, or even depleted, the capacity of this fungus to sense and adapt to engage efficient alternative acquisition strategies to maintain competitiveness is paramount to successful infection. This section focuses on different nutrient acquisition systems known for C. albicans, with a particular focus on carbon- and nitrogen-source sensing.
Carbon assimilation
There are a variety of carbon sources that can be assimilated by C. albicans in the host. Carbon sources that can be used are simple (e.g., glucose, lactose, and galactose) and amino sugars (e.g., N-Acetyl-glucosamine (GlcNAc), a common constituent of glycosylated proteins from the host and a major component of bacterial cell wall peptidoglycan), amino acids, and organic acids (e.g., lactate, acetate, and butyrate). It is commonly accepted that C. albicans is a glucose- insensitive, Crabtree negative yeast. However, several papers including our own (Paper I) suggest that C. albicans is not strictly Crabtree negative; the addition of high glucose (2%) can negatively affect mitochondrial-related functions (e.g., TCA cycle genes, respiration) and triggers the formation of fermentation products like ethanol even when grown under aerobic conditions (76-82). This information needs to be considered carefully as many amino acid catabolic processes like that of proline and glutamate occur in the mitochondria, and that shall be discussed in later part of this thesis.
Of all the carbon sources that C. albicans can access in the host, glucose is preferred and will be the major focus of this section. Contrary to the glucose concentration normally used (2%, ~110 mM) for routine propagation of C. albicans in the lab, the level of glucose in human blood ranges from 0.05% - 0.2% (3-11 mM) and could be even lower in some host niches. Aside from its central role in metabolism, glucose functions both as a ligand for glucose-sensing receptors and as a substrate for glucose transporters localized to the plasma membrane. Glucose sensing in C. albicans occurs via three distinct, yet interconnected mechanisms: the
22 extracellular glucose sensing pathway, the intracellular glucose repression pathway, and the intracellular adenylate cyclase pathway, all of which have been reviewed (51, 83-85).
Extracellular glucose sensing
The presence of extracellular glucose induces the capacity of C. albicans cells to take up glucose. The underlying inducing mechanisms exhibit clear similarities to glucose sensing in S. cerevisiae except for two important differences: 1) Hgt4 is the only orthologue of the two characterized glucose sensors of S. cerevisiae, Snf3 (high affinity) and Rgt2 (low affinity); 2) S. cerevisiae has two transcriptional co-repressors, Std1 and Mth1, whereas C. albicans only has one designated Std1. Briefly, in the absence of glucose, the expression of genes induced by the presence of extracellular glucose is repressed by the Rgt1-Std1 repressor complex, which binds to regulatory motifs in their promoters. Rgt1 binds DNA and Std1 functions as a co-repressor that prevents transcription. Thus, when Rgt1-Std1 are bound together, gene expression is off (Fig. 13). Induction by extracellular glucose relieves the negative regulatory effect of Std1. This is accomplished as follows: extracellular glucose is sensed by Hgt4, a 12-transmembrane protein localized to the plasma membrane that exhibits a high affinity for glucose (micromolar range). This glucose sensor is a unique member of the glucose transporter protein family that binds but does not facilitate glucose uptake. Hgt4 carries a long cytoplasmically oriented C- terminal tails not present in transporters catalyzing glucose uptake. The C-terminal tail is essential for transducing glucose-inducing signals to the downstream repressor factor (reviewed in (51, 83-85)). The C-terminal tail of Hgt4 serves as a scaffold for binding Std1. In the presence of extracellular glucose, Hgt4 undergoes a conformational change that presents Std1 to Yck2, a plasma membrane-localized casein kinase 1, and consequently, Std1 is phosphorylated. The phosphorylated form of Std1 is targeted for ubiquitylation and degradation by the SCFGrr1 ubiquitin-protein ligase complex. As the levels of Std1 becomes depleted, Rgt1 is exposed to phosphorylation by protein kinase A (PKA), which decreases its affinity for DNA binding. Thus, these signaling events lead to the clearing of the repressor complex from the promoters of target genes, enabling their transcription. Examples of regulated genes include hexose transporters (e.g., HGT7, HXT10, and HGT12) and genes involved in glycolysis (HXK2, TYE7, and PFK26) (reviewed in (51, 83-85)). Cells lacking HGT4 (hgt4Δ/Δ) have compromised growth on low glucose, fructose, mannose, and in the presence of antimycin, which specifically inhibits mitochondrial respiratory complex III. In addition, the hgt4Δ/Δ mutant exhibits defective filamentous growth and reduced virulence in a murine systemic infection model (86). In contrary, strains lacking the repressors Std1 (std1Δ/Δ) or Rgt1 (rgt1Δ/Δ) are hyperfilamentous and even capable of suppressing the filamentation defect of hgt4Δ/Δ mutant.
Once taken up, glucose is phosphorylated, which funnels it primarily to glycolysis. In S. cerevisiae, this process is catalyzed by at least three enzymes: hexokinase 1 (Hxk1), hexokinase 2 (Hxk2) and glucokinase 1 (Glk1) with Hxk2 as the predominant kinase. In addition to glucose, these kinases can also phosphorylate other fermentable hexoses like
23 fructose and mannose. The genome of C. albicans encodes at least two hexokinases (HXK1 and HXK2) and two glucokinases (GLK1 and GLK2). C. albicans Hxk1 apparently does not participate in glucose phosphorylation, but instead phosphorylates GlcNAc. Recently, Laurian et al. showed that similar to S. cerevisiae, glucose phosphorylation is performed by Hxk2, and that strain lacking HXK2 have attenuated virulence in both Galleria mellonella and macrophage infection models (87).
Fig. 13. Schematic of glucose sensing by Hgt4. Ref. (86)
Intracellular glucose repression pathway
In S. cerevisiae, the transcriptional repressor Mig1 is mainly responsible for the glucose catabolite repression. Mig is regulated by the Snf1 protein kinase, an AMP kinase. When the intracellular energy is low (AMP/ATP ratio is high), Snf1 is activated through phosphorylation by Sak1, Elm1, and Tos3 (85, 88). Activated Snf1 then deactivates Mig1 through phosphorylation, the phosphorylated form of Mig1 is sequestered in the cytosol preventing the transcriptional repression of genes required for alternative carbon utilization and gluconeogenesis. When glucose becomes available, Snf1 and Mig1 can be simultaneously dephosphorylated by Reg1, which enables Mig1 to re-enter the nucleus and re-exert its transcriptional repressive effect on target genes. Hxk2, the primary glucose-phosphorylating hexokinase in yeast, can form complex with Mig1 preventing its phosphorylation by Snf1. Interestingly, the Snf1 phosphorylation site is lacking in C. albicans Mig1, raising the question regarding the mechanisms mediating its glucose repressive affect in C. albicans. Even more surprising, C. albicans strain lacking MIG1 exhibit no gross defects on either growth phenotypes or virulence, suggesting that glucose repression is dispensable for virulence. However, it should not be discounted that another orthologue of S. cerevisiae Mig1 protein exists in C. albicans (e.g., Mig2). This possiblity remains unexamined; only with this information in hand can the importance (or lack thereof) of intracellular glucose repression pathway in C. albicans virulence be determined.
24 Regulatory Control of Carbon Utilization
In S. cerevisiae, glycolysis is activated by Gcr1 and its co-activator, Gcr2. However, there are no orthologues of these proteins in C. albicans. Instead, glycolysis in this fungal pathogen is controlled by the two key positive regulators, Tye7 and Gal4 (89). In S. cerevisiae, Tye7 also acts as a positive regulator of glycolysis, although to a lesser extent than Gcr1, whereas Gal4 activates the expression of several genes required for galactose utilization (e.g., GAL1, GAL7, and GAL10). In C. albicans, both Tye7 and Gal4 bind promoters and activate the expression of glycolytic genes during growth on fermentable and non-fermentable carbon sources. However, Gal4 appears to exert a more pronounced affect during growth on fermentable carbon sources (89). Strain lacking both TYE7 and GAL4 show severe growth defects on fermentable carbon sources and exhibit attenuated virulence in wax moth and murine systemic infection models (89). Interestingly, in addition to regulating the expression of glycolytic enzymes, Tye7 and Gal4 differentially regulate the expression of distinct sets of carbon metabolic genes. Tye7, for example, activates trehalose, glycogen, and glycerol metabolism, whereas Gal4 activates the expression of at least five genes constituting the pyruvate dehydrogenase complex (e.g., PDB1, PDA1, LAT1, PDX1, and LPD1).
This regulatory rewiring also extends to posttranscriptional circuitry. In S. cerevisiae, utilization of alternative carbon sources is repressed when a preferred carbon source such as glucose is present. This repression, imposed both at the transcriptional and posttranscriptional levels, ensures that the utilization of preferred carbon source is prioritized (88). At the transcriptional level, the expression of genes required for alternative carbon source utilization is repressed through the action mainly of the Mig1 transcriptional repressor, whereas at the posttranscriptional level, the mRNAs are degraded and the enzymes targeted for proteosomal degradation through their specific ubiquitin targeting sites. Interestingly in C. albicans, post- translational control at the protein level is not as pronounced, the enzymes required for the utilization of alternative carbon sources are not subject to enhanced turnover even when glucose is present. This may be explained by the lack of conservation of ubiquitylation sites required to trigger rapid proteasome-dependent degradation (90, 91).
A paradigm example of this is the expression of ICL1, a gene that encodes for the glyoxylate cycle enzyme isocitrate lyase. ICL1 transcription is tightly repressed when cells are grown in the presence of glucose. However, when cells are pregrown on a non-fermentable carbon source where ICL1 is expressed and then subsequently shifted to glucose, ICL1 mRNA is rapidly degraded (90, 91). Thus, the repressive effect of glucose is not at the protein level as the Icl1 protein, which lacks ubiquitylation sites needed to trigger rapid proteasome- dependent degradation (90, 91). The significance of the rather relaxed carbon catabolite repression in C. albicans, due to rewiring of ubiquitylation targets (Fig. 14), is that it provides enhanced metabolic flexibility, allowing for the simultaneous assimilation of glucose and alternative carbon sources. This is believed to give C. albicans a competitive advantage particularly in nutrient-limited niches with highly competitive and complex endogenous microbiota.
25
Fig. 14. Rewiring of ubiquitylation targets allow C. albicans to simultaneously utilize preferred (glucose) and alternative carbon sources. Adapted from: (90)
Nitrogen assimilation
In addition to carbon-based nutrients, C. albicans also require a nitrogen so urce to synthesize the proteins needed to carry out necessary cellular functions and nucleotides for DNA and RNA synthesis. Of the nitrogen sources available in a mammalian host, amino acids are preferred as they can be easily assimilated as both nitrogen and carbon sources (92, 93). In addition to global cross-pathway regulatory networks controling nitrogen source utilization, i.e., general amino acid control (GAAC), nitrogen catabolite repression (NCR), and target of rapamycin (TOR) pathway, extracellular amino acids are sensed by the SPS and Gpr1-Gpa2 sensing pathways. Finally, the Mep1/Mep2 ammonium sensing pathway will be discussed.
Global Crosspathway Regulation
General amino acid control (GAAC)
In response to amino acid starvation, a general response pathway is activated in yeast resulting in increased expression of genes required for amino acid biosynthesis. This global response is controlled by the bZip transcription factor Gcn4 (94, 95). The levels of Gcn4 are regulated at the level of translation in a manner that limits amino acid consumption while the cell is actively synthesizing amino acids (94). GCN4 translation is regulated by the eukaryotic initiation elongation factor 2 (eIF2). Amino acid starvation results in increased levels of uncharged tRNAs. Uncharged tRNAs bind to and activate the Gcn2 kinase, which phosphorylates the α- subunit of eIF2. The phosphorylated eIF2 leads to reduced levels of ternary complex (TC) that reduces the efficiency of protein translation. In S. cerevisiae, Gcn4 regulates more than 500 genes through binding to the promoters containing the consensus UASGCRE sequence motif GAGTCA (94). Like in S. cerevisiae, GCN4-like response has been observed in C. albicans (95). Interestingly, in addition to transcription of genes required for amino acid biosynthesis, Gcn4 is involved in filamentous growth in C. albicans through the Ras1/cAMP/PKA pathway in a mechanism that is not fully understood (95). Nonetheless, these results highlight the role of
26 Gcn4 as a global regulator of both metabolism and filamentation in response to amino acid starvation.
Nitrogen Catabolite Repression (NCR)
Yeast cells have an intricate way of repressing the expression of genes required for the utilization of secondary nitrogen sources when preferred ones are available. This process is known as nitrogen catabolite repression (NCR). In general, ammonia and glutamine are preferred over other nitrogen sources such as proline, arginine, and urea. The genes involved in the utilization of proline (PUT1 and PUT2), arginine (CAR1) and urea (DUR1,2) are subject to repression by NCR. NCR in yeast is controlled by four GATA transcription factors: Gln3, Gat1, Dal80, and Gzf3, which possess zinc-finger DNA-binding motifs recognizing a conserved GATAAG consensus sequence present in the promoter of target genes. Gln3 and Gat1, act as positive regulators of gene expression whereas, Dal80 and Gzf3 act as negative regulators. Gln3 and Gat1 are restricted from entering the nucleus in the cytosol by binding to phosphorylated species of Ure2. Inactivation of Ure2 constitutively derepresses NCR-sensitive genes. Contrary to Gln3 and Gat1, Dal80 and Gzf3 are constitutively localized in the nucleus. Nitrogen regulation through NCR has been described in C. albicans (96, 97). Strains lacking either or both GATA transcription factors, Gln3 and Gat1, are unable to efficiently utilize a number of alternative nitrogen sources due to inability to derepress genes necessary for their catabolic utilization. The functions of Gln3 and Gat1 appear both specific and overlapping depending on the nitrogen source. Work from the Fonzi laboratory has shown that certain amino acids traditionally classified as poor (e.g., proline) in S. cerevisiae (94, 98) were readily utilized by C. albicans lacking the transcription factors, Gln3 and Gat1. Thus, proline utilization does not appear to be under NCR control. Whereas arginine utilization is primarily Gln3 dependent, urea utilization, one of the primary catabolic products of arginine degradation, is only severely impaired in a strain lacking both of these transcription factors. As described earlier, some of the genes required for the utilization of arginine are regulated by factors outside the sphere of NCR. The importance of arginine metabolism is underscored by the fact that when phagocytic cells engulf C. albicans, there is a dramatic increase in the transcription of genes encoding for arginine biosynthesis and catabolism (46). We found that proline utilization is not subject to NCR (Fig. 15).
Fig. 15. Proline utilization is insensitive to NCR. Proline catabolic enzyme (Put2) is still expressed despite growing in the presence of excess preferred nitrogen source (ammonium sulfate) and the lack of the GATA transcription factor, Gln3 and Gat1 (81). The expression of Put2 in the presence of arginine (Arg) and ornithine (Orn) suggest that they are converted to proline.
27
Target of rapamycin (TOR) pathway
When nutrients are abundant, the Ser/Thr kinase Target of Rapamycin (TOR) stimulates anabolic processes and downregulates catabolic processes in order to promote cell growth and proliferation. TOR, which is a member of the PI3-kinase-like protein kinases (PIKK) protein family, was initially discovered in S. cerevisiae through genetic screen look for mutations that confer resistance to the growth inhibitory effect of the macrolide antifungal drug rapamycin (99, 100). There are two known conserved TOR complexes (TORC), TORC1 and TORC2, which are functionally and structurally diverged. Of the two, only TORC1 is sensitive to rapamycin. The TOR complexes are composed of the following key essential components: TOR1 or TOR2 (mTOR in mammals) bound to Kog1 (RAPTOR in mammals), and Lst8 (mLST8) (99, 100). In the yeast S. cerevisiae, TORC1 is activated by amino acids like glutamine and leucine. Cells treated with rapamycin elicit an apparent amino acid starvation response and accompanying arrest of protein synthesis, cell cycle exit and entry to G0, and activation of autophagic processes.
Tuned Regulatory Pathways
SPS extracellular amino acid sensing regulon
Over the past several years, our understanding of the primary sensing mechanism that enables C. albicans to respond to the presence of amino acids in the extracellular environment have largely been defined. Sensing and uptake of amino acids is facilitated by a sophisticated system of amino acid permeases whose expression is linked to a plasma membrane-localized SPS-sensor defined by the three core components Ssy1, Ptr3 and Ssy5 (Fig. 16) (101-103). Upon activation of the membrane-bound sensor Ssy1 (Csy1) by distinct sets of amino acids, the Ssy5 protease cleaves off the N-terminal regulatory domains of the latent cytoplasmic-bound transcription factors, Stp1 and Stp2, which facilitates their efficient translocation to the nucleus where they induce the expression of distinct sets of SPS-regulated genes that facilitate the proper assimilation of external nitrogen source (101). Stp1 regulates the expression of Sap2, the major secreted aspartyl proteinase and multiple oligopeptide transporters that catalyze the uptake of the resulting peptides. STP1 expression is controlled by the GATA transcription factors, Gln3 and Gat1, the positive regulators of NCR(104). STP1 expression is repressed when preferred nitrogen sources are available and derepressed when these nitrogen sources become limited or absent. The second transcription factor, Stp2 is constitutively expressed, and when activated by SPS-sensor signaling derepresses the expression of a subset of amino acid permeases. Csh3, the endoplasmic reticulum (ER) membrane-localized chaperone protein, facilitates the proper folding and functional expression of amino acid permeases, including Ssy1 in the plasma membrane. Strains lacking either Ssy1 or Csh3 fail to respond efficiently to the presence of extracellular amino acids and have impaired capacity to filament in amino acid-based media (102, 103). Although there have been many sets of studies performed to understand the primary sensing mechanism for extracellular amino acids and
28 how it affects this organism’s propensity to undergo morphological switching, little is known about the downstream mechanisms that respond to the presence of these amino acids, and that drive morphological switching. Prior to the work described in this thesis, it is not clear if amino acids induce morphogenesis through direct sensing by the primary plasma membrane- bound Ssy1 sensor, or rather a secondary consequence of due to their internalization. In addition, it remains unclear if the signals from amino acids are transduced through the other signaling pathways known to regulate the yeast-to-filamentous transition in C. albicans. Recent work has implicated Stp2 as being required for the induction of morphogenic switching as it facilitates the uptake of amino acids that are deaminated to form ammonia, which are then extruded to increase extracellular pH (92). This will be discussed in details in the context of the macrophage phagosome.
Fig. 16. Extracellular amino acid sensing in C. albicans is governed by the SPS sensing pathway. The SPS sensor complex in C. albicans is composed of the main plasma membrane-bound sensor Ssy1, the scaffold protein Ptr3, and the protease Ssy5. Stp1 and Stp2 are the effector transcription factors of this pathway. (Left panel, OFF state) In the absence of extracellular amino acids, Stp1 and Stp2 are produced as latent cytoplasmic precursors that are retained in the cytosol due to N-terminal regulatory domains that possess both a cytoplasmic retention motil and nuclear degron, the latter recognized by the putative E3-ubiquitin ligase, Asi3. (Right panel, ON state) In the presence of amino acids, Ssy1 is stabilized in its signaling conformation, which initiates downstream events resulting in the degradation of the inhibitory prodomain of the Ssy5 protease. Thus, activated Ssy5, proteolytically cleaves away the N-terminal regulatory domains of Stp1 and Stp2, facilitating their efficient translocation into the nucleus where they bind upstream activating sequences (UAS) and induce the expression of SPS-regulated genes (SRG). Importantly, and Stp1 and Stp2 induce divergent subsets of genes. Also Stp1 is repressed in cells grown with millimolar concentration of amino acids, whereas Stp2 is constitutively expressed. Activated Stp2 induces expression of amino acid permeases (AAP) genes. AAPs require the assistance of the ER membrane-localized chaperone Csh3 to attain their native structures. In the absence of Csh3, AAPs are retained in the ER. SPS-sensor activated Stp1 derepresses the expression of SAP2, which encodes a secreted protease capable of digesting host proteins, and multiple oligopeptide transporter genes encoding proteins that facilitate uptake of peptides resulting from Sap2 proteolysis. In summary, Stp1 triggers responses for protein utilization whereas Stp2 for amino acid utilization.
29
The Gpr1-Gpa2 Pathway
In yeast, the G protein-coupled receptor Gpr1, and its cognate Gα protein Gpa2, activates cAMP synthesis in response to glucose. Gpr1-initiated signals activate Cyr1 by stimulating GTP-GDP exhange on the Gα protein Gpa2; the active GTP-bound form of Gpa2 is thought to bind to the Gα-binding domain within the N-terminal of Cyr1 leading to enhanced cAMP production (reviewed in (55, 105)). (Fig. 17). Null mutations of the GPR1 or GPA2 orthologues in C. albicans diminish filamentous growth on solid media, but filamentation can be restored by the addition of exogenous cAMP (106). Interestingly, deletion of GPR1 or GPA2 does not affect glucose-induced cAMP signaling, and cells remain responsive to methionine and proline (107, 108). Also, the levels of cAMP in gpr1 null mutants still spike in response to serum or large amounts of glucose (100 mM, or 1.8%), suggesting that Cyr1 can be activated by Gpr1- independent means (108). Gpr1, which has been implicated in amino acid-induced morphogenesis, does not appear to require Ras1. It has been reported that Gpr1 senses the presence of extracellular methionine (108) and glucose (106), however recently, lactate has been proposed to be the primary activating ligand (109). The role of Gpr1 in amino acid- induced morphogenesis remains an open question.
Fig. 17. Gpr1-Gpa2 pathway for amino acid sensing. The G protein-coupled receptor Gpr1 and its cognate Gα protein, Gpa2, activates cAMP synthesis in response to amino acids (i.e., methionine).
The Mep1/Mep2 pathway
Sensing of extracellular ammonium in C. albicans is facilitated by the ammonium permeases Mep1 and Mep2, both containing 11-transmembrane segments. Mep1 and Mep2 are essential for growth in the presence of low ammonium concentrations as sole nitrogen source (110). While both of these permeases facilitate ammonium uptake, Mep2 is a less efficient than Mep1, as determined by ammonium uptake measurements using radioactive methylammonium (110). Interestingly, in addition to its ammonium permease function, Mep2 is reported to be involved in promoting morphogenesis during nitrogen limitation, suggesting that it functions as a transceptor, i.e., combining transport and signaling receptor activities (110). The C- terminal cytoplasmic tail of Mep2, which consists of 74 amino acids, is critical for filamentation but dispensable for ammonium uptake. Of these 74 amino acid residues, only the first 57 residues led to abrogation of filamentation under nitrogen limited condition (111). MEP2
30 expression is regulated by the nitrogen catabolite repression (NCR)-related GATA transcription factors Gln3 and Gat1, which bind to the MEP2 promoter during nitrogen limitation (112). Mep2-dependent filamentous growth is transduced via the MAPK and cAMP- PKA pathway signaling cascades through Ras1 (Fig. 18). This conclusion stems from the fact that a MEP2 hyperactive allele does not rescue the filamentation defect of ras1Δ/Δ or cph1 Δ/Δ efg1 Δ/Δ deletion mutants, but restores filamentation competence when introduced in either cph1 Δ/Δ or efg1 Δ/Δ mutant (110).
Fig. 18. Mep2-mediated filamentation in C. albicans during nitrogen limitation. The C-terminal cytoplasmic tail of Mep2 is required for transducing the signal to either MAPK or cAMP/PKA pathway through Ras1.
The RIM101/pH control pathway
At alkaline-neutral pH, C. albicans activates the RIM101 pathway to activate the genes that are required for adaptation at alkaline pH and at the same time, repress acidic response genes. In addition to alkaline response, the RIM101 pathway is also integrated to a regulatory circuitry that stimulate the morphological transition of C. albicans from yeast to filamentous hyphal form in response to alkaline pH ((113), reviewed in (114, 115)). The RIM101 pathway, which is well-conserved in many yeast and filamentous fungi, is effected by the Cys2His2 zinc-finger transcription factor, Rim101, that is the orthologue of pacC in Aspergillus nidulans (116). In response to alkaline pH, the full-length inactive form of Rim101 is recruited to the endosome where it is activated through proteolytic processing of its C-terminus by the calpain-like cysteine protease, Rim13. Endosomal membrane recruitment of Rim101 is facilitated by Rim20, which acts as an adaptor protein that recognizes and binds the C-terminal inhibitory domain of Rim10. Rim13 on the other hand, is recruited to the endosome by Snf7. The active form Rim101 translocates to the nucleus where it acts both as a transcriptional activator and repressor. In yeast, the upstream sensing component of the RIM101 pathways is thought to be mediated by the sensing complex composed of the Rim21, Dfg16, Rim9, and Rim8 proteins. The first three proteins are localized in the plasma membrane with seven (Rim21 and Dfg16/
31
PalH) and three (Rim9/PalI) transmembrane domains. Rim9/PalI is thought to facilitate the plasma membrane localization of Rim21/Dfg16/PalH (117). The arrestin protein, Rim8, is localized in cytosol directly interacting with the C-terminus of Rim21 and Dfg16/ PalH (116). In response to alkaline pH, the Rim8 orthologue PalF in A. nidulans becomes phosphorylated and ubiquitylated in a strict PalH-dependent manner (118). This activated sensing complex is endocytosed and delivered to the endosome via the multivesicular body (MVB) pathway mediated by ESCRT complexes, which contains Snf7 as a component. Upon loading of the cargo, Snf7 forms a protease complex with Rim101/Rim20 and Rim13, which then facilitates the proteolytic activation of Rim101 (116). C. albicans have all the components of the pathway(113, 119-121), including an additional Rim9 protein that closely resembles that of PalI in A. nidulans due to the presence of C-terminal extension in addition to the Sur7 domain conserved in Rim9 proteins (122). Consequently, these forms are classified as the long and short forms, respectively (122). DNA microarray analysis showed that 514 genes including filamentation genes were affected by shifting C. albicans from pH = 4 to pH = 8 (123). In addition to the RIM101 pathway, a second pH-response pathway mediated by MDS3 has also been shown to regulate C. albicans adaptation to alkaline pH and filamentation mediated through the TOR pathway (124-126). The importance of properly adapting to changes in environmental pH within the host is underscored by fact that cells lacking components of the RIM101 pathway or MDS3 have attenuated virulence in mouse model of systemic infection and keratomycosis (RIM101) ((113, 125, 127), reviewed in (114, 115)).
32 PROLINE METABOLISM
Proline: a unique amino acid
Of the proteogenic amino acids proline is unusual as it has its nitrogen atom covalently locked within a pyrrolidine ring (Fig. 19). The biological function of proline covers a plethora of cellular processes, including osmoregulation, cellular bioenergetics, protein synthesis and structural stabilization, cell signaling, REDOX homeostasis, and stress response (Fig. 19) (Reviewed in: (128, 129)). Proline is one of the most potent inducers of yeast-to-hyphal transitions in C. albicans, ((78, 130-135), reviewed in: (136)), and the results presented in this thesis provide the basis for a mechanistic understanding of how proline induces filamentous growth.
Exogenous proline can be taken up from the host or can be derived from the breakdown of endogenous host proteins. However, due to its distinct structure, proline induces conformational constraints on the peptide bond, protecting it from degradation by most common proteolytic enzymes (137). Therefore, releasing proline completely from peptides for catabolism is thought to have special regulatory and physiological functions. Indeed, the release of proline from peptides is only carried out a by a small subset of a proteases capable of hydrolyzing the bond constrained within the pyrrolidine ring (137). One of the special proteases capable of releasing proline from dipeptides is called prolidase or proline dipeptidase, which hydrolyzes the peptide bond when proline or hydroxyproline is positioned at the C-terminus (137-140). Prolidases are ubiquitious in nature and have been found to exist in both prokaryotes and eukaryotes (137).
Fig. 19. Proline structure and biological functions. Adapted from: Ref. (128)
33
Mitochondria: the site of proline catabolism
Mitochondria are dynamic organelles that have long been known as the power house of eukaryotic cells. Mitochondria produce ATP through oxidative phosphorylation. These organelles are of endosymbiont origin possessing their own genome contained within a double-membrane composed of the outer (OMM) and inner (IMM) mitochondrial membranes. The IMM is folded into finger-like structures called cristae (Fig. 20), which drastically increases the surface area to accommodate the four respiratory electron transport chain (ETC) complexes and the F1/Fo ATPase. The matrix, which contains a gel-like viscous fluid is where many metabolic reactions take place. The proteome that is partially derived from the mitochondrial genome encoding a limited set of about 13 proteins, which are mainly involved in oxidative phosphorylation. Most mitochondrial proteins are nuclear encoded and are imported by the mitochondria recognizing specific mitochondrial targeting sequences (MTS). Beyond ATP production, Fig. 20. Mitochondrial structure. Images was adapted from CUNY, ref. (257). mitochondria participate in many critical cellular functions ranging from ion homeostasis, stress response, cellular REDOX homeostasis, and cell signaling (141-145). Therefore, it is not surprising that mitochondrial dysfunction in humans has been implicated in a broad range of pathologies, including metabolic and cardiovascular disorders, cancer, neurodegeneration, autoimmune diseases, and aging (141- 145). Importantly, human fungal pathogens require mitochondria to obtain energy for virulent growth, underscoring the importance of mitochondria in health and disease
Electron transport chain (ETC)
The ETC is composed of a series of complexes that transfer electrons from reduced electron donors, NADH and FADH2, generated from intermediary metabolism, to electron acceptors via a series of redox reactions generating an electrochemical gradient (proton motive force; PMF) across the IMM (143). The electron transfer initiates from the oxidation of NADH by
Complex I (NADH-ubiquinone oxidoreductase) or FADH2 by Complex II (succinate- ubiquinone oxidoreductase). In both cases, the electrons are transmitted to ubiquinone (CoQ) forming ubiquinol (CoQH2). The electrons from CoQH2 are subsequently transferred to cytochrome c via Complex III (ubiquinol-cytochrome c oxidase reductase). Finally, Complex IV (cytochrome c oxidase) transfers the electrons from reduced cytochrome c to oxygen forming water. The energy released from the REDOX reactions, coordinated by Complexes I, III, and IV, is coupled to the pumping of protons from the matrix to the intermembrane space,
34 which creates a proton gradient across the IMM, i.e., generating the PMF (146). Complex V
(F0F1 ATP synthase) utilizes the PMF to synthesize ATP from ADP and inorganic phosphate (Pi). Together, the four ETC complexes and the ATP synthase catalyze the formation of ATP in the process called oxidative phosphorylation (OXPHOS) (Fig. 21). The ATP produced is exported through a specific carrier, i.e., adenine nucleotide translocator (ANT) that exchanges ATP with ADP. In addition to OXPHOS, the PMF also drives the import of nuclear-encoded mitochondrial proteins and the influx of Ca2+ ions, the latter buffers the calcium concentration in the cytosol. Notably, the mitochondria of the model yeast S. cerevisiae lack Complex I, the oxidation of NADH in yeast is carried out by two alternative peripheral NADH dehydrogenases that are not coupled to proton pumping (147)
Fig. 21. Schematic presentation of OXPHOS. Images was adapted from ref. (143)
Additional Respiratory Pathways in C. albicans
In addition to the well-characterized ETC, C. albicans possesses two additional respiratory pathways, designated the alternative respiratory pathway (AOX) and the parallel electron transport chain (PAR). AOX and PAR are activated when the ETC is inactivated by inhibitors or damaged (148). The AOX pathway, which is absent in mammals and yeast S. cerevisiae, relies upon two nuclear-encoded and cyanide-resistant alternative oxidases Aox1 and Aox2 (149, 150). Both Aox1 and Aox2 localize to the IMM on the matrix face and catalyze the transfer of electrons directly from ubiquinol (CoQH2) to oxygen, thus bypassing Complex III and IV (151). Aox1 is constitutively expressed even at low abundance whereas Aox2 can be induced upon inhibition of ETC by cyanide or antimycin A, or upon treatment with oxidative stressors (e.g., paraquat, hydrogen peroxide and menadione)(149, 150). Importantly, although the alternative oxidases do not directly contribute to energizing the mitochondrial membrane (152), the AOX pathway working in conjunction with Complex I (NADH-ubiquinone oxidoreductase) facilitates ATP formation due to the proton pumping capacity of Complex I (153). Thus, the AOX pathway is capable of supporting biosynthetic processes when classical respiration is inhibited. This alternative pathway is inhibited by salicylhydroxamic acid (SHAM) (Fig.
35
22)(151). Notably, all fungi possessing alternative oxidases also possess Complex I; in the absence of Complex I, the AOX pathway would result in the complete uncoupling of mitochondrial energy production.
The PAR pathway is activated when both the ETC and AOX pathways are inhibited. Here the electrons from NADH formed by reactions within the cytosol are directed to the reduction of ubiquinone by alternative NADH dehydrogenases. These dehydrogenases use FAD as a prosthetic group and are found localized to either the outer or inner face of the IMM. The PAR dehydrogenases on the matrix side oxidize NADH after it is transported across the IMM. In contrast to Complex I, the PAR dehydrogenases reduce ubiquinone in a reaction that is not coupled to proton translocation. Thus, PAR only bypasses Complex I and functions in parallel to the first step of the ETC.
Fig. 22. Alternative respiration in C. albicans. The alternative respiratory pathway is activated upon treating cells with the ETC inhibitors, cyanide (KCN, top left) and antimycin A (top right) that can be inhibited using SHAM. Images adapted from ref. (148, 152).
Mitochondria and ROS production
In aerobic organisms, respiration is not entirely efficient. Thus, actively respiring cells often generate reactive oxygen species (ROS) as a by-product of the electron transport reactions leading to the reduction of molecular oxygen (O2). ROS are not always deleterious, nor all antioxidants good. Although normally thought to be merely toxic by-products of metabolism, ROS are now recognized to have a critical role in cellular physiology by acting as a signaling molecule in gene regulation and stress response (144, 154, 155). Oxidative stress arises in the cell when the capacity to clear excess ROS by the cells own antioxidant defenses becomes limiting, resulting in local and general cellular damage. Since respiration occurs in the
36 mitochondria, it is not surprising that majority of cellular ROS originates from this organelle •- (156). Superoxide (O2 ) is the most abundant and proximal mitochondrial ROS species produced by mitochondria (154). It can lead to formation of other ROS species like hydroxyl radical (•OH), hydrogen peroxide (H2O2), peroxynitrite (ONOO-), and singlet oxygen (1O2). Superoxide and its hydroxyl radical derivative are true radicals in the sense that they contain an unpaired electron. The other ROS species, hydrogen peroxide, peroxynitrite, and singlet oxygen, are highly reactive non-radical derivatives of superoxide. Superoxide in the mitochondria is believed to be primarily produced by electron transfer reactions catalyzed by complex I and III (154). Superoxide generated by complex I are mainly released to the mitochondrial matrix whereas those produced at ubiquinol oxidation sites of complex III can be released in either the matrix or the inner membrane space (Fig. 23) (156, 157). Since superoxide anions are charged, they cannot readily diffuse across membranes; this explains why the deleterious effects of superoxide anions are often restricted to the site where they are produced. However, they can move to one compartment to another. In mammalian cells, superoxide can exit the mitochondria and gain entry to the cytosol via the voltage-dependent •- anion channel (VDAC) (158, 159). O2 is highly reactive and can effectively disrupt the functions of many essential regulatory and structural proteins that contain iron-sulfur clusters •- (Fe/S clusters) (155, 160). Therefore, O2 must be converted quickly to H2O2 in a dismutation reaction catalyzed by the enzyme superoxide dismutase (SOD)(154, 155, 160). In eukaryotes, •- the disproportionation of mitochondrial-generated O2 to H2O2 is mainly performed by the mitochondrial-localized and Mn-dependent SOD2 isoform (155).
.− Fig. 23. Superoxide (O2 ) production in the mitochondria. In normal respiration, molecular oxygen (O2) acts as the final electron acceptor at complex IV. However, O2 can also be prematurely reduced at Complexes I and III leading to the superoxide generation. Unlike the superoxide produced in Complex I, which is mainly released in the matrix lumen, superoxide from Complex III can be released on either side of the inner membrane.
•- Compared to O2 , H2O2 is membrane permeable and generally not very reactive, but still is toxic (160). Under optimal respiratory conditions, it is estimated that the ETC generates •- approximately 1-2% O2 and H2O2 per water molecule formed. On its own, H2O2 exerts its 37 effect through negative regulation of many regulatory enzymes by reacting with oxidant-labile Fe/S clusters and certain amino acid residues. However, the primary reason why the level of
H2O2 in the cell must be efficiently controlled by enzymes like catalase and glutathione peroxidase is that it can quickly react with transition metals to generate the highly toxic hydroxyl radical (•OH) in a process known as the Fenton reaction (155, 160). •OH is very reactive and can cause damage to cells; it reacts with macromolecules like nucleic acids, protein, and lipids, in the process creating additional organic peroxides that enhance damage (160, 161). Furthermore, •OH cannot be cleared enzymatically and therefore its accumulation can have deleterious consequences for the cell (160, 161).
Mitochondria as an antifungal drug target?
Recognition of the role of mitochondria in virulence of C. albicans has increased in recent years, yet this field remains under explored. In the plant field, significant advances have been made as drugs that target ETC complexes already exist to treat plant fungal diseases. The potential of using mitochondria as a target for antifungal drug development against C. albicans is very promising given that several independent studies have collectively shown that there is a strong link between mitochondrial function and virulence. In general, there is a reduction in virulence, or at least reduction in the expression of virulence traits in strains bearing genetic mutations that give rise to defects in mitochondrial structure or mitochondrial-related functions. For example, strains lacking NDH51 encoding a component of Complex I have a striking defect in forming hyphae at 37 °C and low glucose levels, conditions that should normally facilitate robust hyphal growth (162). The Goa1 protein, which associates with Complex I during stress, has been shown to be essential for full virulence in a murine systemic infection model (163). Additional subunits of Complex I, Nuo1 and Nuo2, were shown as well to be important for virulence in a murine systemic infection model (164). Cells lacking the mitochondrial GTPase Gem1 (gem1Δ/Δ) that interacts with the ER-Mitochondria Encounter Structure (ERMES), which supports direct physical contacts between the ER and mitochondria, was hypovirulent in a worm (Caenorhabditis elegans) infection model. In addition to virulence, the role of mitochondria in antifungal drug resistance is also interesting. Accumulating evidence suggests that mutants lacking one component of Complex I exhibit reduce tolerance to azole drugs. Clearly, the mitochondria is an interesting organelle that can be exploited for drug development.
Proline Metabolism: Catabolism and Biosynthesis
The four-electron catabolic conversion of proline to glutamate is carried out by the successive actions of proline dehydrogenase (PRODH; EC 1.5.5.2) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH; EC 1.2.1.88). PRODH and P5CDH are highly conserved enzymes throughout eukaryotes and bacteria (Fig. 24). PRODH shares a catalytic core domain of a
38 distorted (αβ)8 barrel domain, which is also known as the TIM (triosephosphate isomerase) barrel domain (165). P5CDH, on the other hand, belongs to the ALDH (aldehyde dehydrogenase) superfamily characterized by the classical ALDH fold, consisting of a NAD+- binding domain, catalytic domain and dimerization domain (165). In eukaryotes, PRODH and P5CDH are nuclear-encoded mitochondrial proteins that are imported to the mitochondrial matrix. PRODH remains associated with the IMM. In Gram-positive bacteria, PRODH is peripherally attached to the cytoplasmic membrane, whereas P5CDH is localized in the cytosol (165). Interestingly, in Gram-negative bacteria, proline utilization is carried out by PRODH and P5CDH fused into a single bifunctional protein named proline utilization A (PutA) (166). PutA is comprised of 1000-1300 amino acids and has the PRODH domain linked N-terminally to the P5CDH domain (166). In some Gram-negative bacteria, such as Escherichia coli, PutA acquires an additional transcriptional repressor function making it a “trifunctional” protein. This regulatory function is due to the presence of a DNA-binding domain (ribbon- helix-helix motif) at its N-terminus. The E. coli PutA autoregulates its own expression and of putP (a high affinity, Na+ proline transporter) (166).
Fig. 24. Phylogenetic tree illustrating the diversity and structures of proline catabolic enzymes in bacteria and eukaryotes. Branches 1 and 2 correspond to PutAs, which are bifunctional enzymes found only in bacteria, whereas Branch 3 correspond to monofunctional enzymes found in both bacteria and eukaryotes. The structures shown in Branch 1 correspond to the DNA-binding domain (left) and PRODH domain (right) of PutA found in E. coli, whereas structures illustrated in Branch 3B correspond to Thermus aquaticus PRODH (top) and P5CDH (bottom). Image was adapted from (167).
Proline catabolism to glutamate (Fig. 25) starts with the enzymatic oxidation of proline to P5C by PRODH which contains a non-covalently bound FAD as cofactor (for latest reviews on proline metabolism, refer to: (129, 165, 168)). In eukaryotes, cytosolic proline, derived either
39 from biosynthetic reactions or from catabolism of arginine or ornithine, is imported into the mitochondria via a currently unidentified mitochondrial transporter. PRODH transfers two electrons from proline to FAD to generate P5C and the reduced flavin cofactor (FADH2). P5C tautomerizes spontaneously in a non-enzymatic reaction forming glutamic-γ-semialdehyde (GSA). The prevailing pH strongly affects the equilibrium between P5C and GSA; P5C formation is favored when the pH is > 6.5. P5CDH catalyzes the oxidation of GSA to glutamate reducing NAD+ to NADH. Glutamate is subsequently enzymatically converted by glutamate dehydrogenase (GDH EC 1.4.1.2) to α-ketoglutarate (α-KG) and ammonia (NH3), a reaction that also requires NAD+ as a co-factor (81, 129). α-KG then enters the TCA cycle as a key intermediate. The reduced cofactors (FADH2 and NADH) generated by proline catabolism are oxidized by the ETC of mitochondria or of the cytoplasmic membrane of prokaryotes to generate ATP. PRODH, which is tethered in the inner mitochondrial membrane, directly couples the oxidation of proline to the reduction of ubiquinone (CoQ) in the ETC. One molecule of proline can be completely oxidized to generate approximately 30 ATP equivalents (169, 170), reinforcing the idea that proline can be an important energy source for the fungal cell, especially under nutrient-limited conditions.
Fig. 25. Overview of proline metabolism. Refer to the text for details. The following enzymes are present in C. albicans genome - PRODH = PUT1 (C5_02600W), P5CDH = PUT2 (C5_04880C), GDH = GDH2 (C2_07900W), P5CR = PRO3 (C4_00240), OAT = CAR2 (C4_00160C), ARG = CAR1(C5_04490C), GK = PRO1 (CR_10580), GPR = PRO2 (C3_07220C)
Proline can be synthesized in the cytosol from glutamate (Fig. 25) via the sequential action of the P5C synthase (P5CS) and P5C reductase (P5CR, EC 1.5.1.2) (for latest reviews on
40 proline metabolism, refer to: (129, 165, 168)). In mammalian cells, P5CS, which catalyzes the rate-limiting step of proline biosynthesis, is a bifunctional enzyme carrying both γ-glutamyl kinase (GK, EC 2.7.2.11) and γ-glutamyl phosphate reductase (GPR, EC 1.2.1.41) activities. In prokaryotes and yeast, GK and GPR are separate monofunctional enzymes homologous to the GK and GPR domains of P5CS, respectively. The conversion of glutamate to GSA by P5CS requires ATP and NADPH as cofactor. GSA tautomerizes into P5C in a spontaneous cyclization process, which is then reduced to proline by cytosol-localized P5CR, a reaction coupled to oxidation of NADPH. Alternatively, proline can be derived from ornithine via the catalytic activity of ornithine-δ-aminotransferase (OAT, EC 2.6.1.13). Ornithine is converted into GSA in a reaction that transfers the δ-amino group from ornithine to α-KG yielding, in addition to GSA, glutamate. In S. cerevisiae, OAT (Car2) is found in the cytosol whereas in other eukaryotes, like mammals and plants, OAT is localized to the mitochondria. As shown in Fig. 25, P5C, which is in tautomeric equilibrium with GSA, is a strategic intermediary metabolite that acts as an essential carbon bridge in the cycling of glutamate and P5C in and out of mitochondria, respectively, and the tricarboxylic acid cycle.
Proline-P5C Cycling
Proline-P5C cycling in and out of mitochondria has been well documented in plants and animals. As proline is catabolized in the mitochondria and the activity of PRODH exceeds that of P5CDH, P5C transiently accumulates and is transported out to the cytoplasm via an as yet to be defined carrier protein. In the cytoplasm, P5C is reconverted to proline by P5CR, a reaction driven by the coupled oxidation of NADPH. Collectively, this process is known as the Proline-P5C cycle (Fig. 25). An important aspect of this cycle is that it enhances the production of ROS, which is thought to result from a combination of two factors. The first, is the result of increased PRODH activity leading to an increased flux through the ETC, which enhances ROS production. The importance of proline as a substrate for ROS formation is supported by several studies showing increased ROS formation when proline is used as substrate by isolated Drosophila mitochondria (171). The levels of ROS produced was found to be proportional to the amount of proline used in the experiments. Secondly, P5C is inherently toxic. Genetic modifications that down-regulate or completely disrupt P5CDH function, or that lead to the overexpression of PRODH activity, result in the accumulation of P5C. When proline is made available, these modifications induce cell death through increased ROS production in diverse organisms, including bacteria, yeast, plants, and mammals (172). Disruption of P5CDH activity in plants results in the hypersensitivity response (HR) towards excess proline and elevated ROS production. Additional examples are that yeast S. cerevisiae and human fungal pathogen Cryptococcus neoformans lacking P5CDH (put2Δ) exhibit enhanced ROS production and arrested growth when exogenous proline is added to culture media (173, 174). Similar observations of increasing ROS formation were observed in Arabidopsis and Tobacco plants (175). Consistently, plant and yeast cells exposed to P5C, exhibit a hypersensitivity phenotype resulting in enhanced ROS formation and cell death (174, 176).
41
Proline catabolism in C. albicans
Accumulating evidence implicates proline in the virulence of some human pathogens like Staphylococcus aureus (177, 178), Helicobacter pylori(179), Mycobacterium tuberculosis (180, 181), Cryptococcus neoformans(173), Trypanosoma cruzi (182-188), and even in the pathogenesis of cancers (129, 168, 189-192). It is therefore tempting to speculate that proline has a more universal role in human diseases. The human fungal pathogen C. albicans can utilize proline and therefore is able to express catabolic enzymes for this purpose. The genome of C. albicans encodes PUT1 (C5_02600W) and PUT2 (C5_04880C). Similar to the yeast S. cerevisiae, PUT1 encodes for the PRODH, whereas PUT2 encodes for P5CDH (193, 194). Using a Put2-GFP reporter strain, we confirmed that C. albicans Put2 localizes to the mitochondria (Fig. 26a). Put2 protein is constitutively expressed in low abundance, but is hyperinduced in the presence of proline (Fig. 26b). As in S. cerevisiae, the induction is dependent upon the transcription factor Put3 (PUT3, C1_07020C), which positively regulates the expression of PUT1 and PUT2 genes in the presence of proline (195-197). Inactivation of these genes using CRISPR/Cas9 abolished the growth of put1-/-, put2-/-, put1-/- put2-/- mutants in minimal media containing proline as the sole nitrogen source Fig. 26c. The growth of put3-/- was severely impaired but not completely abolished, which is consistent with the basal level of expression we observed (Fig. 26b). In summary, we identified all the components of the proline catabolic pathway in C. albicans that is a main focus of this thesis. a c
b
Fig. 26. Proline catabolism in C. albicans. a) C. albicans Put2 (P5CDH) is localized in the mitochondria. Confocal images of Put2-GFP signal colocalizing with the mitochondrial marker, Mitotracker Red; b) Put2 induction by proline is Put3-dependent. (top) Schematic diagram of Put3-dependent induction of PUT1/PUT2 expression in the presence of proline; (bottom) cells expressing Put2-GFP were grown to log phase in SD medium at 30°C before spiking with 10 mM of proline (or ddH2O for uninduced control) and then harvested for whole cell protein extraction after 1 h. Put2-GFP and α-tubulin (loading control) were probed by immunoblotting. c) Proline utilization is strictly dependent on PUT1 and PUT2. Cells of the indicated strains from YPD pre-culture were collected, washed, and then adjusted to OD600 = 1 before making a 10-fold serial dilution in ddH2O. Five microliters were spotted on buffered minimal media containing the indicated nitrogen source and 2% glucose as carbon source and then incubated for 3-4 days at 30°C. Growth in YPD was used as non-selective growth control.
42 C. albicans interactions with innate immune cells
Pathogen Recognition
Phagocytes such as macrophages, neutrophils, and dendritic cells are among the first line of host defenses encountered by C. albicans, and believed to be the most effective in controlling and clearing C. albicans infections (reviewed in (198, 199)). Innate immune cells recognize specific fungal surface antigens, collectively known as pathogen-associated molecular patterns (PAMPs), via specific plasma membrane-localized pattern recognition receptors (PRR) (200, 201) (Fig. 27). Pathogen recognition triggers the activation of intracellular signaling cascades that result in the production of proinflammatory cytokines that are crucial to both innate and adaptive immune responses (reviewed in (199, 202, 203)). The Toll-like receptors (TLRs) and the C-type lectin receptors (CLRs) are well-studied PRRs that recognize C. albicans PAMPs. TLRs, characterized by their leucine rich repeats (LRR) domains, can be cell membrane- associated such as TLR-4 and TLR-2, which recognize mannans on the cell wall of C. albicans, or intracellular receptors such as TLR-9, which senses fungal DNA (reviewed in (199, 201- 204)). TLR-4 specifically recognizes the short linear O-linked mannans resulting to production of cytokines like IL-8 and tumour necrosis factor (TNF) whereas TLR-2 recognizes phospholipomannan leading to production of transforming growth factor β (TGF-β), IL-10, and TNF cytokines (199). The role of TLR-4 in host defense against C. albicans infection is controversial, and only TLR-2 was shown to be significantly involved in eliciting an antifungal response (199).
C-type lectin receptors (CLRs), represent the second major family of PRRs that recognize C. albicans PAMPs, mainly polysaccharide structures. CCRs are mainly found on the membranes of antigen-presenting cells (APC) and are characterized by their C-type lectin domain (CLD). Dectin-1 recognizes β-glucan resulting in the production of IL-10 and IL-2 in macrophages and IL-23 and IL-6 in dendritic cells, whereas the mannose receptor (MR) in both macrophage and dendritic cells recognize N-linked mannan resulting in the production of proinflammatory cytokines, such as TNF and IL-1β (reviewed in (199, 201-204)). Interestingly, C. albicans can exploit strategies to avoid recognition by phagocytes through shielding of their PAMPs, such as β-glucan, by the outer cell components from recognition by Dectin-1, and thus prevent phagocytosis and cytokine release (205). Exposing the β-glucans in C. albicans via heat- killing, which disrupts cell wall architecture, enhances cytokine production in human monocytes (206).
In addition to plasma membrane-bound TLRs and CLRs that recognize extracellular fungal PAMPs, mammalian host defense has additional intracellular recognition receptors of the NOD-like receptor (NLR) family characterized by LRR and a nucleotide-binding domain. One of these NLRs is the NLRP3 (NLR family pyrin domain containing 3) inflammasome, a large multi-protein complex that activates caspase-1 upon recognition of fungal PAMPs
43 resulting in the processing and secretion of the proinflammatory cytokines, IL-1β and IL-18 (reviewed in (203, 207)).
Fig. 27. Pathogen recognition by innate immune cells. Main pattern recognition receptors (PRRs) in innate immune cells for the specific recognition of C. albicans pathogen-associated molecular patterns (PAMPs). Image adapted from Ref. (201)
Phagocytic cell types
Neutrophils
Polymorphonuclear leukocytes (PMNs), or simply neutrophils, are the most abundant leukocytes in circulation. PMNs play a critical role as an effector immune cell in controlling and clearing both mucosal and disseminated fungal infections (208). Patients with neutropenia or neutrophil-linked defects such as those with chronic granulomatous disease (CGD) are more susceptible to invasive fungal infections (208, 209). PMNs are recruited to the site of infection through proinflammatory cytokines, such as IL-6, IL-8, and TNF-α, that are produced by inflamed tissues or through pathogen recognition (reviewed in (201)). In addition to these proinflammatory cytokines, the hematopoietic growth factors, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM- CSF), are also important in recruiting and activating PMNs (reviewed in (201)). PMNs are able to kill C. albicans both intra- and extracellularly, and have been shown to be the only immune cell type in the blood to inhibit hyphal formation (210) . Phagocytosed fungal cells are efficiently killed intracellularly by fusion of the phagosome with the lysosome to form the mature phagolysosome (211, 212). In addition to intracellular killing, extracellular killing of fungal cells has been reported specifically for PMN. Extracellular killing is mediated through the
44 release of neutrophil extracellular traps (NETs), which have potent antifungal properties due to the presence of calprotectin, which is active in the presence of oxygen (213, 214). Recent transcriptomic analysis revealed that proline catabolism, which is the subject of this thesis, is activated in C. albicans cells during interaction with neutrophils (215) and therefore, it is of keen interest to determine whether proline catabolism is essential for C. albicans survival during interaction with neutrophils.
Dendritic cells (DCs)
DCs are a heterogenous population of professional antigen-presenting cells, which play an important role in bridging innate and adaptive immunity (reviewed in (199, 201-203)). DCs continuously monitor and patrol the skin, tissues, and mucosal surfaces and phagocytize pathogens like C. albicans. The strategic distribution of DC places them at prime positions to initiate adaptive immune responses against C. albicans. Phagocytosis depends on PAMP recognition via the MR and DC-SIGN PRRs (reviewed in (199, 201-203)). Following uptake, DCs process the pathogen and undergo a maturation process that leads to the expression on the cell surface of the major histocompatibility complex (MHC) containing pathogen-specific antigens. Mature DCs leave the peripheral tissues to migrate to the secondary lymphoid organs where they present the antigens to circulating naïve T-cells, priming effector T-cell response. DCs can specifically discriminate between the yeast and hyphal forms of C. albicans and can prime distinct T helper cells for differentiation. For example, ingestion of yeast form favors Th1 differentiation whereas ingestion of hyphal forms favors Th2 differentiation (216). The unique ability of DCs to initiate specific adaptive T cell response based on the recognition of different fungal morphologies of C. albicans, makes DCs attractive targets for antifungal vaccine development.
Monocytes/macrophages.
Monocytes/macrophages are essential components of innate immunity. They provide a defensive function against pathogens like C. albicans through ingestion and intracellular killing and through secretion of both pro-inflammatory and antimicrobial mediators (reviewed in (199, 201-203, 217)). When their capacity to clear infection is overwhelmed, macrophages orchestrate an inflammatory response mediated through PRR, a response aimed at recruiting other cell types to control the infection. Once an infection is controlled, macrophages downregulate the inflammatory response to minimize further tissue damage. Monocytes are generated from bone marrow haemopoietic stem cells and released to the bloodstream where they migrate to peripheral tissues to differentiate into macrophages in response to CSF1 and GM-CSF (217). They can also develop into distinct functional phenotypes in a process known as macrophage polarization. Blood monocytes replenish resident macrophage populations with high turnover. Depending on the presence of specific cytokines, macrophages can be polarized into classically activated (M1) and alternatively activated (M2) macrophages. M2 macrophages can be further divided into four subcategories (M2a, M2b, M2c, and M2d) diverging with respect to function, secreted cytokines, and cell surface markers. M1 macrophages are
45 polarized by lipopolysaccharide (LPS) and Th1 cytokines, such as IFN-γ and TNF-α, whereas M2 macrophages are polarized by IL-4, IL-13, IL-10, IL-33, and TGF-β(218). Interestingly, human M1 and M2 macrophages have contrasting phagosomal pH: M2 phagosomes are more acidic than M1 phagosomes. The differences in acidification rates are the consequences of delays in the fusion with endosomes and lysosomes in M1 (219) Interestingly, macrophages can enhance their capacity to clear re-infection by C. albicans and provide cross protection against other pathogens through an epigenetic reprogramming event that increases its proinflammatory cytokine release upon secondary exposure to β-glucan and chitin in a process known as trained immunity (TI) (220-224).
The Phagosome: The kill zone
Upon recognition of fungal cells by PRR, a signaling cascade in macrophages is activated that results to actin remodeling. This process leads to the formation of a phagocytic cup surrounding the fungal cell. Even at this early stage of phagosome formation, the NADPH oxidase complex, which generates the superoxide burst, can already be observed at the phagocytic cup (reviewed in (200, 225)). Fungal cells are thrust into a hostile microenvironment as they become surrounded by membrane protrusions simultaneously forming the phagosomal compartment (Fig. 28). Phagosomal maturation involves interaction with many intracellular organelles resulting in a compartment that is replete with potent hydrolytic enzymes, oxidative and nitrossative stressors, cationic peptides, and in macrophages, an acidic pH ((200, 219, 225, 226)). Among the innate immune cells, the macrophage, by virtue of the progressive acidification that occurs in the phagosome during maturation, is dedicated to fully destroying phagocytosed fungal cells. The acidification of the phagosomal lumen occurs as a result of the proton (H+)-pumping action of V-ATPases that are recruited by fusion with endosomes and lysosomes. Acidification enables the activation of acidophilic hydrolytic enzymes, which promote and enhance fungal killing. In addition to the low pH, the phagosomal milieu is thought to represent a nutrient poor environment. Consistently, the transcriptional state of C. albicans cells within phagosomes is characterized by derepression of genes involved in gluconeogenesis and the expression of oligopeptide transporters, respectively (46), hallmarks of low nutrient conditions. Furthermore, phagocytosed C. albicans cells exhibit increased expression of both arginine biosynthesis and catabolic genes (46, 227). Based on the pattern of gene expression, it was proposed that urea, one of the catabolic by-products of arginine, is converted to CO2, a key stimulatory molecule for the induction of hyphal formation (227). Interestingly, the expression pattern implicated the enzyme Dur1,2 in catalyzing environmental alkalization, providing an additional signal for hyphal formation (See next section).
46
Fig. 28. Phagosome maturation. Macrophages recognize fungal cells through their cell surface PRR. Once recognition takes place, a fungal cell is enclosed by membrane protrusions forming the phagosome, which matures by the fusion of endosomal pathway-derived vesicles, and consequently becomes highly acidic due to the proton pumping activity of V-ATPases. Inside the phagosome, the fungal cell is subject to harsh conditions due the presence oxidative and nitrosative stressors, low pH, and potent hydrolytic enzymes, which facilitate its killing.
Phagosome evasion mechanism
Although a phagocytized C. albicans cell is thrust into a hostile microenvironment evolved to destroy it, fungal cells can in some instances survive and evade killing, as summarized in Fig. 29. Hyphal formation is the best characterized mechanism for escaping from macrophages. The elongating fungal cells eventually disrupt the macrophage membrane resulting in lysis. However, not all macrophage cell death results from mechanical lysis, rather hyphae formation can also induce pyroptosis, a type of lytic (necrotic) programmed cell death that results to the release of intracellular components, including the inflammatory cytokines interleukin-1β (IL-1β) and IL 8, which trigger a potent inflammatory response (reviewed in (198, 228-230)). Pyroptosis is fundamentally distinct from other types of cell death like apoptosis (programmed, non-lytic, and non-inflammatory) and necroptosis (programmed, lytic, and inflammatory) due to its exclusive dependence on Caspase 1 for activation of the NLRP3 inflammasome((231), reviewed in (198, 228)). Recently, it was shown that in addition to pyroptosis, C. albicans can kill macrophages at a later time during the infection process via glucose depletion (232). Though hyphae can mediate macrophage killing via pyroptosis and glucose depletion, physical disruption through mechanical piercing of growing hyphae is the primary mechanism by which C. albicans escape the macrophage (reviewed in (198, 204)). Interestingly, not all mechanisms used by C. albicans to escape macrophage result to killing; for example, fungal cells can facilitate their own expulsion from macrophage keeping the host cell intact in a process known as vomocytosis (233). Fungal cells inside the phagosome can also exploit an array of protective mechanisms to directly neutralize and counteract various stresses, such as the expression of superoxide dismutases (SODs) on their surface that convert
47
• the reactive superoxide (O2 −) to the less harmful H2O2, which can then be converted to H2O by catalase (234, 235).
Fig. 29. Evasion mechanism of C. albicans from macrophage. Image adapted from ref. (18)
How hyphal formation is initiated upon phagocytosis is still poorly understood. An interesting mechanism proposed for C. albicans is via ammonia (NH3) extrusion, which has been proposed to promote morphogenesis by neutralizing the acidic phagosomal lumen. Alkalization is also advantageous due to the inhibition pH-sensitive proteolytic enzymes (92, 93). Ammonia is thought to be produced through deamination of amino acids taken up by plasma membrane-localized permeases. Permease expression is regulated by the SPS (Ssy1- Ptr3-Ssy5) amino acid sensing regulon, which controls the promoter binding capacity of the Stp2 transcription factor (Refer to pp. for review) (92, 101). C. albicans cells lacking Stp2 cannot derepress a subset of amino acid permeases and fail to form hyphae and fail to escape from macrophages. These findings are consistent with the suggestion that phagosomal alkalization is dependent upon amino acids (92). Alternatively, ammonia formation has been proposed to be catalyzed by the enzyme urea amidolyase (Dur1,2), which is localized in the cytosol. The ammonia released by this reaction is postulated to be exported into the phagosome via the Ato proteins. In both scenarios, the extruded ammonia effectively neutralizes the protons pumped into the phagosome by the V-ATPase (92, 93, 236). These possibilities are not compatible with the notion that the phagosome represents a nutrient poor environment.
Recent work by Westman et al. (2018) contradicts the notion of ammonia-dependent alkalization of the phagosome. Using dual-wavelength ratiometric imaging to measure phagosomal pH, alkalization appears to be the consequence of the transient and reversible rupture of phagosomal membrane during hyphal expansion. Thus, alkalization is not the stimulus that triggers hyphal initiation (237). The authors of this paper also questioned the role of alkalization based on calculations showing that the rate of ammonia extrusion by C. albicans is almost 100-fold lower than the capacity of the V-ATPase to pump-in protons into the lumen, suggesting that at least theoretically, the phagosomal microenvironment will remain acidic if the phagosomal compartment remains intact. Also, the data convincingly
48 demonstrated that hyphal formation starts prior to a measurable change in pH when the phagosomal compartment is still relatively acidic (pH ~ 4.5-5). This effect on phagosomal alkalization seems to be restricted to filamenting cells as even the non-filamenting but replication-competent mutant cph1Δ/Δ efg1 Δ/Δ failed to alkalinize the phagosome unless treated with GPN (glycyl-L-phenylalanine-beta-naphthylamide), a drug that causes the permeabilization of the phagosome via osmotic swelling. It is possible that phagosomal size and integrity are both maintained despite replicating. This is consistent with our findings using time-lapse live cell imaging (Fig. 30). In summary, emerging data clearly suggest that ammonia derived from metabolism does not provide the stimulus that triggers hyphal formation in the phagosome.
Fig. 30. Phagosome stretching (yellow broken line) as a C. albicans cph1Δ/Δ efg1Δ/Δ cell constitutively expressing RFP (RED) replicates inside RAW264.7 macrophage. The phagosomal membrane tightly surrounds a cph1Δ/Δ efg1Δ/Δ put1-/- expressing Put2-GFP (Green). The inactivation of PUT1 prevents the utilization of proline (Silao, unpublished data).
Many studies that apparently demonstrate the inability of null mutant strains to escape from phagosomes of macrophages rely on endpoint microscopy and fixed cells. Often, the growth defects reported are general in nature, meaning the cells had already acquired a defect as a result of the precise genetic modification made and is not in any way linked to the phagosome-specific response. This in effect creates an obscured picture of the real phagosomal microenvironment. Furthermore, germ tube formation in the phagosome is very quick, occurring 15-20 min after phagocytosis, which is not captured in most endpoint studies. The macrophage cell culture media also represents a complicating factor to control as the media effectively induces hyphal growth of C. albicans. Therefore, in the microscopy studies documented in this thesis, fixed cell microscopic examinations were complemented with timelapse microscopy in order to precisely follow hyphal induction of C. albicans during co- culture with macrophages. The initial stages of phagocytosis assays in most experiments were carried out in isotonic buffer solution (i.e., HBSS) to minimize the rapid onset of hyphal growth. This proved helpful in distinguishing media versus bona fide phagosomal responses.
49
Genetic Modification of C. albicans
C. albicans is for all practical purposes, an obligate diploid, and as a consequence, the classical forward genetic methods used to study S. cerevisiae cannot be readily applied to C. albicans. The information available regarding C. albicans has primarily been obtained through the use of reverse genetic approaches that rely on directed mutagenesis using standard molecular biological transformation-based strategies to inactivate specific genes. Here, the biological function of a gene is elucidated by examining the changes in the phenotype of cells carrying null alleles, or knockout mutations. Since C. albicans is diploid, creating deletion mutants is quite cumbersome, it requires at least two rounds of gene disruption to inactivate both alleles of interest. This becomes even more problematic when there is aneuploidy (extra copies of one or more chromosomes). The complication of aneuploidy was, for example, observed during the creation of a stp2 null mutant strain of C. albicans, which required three rounds of gene disruption (101). The rather limited number of genetic strains and nutritional and dominant selection markers available for C. albicans hampered early work. Of importance, some of the nutritional markers used for gene disruption methods, such as the Ura3 blaster method, have been shown to significantly alter the virulence of C. albicans. For example, the ectopic expression of the URA3 marker placed at different genomic loci has been implicated in reduced virulence. At one point this raised uncertainties regarding the validity of virulence assays conducted using strains that used URA3 as a marker (238). Virulence is restored when the URA3 is reintroduced back to its natural locus. Also, negative selection by growing cells in FOA for excision of URA3 marker, has been shown to cause chromosomal alterations, and hence represents an additional drawback in using this tool (239).
The SAT1 flipper cassette, has recently been considered the method of choice for gene disruption in C. albicans, partly owing to the use of the nourseothricin, a recyclable dominant selection marker that can be excised by FLP/FRT site-specific recombination system (240). Despite the benefit of this technique, creating deletion mutants remains cumbersome, as it has to be repeated at least twice to inactivate both gene copies. The development of the CRISPR/Cas9 gene-editing tool optimized for C. albicans has overcome several limitations as it allows simultaneous and efficient inactivation of multiple alleles of a gene (or in some case even a family of genes) (241). In this system, the Candida-compatible Cas9 endonuclease is directed by a specific synthetic guide RNA (sgRNA) to cleave at a specific and desired genomic region. The 20-bp sgRNA guide sequence is placed 5’ of the protospacer adjacent motif (PAM, Fig. 31a). The repair template (RT; also known as the donor DNA), which contains the desired sets of mutations, is used to fix the cleaved DNA through homologous recombination. SSY1 gene inactivation provides an example to illustrate the use of CRISPR/Cas9 as major tool in this thesis (Fig. 31b, 31c).
50 Fig. 31. CRISPR/Cas9 mutagenesis. a) Schematic diagrams for the Candida-optimized CRISPR/Cas9 gene-editing method and the solo vector expression system in plasmid pV1093. Adapted from Vyas et al, 2015, Sci Adv. (241); b) Schematic diagram of CRISPR/Cas9-mediated inactivation of SSY1, a gene encoding for a primary plasma membrane-bound amino acid sensor. Shown are the 20-bp sequence recognized by the Cas9 endonuclease, the position of the repair template containing the stop codon and restriction site, and the position of primers used for PCR verification of ssy1 null mutants; c) (top) ssy1 mutation is characterized by hypersensitivity to MM, an inhibitor of branched-chain amino acid biosynthesis. NouR transformants were pre-screened in YPD-MM together with the ssy1Δ/Δ knockout control strain (ΥJA64); (middle) NouR and MMHS clones were selected for further verification by PCR-RFLP with wild type as control (Only clone 1 was shown); (bottom) ssy1-/-CC9 verified clones expressing a HA- tagged Stp2 were tested for Stp2 cleavage as ssy1 mutants are unable to process latent Stp2 even in the presence of inducing amino acid. Whole cell protein extracts were prepared from log-phase SD culture induced with 1 mM glutamine for 30 min at 30°C and immunoreactive Stp2 probed by immunoblotting.
51
Aims of the thesis
The overall aim of this thesis is to provide a mechanistic understanding on how proline catabolism is regulated in C. albicans and how it affects pathogenesis. Understanding the mechanisms by which C. albicans utilizes proline is important for developing new strategies against this opportunistic fungal pathogen.
Specifically, the focus of this thesis is to address the following key aims:
o Characterize proline catabolism in C. albicans and define the mechanistic basis for understanding how proline specifically triggers filamentous growth.
o Place our understanding of proline catabolism in C. albicans in the context of host- pathogen interactions.
o Explore other virulence-related traits linked to proline catabolism that underlie the success of C. albicans as an opportunistic human pathogen.
52 Results summary
Paper I. Mitochondrial proline catabolism activates Ras1/cAMP/PKA-induced filamentation in Candida albicans
A major goal of this study was to characterize how proline induces filamentous growth in C. albicans. Using a combination of biochemical and genetic approaches, we have shown that proline catabolism is required and that it triggers morphogenesis via ATP-mediated activation of the Ras1/cAMP/PKA signaling pathway. Experimental support for this conclusion is documented in Paper I and includes the following: 1) proline uptake from the extracellular environment is required; the functional expression of the amino acid permeases facilitating proline uptake depend on the ER-chaperone Csh3 to fold; 2) proline-induced morphogenesis occurs in a Ras1- and Efg1-dependent manner; 3) intracellular ATP levels are high in proline- grown cells as compared to non-inducing amino acid controls, and proline induced cells possess elevated levels of active Ras1 (Ras1-GTP) and NADH; 4) inhibition of ATP generation by methylene blue (MB) treatment arrests proline-induced filamentation; 5) proline metabolism is required to induce switching as genetic disruption or biochemical inhibition of key catabolic enzymes significantly reduce filamentation; and 6) the SPS-sensor of extracellular amino acids affects filamentation indirectly, and does so by inducing the expression of amino acid permeases that catalyze proline uptake. Although C. albicans is thought to be a Crabtree negative fungus, the magnitude of proline-induced morphogenesis is influenced by glucose in a manner reminiscent of the Crabtree effect. We provide evidence that high glucose levels negatively affect mitochondrial function, which correlates with reduced proline utilization. Contrary to existing literature (81, 227), we show that proline catabolism is the primary and prioritized route for arginine catabolism, which occurs independent the deamination of urea catalyzed by Dur1,2. Consistently, we report that the expression of DUR1,2 is under nitrogen catabolite repression (NCR) and is not expressed when proline is present. Finally, we observed that proline is actively utilized by phagocytized C. albicans cells inside macrophages, and that proline catabolism is essential for escape by hyphal formation (81). In striking contrast to S. cerevisiae, the expression of proline catabolic genes (e.g., PUT2) is insensitive to NCR; the deletion of transcription factors GLN3 and GAT1 did not affect the expression of Put2. Together these findings indicate C. albicans prioritizes the use proline as a preferred nitrogen source.
Paper II. Proline catabolism contributes to reactive oxygen species homeostasis in Candida albicans
In Paper II we further document the role of proline catabolism in promoting virulence of C. albicans. The experiments, based on insights derived from Paper I, yielded data that strengthen the notion that proline is an important energy source during virulent growth. Mutant cells carrying deletions in the proline catabolic enzymes have significantly lower intracellular ATP
53 levels compared to wild type cells. The ability to catabolize proline is essential for growth in 3D microenvironments; put1 and put2 mutants exhibit reduced capacities to invade solid substrates in a 3D collagen transmigration assay, reflected by reduced hyphal growth and fitness when grown on a PureCol EZ gel matrix (Fig. 32). Using Phloxine B as a viability stain, the proline catabolic mutants, particularly put2-/-, appear to undergo cell death faster in rich complex media when grown in the presence of glycerol and at 37 °C, presumably linked to low ATP and the toxic effects of the intermediate P5C. Consistently, in defined minimal media supplied with exogenous proline, the put2-/- mutation becomes lethal when grown under respiring conditions (e.g., with glycerol as the primary carbon source). Consistent with derepression of mitochondrial function, this lethality can be circumvented when glycerol is replaced with high glucose (2%). However, even in the presence of glucose, the put2-/- mutant still shows growth impairment and generates excessive mitochondrial superoxide (Fig. 33). This is likely due to the accumulation of P5C, which may promote P5C-proline cycling leading to increased ROS, and/or impair growth simply due to its inherent toxicity. Data was obtained to support this notion, specifically, put1-/- mutations suppress the negative effects associated with put2-/- mutations. A strain lacking both PUT1 and PUT2 phenocopies the put1-/- and not put2-/- single mutants. Also, we were not able to rescue the growth defects of put2-/- even by treating cells with ROS scavengers, e.g., Mito-TEMPO, N-acetylcysteine or TIRON. These latter findings suggest that in addition to increasing ROS, the accumulation of P5C, resulting from the loss of PUT2, leads to a hypersensitivity phenotype similar to that demonstrated in yeast (174). Finally, the proline catabolic mutants exhibit reduced virulence in various infection models; the put2-/- mutant is avirulent in Drosophila and in a 3D skin infection model and exhibits significantly attenuated virulence in a systemic murine tail-vein infection model.
a b
Fig. 32. Collagen invasion assay. a) Schematic of collagen invasion assay; b) Cells from log phase YPD culture were collected and washed before spotting a 1-μl aliquot of OD600 = 0.1 on top of a pre-solidified PureCol EZ gel matrix in a PET-membrane transwell sitting on top of complete sterile medium. Fungal cells of the indicated strains were allowed to invade the matrix for 14 days in a humidified chamber before analyzing the medium for CFU. Box and whiskers plot derived from seven biological replicates (**P <0.01, ***P <0.0001 by Kruskal-Wallis test with Dunn’s multiple comparison test).
54
Fig. 33. put2-/- is sensitive to excess proline. a) Growth curves for the indicated strains grown in SD medium with and without 10 mM proline using the BioScreen analyzer; b) C. albicans put2-/- cells from 72 h-old SD cultures with and without proline supplementation were collected and then stained with Mitosox. Confocal images were acquired using LSM800, 63X/oil using 561 nm excitation laser.
Paper III. Glutamate dehydrogenase (Gdh2)-dependent alkalization is dispensable for survival and escape of Candida albicans from macrophages
In the course of genetically dissecting the arginine catabolic pathway (Paper I; (81)), using alkalization as qualitative readout for growth, we observed that dur1,2-/- cells lacking urea amidolyase remained fully competent to alkalinize a basal medium containing arginine as sole nitrogen and carbon source. In striking contrast, the proline catabolic pathway mutants (i.e., put1-/- or put2-/-) lacked the ability to alkalinize the same medium. Thus, Dur1,2 activity is not linked to the alkalization of the extracellular environment when arginine is used as the carbon and nitrogen source, but rather alkalization is strictly linked to proline catabolism. These findings are contrary to previous reports (93, 236). Interestingly, a reaction that releases
NH3 occurs just downstream of Put2, i.e., the deamination of glutamate to α-ketoglutarate catalyzed by the enzyme glutamate dehydrogenase (Gdh2). In paper III, we confirm that this enzyme is responsible for the release of the ammonia, endowing C. albicans with the capacity to increase the extracellular pH in the presence of amino acids. Mutants lacking GDH2 fail to alkalize media containing amino acids under all non-repressing conditions of mitochondrial activity tested (Fig. 34). A completely unexpected finding is that the inactivation of GDH2 did not affect the induction of filamentous growth of C. albicans, either in vitro in filament-inducing media, or importantly, in situ in the phagosome of primary murine macrophages (Fig. 34). In all of these conditions, the gdh2-/- mutant behaved similar to wildtype. These results indicate that phagosomal alkalization is not the defining event that induces hyphal growth of C. albicans cells phagocytized by macrophages. Our results are well aligned with recent findings by Westman et al. (237) showing that phagosomal alkalization results as the consequence of hyphal expansion, which physically disrupts the integrity of the phagosomal membrane. The fact that hyphal growth is induced prior to alkalization is consistent with our observations.
55
a
b
Fig. 34. Gdh2 catalyzes environmental alkalization but is dispensable for hyphal escape from macrophage. a) (left) Amino acid-dependent environmental alkalization is dependent on Gdh2 but nor Dur1,2. Cells of the indicated genotypes were spotted on the surface of solid YNB+CAA+BCP (i.e. YNB+CAA; pH = 4.0, unbuffered) with or without an additional source of carbon and were allowed to grow for 72 h at 37 °C. Representative results of at least 3 independent experiments are shown (i.e., yellow = acidic; purple = alkaline); (right) ammonia release assay. The indicated strains were grown on solid YNB+CAA with 0.2% glucose (pH = 7.4) for 72 h at 37 °C and the volatile ammonia released were captured using 10% citric acid placed directly underneath the growing colonies. Results presented are the average of at least 3 independent experiments (Error bar, 95% CI; **** P£ 0.0001 by one-way ANOVA with Dunnett’s multiple comparison test); b) Snapshot of timelapse microscopy (2 h) showing both wildtype (WT; PADH1-GFP; CFG273) and gdh2/- (PADH1-RFP, CFG275) mutants escaping from primary BMDM (MOI of 3:1; C:M). Green arrow indicates BMDM containing WT cells only, red indicates gdh2-/- cells only, white arrow indicates both strains in the same BMDM. The observed growth of WT and gdh2-/- cells within a single macrophage is schematically illustrated (right upper panel). Candidacidal activity of BMDM (right lower panel); CFUs recovered at 2 h were compared to the CFUs in the starting inoculum (P = 0. 2359 by Student’s t-test).
56 Synthesis
Accumulating evidence suggests that proline catabolism plays an important role in the pathogenesis of many human diseases ranging from bacterial infection to cancer (128, 129, 173, 188, 190, 242-245). This thesis describes the role of proline catabolism in the opportunistic human fungal pathogen C. albicans. Our results indicate that under physiologically-relevant host conditions, proline catabolism is important for C. albicans for three key functions: 1) energy generation, 2) ROS homeostasis, and 3) as a major catabolic route for amino acid- mediated environmental alkalization.
C. albicans cells within the phagosome of a macrophage are under immense stress given the limited availability of nutrients, oxidative burst, the presence of active hydrolytic enzymes and low pH. Under these severe conditions, survival of fungal cells must depend on their ability to generate ATP; polarized filamentous growth requires ATP to drive actin polymerization and maintenance of intracellular pH requires ATP to promote proton export by the Pma1 ATPase. We have shown that proline utilization is essential for C. albicans to survive and escape from the macrophages (81). This requirement is strictly specific to life in the phagosome and to 3D growth, as proline catabolic mutants retain the ability to grow and form hyphae in complete cell culture media containing serum. The idea that proline provides the fungal cell with sufficient ATP to both signal to induce and support hyphal growth in the microenvironment of the phagosome is plausible and supported as follows: 1) proline can be oxidized completely to generate 30 equivalents of ATP, which is roughly equal to that produced during complete oxidation of glucose. The direct generation of reduced electron carriers (NADH and FADH2) from the first two steps of proline catabolism, plus the reducing equivalents obtained by the further catabolism of glutamate involving the TCA cycle, allows for efficient production of ATP; 2) the phagosome is glucose poor (46), which relieves the mitochondria from carbon source repression, allowing mitochondrial localized metabolic processes (e.g., proline utilization) to operate optimally; 3) proline catabolic pathway is essential for the catabolism of arginine, which based on transcriptional profiling, is expected to be both synthesized and catabolized by the fungus in the phagosome (46, 81); 4) proline utilization is activated and expressed following phagocytosis (Fig. 35, top), suggesting that proline does not accumulate to counteract stress but to generate energy to enable escape. These processes are not restricted to filamenting cells as non-filamenting cph1Δ/Δ efg1Δ/Δ mutants also induce the expression of proline catabolic enzymes following phagocytosis (Fig. 35, bottom); 5) proline utilization is insensitive to NCR (81, 197) enabling C. albicans to overcome a regulatory hurdle that would prevent them from freely utilizing proline when its available.
The proposed mechanism by which ATP mediates Ras1/cAMP/PKA pathway activation (56) provides an excellent framework to understand how nutrients can trigger hyphal growth in C. albicans. Published data suggests that amino acids serve as primary nutrients in fungal cells; strains lacking STP2 (stp2Δ/Δ), which cannot induce amino acid uptake, fail to form hyphae and escape macrophages (92). Amino acids were thought to be used primarily to generate ammonia to neutralize the phagosomal pH (92). However, our data
57 in Paper III show that hyphal formation is clearly uncoupled from alkalization; a gdh2-/- mutant, lacking the capacity to alkalinize the environment in the presence of amino acids, remained competent to form hyphae. This finding provides the most compelling illustration of the dispensability of alkalization for hyphal-dependent escape from macrophages. Furthermore, our data do not align well with the suggestion that alkalization is required for hyphal induction; amino acid uptake is dependent on proton (H+) symport, consequently, uptake becomes less efficient when pH is neutral or alkaline, which would hamper the uptake of amino acids. Though we are still uncertain regarding the precise origin of proline, our data suggest that it is available and is catabolized following phagocytosis (Fig. 35) (54). These findings are well aligned to a recent report showing that hyphal formation initiates prior to detectable alkalization of phagosomes (237).
10 min 30 min 50 min 70 min 150 min
Fig. 35. Proline catabolism is activated shortly after engulfment by macrophages (Silao, unpublished data). C. albicans wildtype (top) and cph1Δ/Δ efg1Δ/Δ mutant (bottom) expressing Put2-GFP fusion protein were co- cultured with RAW264.7 murine macrophage cells and images were captured at the indicated time. Lysotracker (red) was used to visualize lysosomes and phagosomes.
The unabated growth of strains lacking GDH2 in the phagosome is remarkable and was a total surprise. Based on our in vitro observation that a gdh2-/- mutant is unable to grow using amino acids as the sole nitrogen and carbon source unless supplemented with a small amount of additional carbon source (e.g., glucose, glycerol, or lactate), and the assumption that phagosome is glucose poor, we anticipated that the gdh2-/- mutant would show a striking growth defect inside the phagosome. However, we observed the opposite, the mutant clearly escaped the phagosome at rates indistinguishable to wildtype. We attribute the inability of gdh2-/- to grow using amino acids alone to the shortage of acetyl-CoA formed by glycolysis to prime the TCA cycle. Since the gdh2-/- mutant grew well inside macrophage phagosomes, the question now is where does acetyl-CoA come from? Most glucose inside macrophages is phosphorylated due to the action of hexokinase, and therefore cannot be efficiently transported into the lumen of the phagosome. We are currently considering whether the endocytic pathway, which allows free nutrients including glucose, from extracellular environment to enter cells via endocytic vesicles, provides the source of glucose. The fusion of nutrient containing endocytic vesicles with maturing phagosomes could supply the glucose.
58 The use of time-lapse microscopy should enable a more rigorous assessment of the requirement of a specific nutrient acquisition pathway for hyphal escape.
The utilization of amino acids to support the bioenergetic demands of hyphal growth within the phagosome is an interesting aspect that needs to be further examined in order to better understand the spatio-temporal dynamics of hyphal formation of this fungal pathogen within this innate immune cell. The role of proline catabolism in virulence of C. albicans is not restricted to survival in macrophages. Mutants unable to catabolize proline exhibit attenuated virulence in other infection models (i.e., Drosophila, 3D skin model, neutrophils, and systemic murine infection). Interestingly, C. albicans has evolved in symbiosis with humans and is therefore exposed to the same physiological conditions as human cells. Their capacity to establish disease and aggressively infect a variety of host tissues in human host arises as a consequence of their sensing and responding to changes in the host environment, resulting in altered metabolism. Proline is available in the host in the contest of proline-rich proteins (PRP), like collagen in the extracellular matrix and in connective tissues. Proline can readily be mobilized from these sources via two possible for mechanisms (Fig. 36). The first depends on the induced expression of proteolytic enzymes by the fungus, such as the secreted aspartyl proteases (Saps) and/or matrix metalloproteases (246, 247), that are subject to strict regulatory controls. Alternatively, proline can be mobilized by the action of host proteases that are expressed as a result of host-mediated responses towards different stresses. For example, in elder patients with sarcopenia (muscle wasting), there is a striking increase in plasma proline concentrations (245). Interestingly, there is a good correlation showing that patients suffering from sarcopenia have higher risk of blood-borne C. albicans infections following liver transplantation (248). The existence of a limited number of specialized proteolytic enzymes that are capable of hydrolyzing the proline peptide bonds supports the idea that proline has an important regulatory function or, alternatively, its catabolic utilization is reserved for discrete sets of pathophysiological roles in a process called Ecophagy (249).
Fig. 36. Model for proline acquisition of C. albicans in the host. a) Proline is actively acquired by the fungus through expression of proteolytic enzymes (Saps and MMPs); b) the fungal cells passively acquire proline from host- dependent protein degradation.
When using proline as an energy source, the catabolic enzymes must operate in synchrony as perturbation in these highly regulated catabolic steps can have deleterious
59 consequence for the fungus. The sensitive phenotype (e.g., smaller colonies, slower growth rate, higher cell death, and higher ROS) displayed by put2-/- in the presence of excess proline is consistent to the occurrence of proline-P5C cycling in C. albicans, which in other organism function as a redox shuttle between the mitochondria and the cytosol (Fig. 37). The increased sensitivity is likely due to the accumulation of toxic intermediate (P5C) resulting to enhanced ROS formation. Although we have been unable to measure the P5C, we speculate that similar to the findings in S. cerevisiae, C. albicans put2-/- mutant would also accumulate P5C (174). Contrary to increase in ROS production, it appears that P5C accumulation is the primary contributor to put2-/- sensitivity as the addition of ROS scavengers, did not fully rescue the growth defect of in proline medium containing glycerol. Studies in yeast suggested that P5C directly inhibits mitochondrial respiration (likely Complex II) in a dose dependent manner resulting to superoxide burst and cell death (174). The rate of P5C formation in our C. albicans put2-/- mutant is likely dependent upon mitochondrial activity that is subject to glucose repression. This potentially explains why the put2-/- mutant failed to grow in minimal proline medium using glycerol as a carbon source but managed to grow relatively well when glycerol is replaced with 2% glucose. However, even at this level of glucose, a time-dependent emergence of hypersensitive phenotype is observed, in this case after 48 h, when glucose becomes depleted. The data also suggest that similar to yeast, PRODH (Put1), in C. albicans does not contribute significantly to ROS formation; there is no difference observed between wild type and the put1-/- mutant in terms of superoxide levels. In addition, the introduction of put1-/- mutation to put2-/- (i.e., put1-/- put2-/-) suppressed the put2-/-phenotype. The significant attenuation in the virulence of put2-/- mutant in different infection models (Paper II) suggests that proline is present in substantial quantity and is actively assimilated under non-repressing conditions (e.g., glucose < 0.2%, approx. 11 mM) resulting in put2-/- sensitivity, which is deleterious to fungal cells. The proline level in whole blood of mice for example is roughly 269 µΜ (250) which is 37-fold lower than what is used in the laboratory (10 mM).
In summary, the data presented in this thesis strongly support the idea that proline catabolism plays an important role in the pathogenesis of C. albicans. We have been able to rationally investigate using different approaches the mechanisms by which proline could signal growth, proliferation and development of C. albicans that are relevant to the onset of disease progression in the human host. Our findings indicate that proline is used as an important energy source when other main sources of energy such as glucose is limited. However, the fungus must thread carefully in using proline as energy source as asynchronous activities of the catabolic enzymes can become deleterious to the cell. Further studies are warranted to determine the applicability of this pathway as a potential drug target against this important fungal pathogen.
60
Fig. 37. Model of Proline-P5C cycling resulting to ROS formation in C. albicans. The direct generation of reduced electron carriers (i.e., FADH2 and NADH) from the first two steps of proline catabolism mediated by Put1 and Put2, respectively, including that of conversion to glutamate and TCA cycle, allows for the rapid production of ATP via the electron transport chain. When Put1 activity exceeds that of Put2 such as in the case of the put2-/- mutant, the toxic intermediate P5C can accumulate requiring it to be transported out of the mitochondria to the cytosol where it can be converted by Pro3 to proline again forming the proline-P5C cycle. The presence of excess proline increases the rate of this cycling, which operates maximally under full respiratory metabolism. P5C has been shown to induce superoxide burst by directly inhibiting mitochondrial respiration. In addition to proline, it is predicted that arginine or ornithine (grey box) can induce the same effect as these amino acids can be directly catabolized to proline. How proline is imported into the mitochondria or how P5C is exported to the cytosol (red question marks) is yet to be deciphered.
61
Future direction
1. Where is proline coming from?
As shown in Fig. 38 and Paper I, C. albicans cells phagocytosed by macrophages resulted to elevated expression of Put2-GFP. This increase in expression is mediated by the transcription factor Put3, which binds exogenous proline resulting to derepression of PUT2. We are currently investigating the possible sources of the inducing proline to get a better picture of the spatio-temporal dynamics of proline utilization in C. albicans during interactions with macrophages. To facilitate this, we will carry out a series of timelapse live cell imaging experiments using a C. albicans reporter strain expressing both inducible Put2-GFP and constitutively expressed RFP placed under the control of the strong ADH1 promoter (PADH1-RFP). To enable a more rigorous quantitative analysis of proline catabolic activity, we will use Put2- GFP/RFP ratio as analytical readout for induction. We speculate that proline is derived either from internalized stores within fungal cells, or is obtained from the host macrophages. To genetically dissect this event, we have created reporter strains in wildtype (WT; filamenting) and cph1Δ/Δ efg1Δ/Δ (non-filamenting) genetic backgrounds lacking CSH3, CAR1, STP1, or PUT3. The strain lacking CSH3 (csh3-/-) exhibits greatly impaired amino acid uptake. If this strain exhibits reduced levels of GFP, it is likely that proline originates from the extracellular environment, Fig. 38. Quantification of proline-induced expression of Put2 using the Put2-GFP/ likely from the phagosomal milieu. Alternatively, RFP reporter strain. if the strain exhibits induced GFP, then proline is likely derived from internal stores. Similarly, the lack of induced expression in a car1-/- strain will indicate that proline comes from arginine. The impaired expression in a stp1-/- strain will indicate that proline comes from oligopeptides derived from the degradation of host proteins. The put3-/- strain will act as control.
2. How is proline imported to the mitochondria?
A major outstanding question is how proline is imported from the cytosol to the mitochondria. To date, no mitochondrial carrier protein has been identified that carries out this specific function. In S. cerevisiae, FAD biosynthesis is reported to occur in the cytosol where the FAD synthetase is compartmentalized (251, 252). FAD is then imported to the mitochondria via the protein Flx1, located at the inner mitochondrial membrane (IMM) and then covalently linked
62 to the histidine residue near the amino terminus of the 70 kDA flavoprotein comprising the succinate dehydrogenase complex (253). Interestingly, it was demonstrated that PRODH co- localizes with Complex II (254) suggesting that the two can directly interact. In E. coli, the PutA catalyzed oxidation of proline is coupled to reduction of ubiquinone (CoQ) by directly transferring electrons from
FADH2 (255). Given this information, we hypothesize that proline together with FAD, are imported to mitochondrial matrix through Flx1 where both are used Fig. 39. Model for mitochondrial import of proline as as substrates by PRODH (Put1); PRODH mediated by the putative transporter, Flx1. then couples the oxidation of proline to the reduction of ubiquinone (CoQ) in Complex II of the ETC (Fig. 39).
3. Is alkalization really required for virulence?
We demonstrated that Gdh2-mediated alkalization is not essential for escape from macrophages. However, this result also raises the question whether alkalization in general is really required for virulence. Is the requirement for alkalization niche-specific? Many niches in the host have a rather dynamic pH and examining the virulence of gdh2-/- mutant in different infection models is important to address the exact role of alkalization in virulence.
4. How do proline catabolic mutants rewire metabolism to generate energy?
In the absence of a functional proline catabolic pathway, how does C. albicans rewire its metabolism to generate energy and respond to oxidative stress in the absence of an intact proline catabolic pathway? What substrates are being utilized to generate the reducing equivalents?
5. Is proline catabolism essential for virulence of other Candida species?
C. glabrata is the second most frequent cause of invasive candidiasis in the US and most of Europe. Unlike its diploid relative C. albicans, C. glabrata is a haploid yeast that is unable to undergo dimorphic transition (i.e., yeast-to-hyphal growth)(256). Although it cannot form true hyphae which is a known virulence factor in C. albicans, C. glabrata is still considered a successful human pathogen, which it partly owes to its high intrinsic stress tolerance and able to persist inside the macrophage phagosome. How is intracellular replication of C. glabrata induced and supported inside the macrophage phagosome? Specifically, is it driven by proline metabolism?
63
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80 Acknowledgement
Having reached this significant milestone of my PhD journey, there are a number of people that I really would like to thank for all the help and support: First and foremost, I would like to extend my deepest gratitude to my supervisor, Per Ljungdahl, for that you took the risk of taking me in your lab as one of your PhD students. I really appreciate the independence given at work, and for all the valuable “homilies” and discussions, whether scientific or personal, shared in the past 5 years. The simple gesture of you going to the lab for random discussions (per my record, 3.5 h is the longest) and for offering me a ride home when I was working late, is so much appreciated! Your thoughtfulness and generosity are also very commendable. For sure, my PhD journey wasn’t ideal, far from it actually, but you definitely empowered me and gave me all the motivations I needed to keep me excited of coming back to the lab for more experiments. Again, thank you! To Ate Che (and kuya Amon), thank you for serving as my primary inspiration to do Science. I can still remember the time 20 years ago when you were quizzing me about mitochondrial structures while you were reviewing for your postgrad exam. Guess where fate has led me! To the past and present members of the Ljungdahl lab: To Kicki, thank you very much for being so kind and supportive of my work. You were there to help me from the very beginning until the end, and I could not imagine doing all these works without your help. Thank you also for acting as my “sense barometer” whenever I try to discuss my sometimes premature and jumbled ideas. To Ioanna (ma prend), thank you for being such a good friend and for always having my back! You certainly brighten up the lab when you came. You are one of those few people I know who can make someone feel so easy and secured because of your fun personality but at the same time, you can also make them uneasy when you ask questions that are often very thoughtful and insightful, an iron fist in a velvet glove! To Dr. Mel, I am very thankful that Per brought you in the lab. Your help on macrophage and microscopy techniques is so invaluable that we wouldn’t have gone this far with our work had you not come to the lab. Maraming salamat! To Andreas (ma ader prend), it was a pleasure meeting you! Thank you for all the fun and helpful discussions, and for helping me out when I was starting in the lab (My notebook would attest to that). To Antonio (amigo!), I really appreciate the time you have given whenever I needed to discuss something to help me organize my thought and find my research direction. To Benedetta, it was nice meeting you! Thank you for the excellent Italian lasagna and for all the horror “sulfur” stories. Keep the Candida passion burning! To Marina and Georgia, thank you for all the support, excellent discussions, and for showing me the polar spectrum of being Greek! J To Vanessa, and also to my former students, Axel and Nico, thank you for all the help with my work and it was a pleasure mentoring you both. To Doc Nic, thank you for very much for all the guidance and for being so helpful during my postgrad application. You were so patient and considerate, you didn’t let down. To Doc Pol, thank you for all the interesting scientific discussions! You are a rare breed, it was a pleasure meeting you here in Stockholm! Gratitude is also extended to the members of the Marie Curie-ITN ImResFun consortium especially to Karl Kuchler and Patrick Van Dijck, and to my fellows Raju, Emilia,
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Nathalie, Tanmoy, Andreas, Leo, Nitesh, Mariana, Cat, Claudia, Ernst, Deb, and Vicent, for the science and friendship. The opportunity to meet you all again is one of the few things that I looked forward to whenever we engaged in those fun, yet oftentimes, taxing meetings. To my extended ImResFun friends, Sabrina and Filomena, thank you for being such a nice company during conferences and for being such efficient collaborators! Can’t wait for more Dr. T’s! I would also like to give my thanks to the members of Claes Andreassoń , Martin Ott, Sabrina Buttner̈ , Anki Östlund, Roger Karlsson, Ann-Christin Lindås, Martin Jastroch, Ulrich Theopold, and Klas Udekwu labs for all the scientific discussions and support. To Claes for all the valuable advice and for being our walking “cloning encyclopedia”. To Lukas, Andreas and Carlotta for all the excellent discussions and for sharing your insights in mitochondrial biology; To Lukas, I really appreciate that you look at my work with the same enthusiasm as me. To Stina and Chris, thank you for all the help with microscopy and for personally inviting me to attend microscopy seminars! To Björn and Paulina for the help in genome sequencing. To the past and present members of F4 north corridor, especially to Toni, Nandu, Naween, Joydeep, Melania, Matthias, Bejan, Fredrik, Anna, Janny, Mahsa, and Yuan, thank you as well for all the good company, parties, and karaoke nights! To my friends in Nestlé especially my ATG family, Ms. Gi, Joanna and my kumpares Erick, Louie, Ric, thank you for all the support and for always keeping in touch! To Sir Jeng Duaqui, thank you for believing in me and for vouching for my return to Nestlé that made this very long study leave possible. To my Pinoy friends in Sweden especially to Walter/Chriz, Dennis/Jen, Elmer/Ethel, and Casey, thank you for making my adjustment in Sweden a whole lot easier! To my family back in the Philippines, thank you for all the love and encouragement! Ma/Pa and to my siblings, Amie, Joy, May, Bok, Mac, and Ja, I would have loved to see you all here together and celebrate with me in this very important milestone of my life but maybe fate dictates that now is not the time. I’ll definitely see you soon! This acknowledgment would not be final without me thanking the reason why I wanted to do my very best in all the endeavors that I do. To my wife, Joan, and son, Lukas, thank you for the love and inspiration, and for agreeing to come with me here in Sweden to pursue my dream. Thank you, Piglet, for all the support and for creating a warm and welcoming home that makes it possible for me to decompress at the end of a busy and sometimes stressful day.
May this suffice.
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