Regulation of nitrogen catabolism is of immense interest in the kingdom Fungi, as nitrogen source utilisation influences key aspects of fungal biology including development, secondary metabolite production and pathogenesis. In the basidiomycete pathogen Cryptococcus neoformans that causes over 625,000 deaths annually, relatively little is known about nitrogen regulation. In view of this lack of knowledge, this thesis is articulated through four major aims to investigate the interplay between nitrogen catabolism and virulence in C. neoformans. The first two aims scrutinise the wiring of the global nitrogen regulatory circuit, while the last two aims examine specific nitrogen catabolic pathways of medical relevance.
In all species of fungi studied thus far, GATA transcription factors are the central regulators of nitrogen assimilation as they globally activate the expression of permease and catabolic enzyme-encoding genes required to degrade most complex/secondary/non-preferred nitrogenous compounds. The first aim is to characterise the function of the nitrogen-responsive GATA factor in C. neoformans. Here, the C. neoformans Gat1/Are1 factor was shown to regulate multiple key virulence traits, in addition to its evolutionary conserved role of controlling nitrogen utilisation. These virulence attributes include the formation of antiphagocytic capsule, deposition of antioxidant melanin in the cell wall, production of infectious basidiospores, and the ability to grow at high temperature.
Given the importance of the GATA factor Gat1/Are1 in pathogenicity, gaining insights into the molecular mechanism governing the regulation of Gat1/Are1 activity in response to nitrogen availability is pivotal. In certain fungal species, GATA factor activity is inhibited through interaction with co-repressor Nmr proteins when readily assimilated (preferred) nitrogen sources such as ammonium or glutamine are present. This regulatory phenomenon, nitrogen metabolite/catabolite repression, enables preferential utilisation of metabolically favoured compounds thereby conserving the organisms’ energy to maximise fitness. The second aim is to characterise the function of a potential Nmr homolog in C. neoformans. Here, unique evidence of divergence between different fungal species in the evolution of Nmr-related proteins was demonstrated. In addition to its functionally conserved role of inhibiting GATA factor activity under repressing conditions, the C. neoformans Nmr homolog Tar1 also positively regulates GAT1/ARE1 expression under non-repressing conditions.
The transcriptional studies of catabolic genes from the proline degradation pathway were proven to be a fertile area of research for investigating the roles of Gat1/Are1 and Tar1 in modulating nitrogen metabolite repression. The third aim is to formally characterise the functions of these enzymes. Degradation of the multifunctional amino acid proline is associated with mitochondrial oxidative respiration. The two-step oxidation of proline is catalysed by proline oxidase and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase, which produce P5C and glutamate, respectively. In the C. neoformans genome, there are two paralogs (PUT1 and PUT5) that encode proline oxidases and a single homolog (PUT2) that encodes P5C dehydrogenase. Here, the expression of all three putative catabolic genes was shown to be inducible by the presence of proline, however, only Put5 and Put2 were required for proline utilisation. Additionally, Put2 was required to prevent excessive mitochondrial superoxide production probably by limiting the P5C-proline cycling that delivers electrons to the electron transport chain and to O2. Intracellular accumulation of reactive oxygen species is known to be a critical feature of cell death; consistent with this fact, the put2Δ mutant exhibited a slight, general growth defect. Further, the put2Δ mutant was avirulent during murine infection, making this the first report highlighting the importance of P5C dehydrogenase in enabling pathogenesis of a microorganism.
Multiple lines of evidence have also suggested that the enzymes of another catabolic pathway, uric acid, may be important for pathogenesis. The ecological niche of C. neoformans is uric acid-rich pigeon guano, and uric acid is also a well-known inducer of the virulence factor capsule formation. Assimilation of uric acid necessitates several enzymes including another bona fide virulence factor, urease, the only enzyme of the pathway that has been characterised. Since uric acid is the end product of purine metabolism in humans and it acts as an immune enhancer, uric acid utilisation by C. neoformans during infection may potentially serve as a method to modulate host immune response. The fourth aim is to characterise the functions of all the uric acid catabolic enzymes. Here, complete degradation of uric acid to ammonia was shown to require: URO1-encoded urate oxidase, URO2-encoded HIU hydrolase, URO3-encoded OHCU decarboxylase, DAL1-encoded allantoinase, DAL2,3,3-encoded allantoicase-ureidoglycolate hydrolase, and URE1-encoded urease. Further studies however, are required to verify the roles of the C. neoformans uric acid catabolic enzymes during infection, ideally using a modified animal model system that lacks these biological enzymes for improved mimicking of human pathogenicity. Overall, these studies lay an excellent foundation for future research into dissecting the link between nitrogen sensing and elaboration of virulence composite in C. neoformans.