| Summary: | Cryptococcus neoformans is an encapsulated fungal pathogen that causes cryptococcosis, a life-threatening disease which affects an estimated 1 million people worldwide annually. Iron acquisition is an important but poorly understood aspect of the pathogenesis of C. neoformans. In particular, no heme uptake system has thus far been characterized in this fungus, although it has been shown to utilize heme as an iron source. A previous study identified the transcript for the extracellular mannoprotein CIG1 as the most abundant message in iron-starved cells with marked down-regulation by iron repletion, thus suggesting a possible iron-related role for Cig1. In the current study, it was found that deletion of CIG1 resulted in an extended lag phase in low iron medium with heme added as the sole iron source. Additionally, the cig1Δ mutant was more resistant to toxic heme analogs than the wild-type or complemented strains implying a role for Cig1 in heme uptake. Western blot analysis and immunofluorescence microscopy identified Cig1 at the cell surface and in association with extracellular vesicles. A heme pull-down experiment, absorbance spectroscopy and isothermal calorimetry also demonstrated that Cig1 is a potential heme-binding protein. Importantly, deletion of CIG1 led to attenuated virulence in a mouse infection model in absence of the high-affinity iron uptake system. More detailed studies on Cig1 revealed that the length of the lag phase of a cig1Δ mutant in low iron medium supplemented with heme was dependent on the inoculum size in support of a cell density-dependent heme acquisition system. Similarly, growth at acidic pH rescued the heme defect of a cig1Δ mutant indicating the presence of a Cig1-independent pathway at low pH. The transcription factor Rim101 may function in this pathway. Finally, expression of a Cig1 truncated polypeptide established a role for Cig1 in secretion and cell wall integrity. In this context, a strain overexpressing CIG1 produced an enlarged capsule and secreted more extracellular vesicles than the wild-type strain. Overall, the data presented in this thesis have contributed to a better understanding of heme uptake and secretion in C. neoformans and the results may facilitate the development of new strategies to treat cryptococcosis.
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