Development Of Tools For Molecular Study Of Cryptococcus Neoformans

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Abstract

During 2004-2005, we applied the ATMT method to all four serotypes of C. neoformans and studied the effect of pyrimidine or purine starvation on the transformation efficacy. Unlike in Saccharomyces cereviciae where adenine mutants transform at a significantly higher rate by ATMT, adenine or uracil auxotophs of C. neoformans did not show any increases in transformation frequency by ATMT. Although the transformation frequencies were higher in pyrimidine auxotrophs of certain strains which indicated that such increases may be strain dependent. During 2005-2006, we screened 30,000 insertional mutant clones for various identifyable phenotypes such as growth under low oxygen, capsule formation and azole resistance. A majority of the transformants harbored a randomly integrated single copy of T-DNA and were mitotically stable. In 2006-2007, we screened the insertional library for their sensitivity to chobalt chloride, a hypoxia mimetic used in the mammalian system. The primary screening of the mutants sensitive to CoCl2 was carried out by spot assays of the library consisting of 30,000 clones on YES+0.6mM CoCl2 and YES+G418 agar plates. Mutants that failed to grow on CoCl2 medium were selected for further analysis. More than 25 mutants were selected based on their sensitivity to CoCl2. Sequence analysis revealed the T-DNA to have been inserted in a wide array of genes. Some of these genes are involved in mitochondrial function and the sterol regulatory pathway (SREBP, SCAP).In the mammalian system, cobalt chloride is known to affect mitochondrial function. In these cobalt chloride sensitive mutants, we checked the mitochondrial integrity by using different mito tracker dyes such as CMXRos and Syto18. We analyzed if cobalt chloride sensitive mutants show any perturbation in mitochondrial membrane potential. Confocal microscopy results showed an inability of many mutants to retain the mito-tracker dye indicating the presence of dysfunctional mitochondria. Based on these observations we grouped cobalt chloride sensitive mutants into 2 groups, mutants showing defect in mitochondria and mutants without mitochondrial defects. Mutants in both groups were further characterized. Based on plate assays, these mutants exhibited sensitivity on media containing hydrogen peroxide and sodium nitrite at pH 4.0. Inability to grow on the media containing these ROS generating agents is a classical phenotype of ROS sensitivity. Interestingly the ROS sensitive phenotype is not restricted to the mutants with defective mitochondria. The mutants that did not show mitochondrial defect were also sensitive to ROS generating agents. These results indicate that cobalt chloride affects different cellular functions.