Development Of Tools For Molecular Study Of Cryptococcus

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Abstract

Although there is much interest in Cryptococcal research, tools for genetic analysis in C. neoformans are less well developed than in Saccharomyces cerevisiae and Schizosaccharomyces pombe. By the end of 2003, the genomic sequence of C. neoformans will be finished and the function of genes can be analysed readily by using the sequence data as well as by gene disruptions. In order to effectively identify the genes disrupted randomly and elucidate their functions, a tagging system in deletion construct will be needed. There are several tagging systems that have been successfully used in bacteria as well as in fungi such as transposon or signature tagging methods (STM). These methods have not been successfully used in C. neoformans due to the lack of controlable transposon in C. neoformans as well as a lack of randomness in STM system. In 2003,we have investigated Agrobacterium mediated transformation system in C. neoformans in serotype D and found that single insertions occur randomly in most transformations. The frequency of transformation could be elavated significantly by using adenine auxotrophs. The Agrobacterium mediated transformation, therefore, will be most useful for gene disruptions at a large scale in C. neoformans. We also investigated the molecular bases of urease production in C. neoformans. Urease activity is one of the hallmarks of C. neoformans and clinical microbiology laboratories routinely use urease activity of yeasts as one of the diagnostic criteria for C. neoformans. Clinical isolates produce large amounts of urease and urease negative isolates causing infection in humans are extremely rare. Urease is a metalloenzyme that enables the hydrolysis of urea to ammonia and carbamate and under physiological conditions results in an increase in pH. The latter property is used as a detection tool for the tentative identification of C. neoformans. Such pH increases caused by urease, in other organisms, have been associated with survival in animal tissues and the cause of tissue damage induced by the production of ammonium hydroxide. Furthermore, urease is postulated to play a pivotal role in allowing C. neoformans to convert urea to usable nitrogen in its ecological niche. The exact role of urease in Cryptococcal pathogenesis, however, is unknown. In bacterial pathogenesis, the enzyme appears to alter host immune function by increasing microenvironmental pH at the infection site. Inhibition of urease activity has been reported to correlate with significant decreases in eliciting immune defense cells in the lungs of mice. Further investigation into the role of urease in crytptococcosis and other fungal infections is warranted even though urease-negative mutants are still pathogenic in animal models. We have recently obtained a urease-negative, serotype A isolate of C. neoformans from an immunocompromised patient. Using electroporative protocols we successfully complemented the urease-negative phenotype of the clinical isolate with a genomic library of the strain H-99, a clinical serotype A isolate of C. neoformans. Several thousand transformants were screened based on a selectable nutritional marker. An analysis of the episomes harbored by the tansformants identified a new gene of Cryptococcus neoformans that complemented the urease deficiency of the fungus. A sequence analysis of this gene, named CnSUDA, exhibited significant homology to a conserved family of suppressor proteins known to be involved in chromosome scaffolds facilitating mitotic segregation in other organisms. Preliminary data suggests that expression of SUDA allowed the secretion of urease by the organism. The interaction of scaffold proteins with histones in the nucleus is known to affect several functional aspects of cell physiology from structural aspects involving chromosomal condensation during cell division to gene regulation via transcriptional control. It is possible that SUDA may affect the structural integrity of the cell wall influencing secretion of urease.