Cryptococcus neoformans (Cn), a facultative intracellular yeast, is a serious cause of meningoencephalitis in normal and immunocompromised patients. The susceptibility of immunocompromised patients to cryptococcosis may be related to intrinsic defects in the functions of phagocytic cells combined with impaired T cell functions. The host immune defense against Cn has been extensively studied. However, few studies are available defining how Cn protects itself against human phagocytes. There is preliminary evidence that CuZn-SOD (product of SOD1 gene) act as antioxidant at permissive temperature of 37 degreeC. In addition, a significant reduction in Cn neutrophil killing by exogenous addition of SOD suggests a possible role for CuZn-SOD against phagocytic killing. Therefore, the central hypotheses to be tested in this project are that; 1) CuZn-SOD is an accessory pathogenic factor that enables Cn yeast to survive intracellular killing by phagocytes and, 2) the regulation, expression and cellular localization of CuZn-SOD is of critical importance in Cn response to host oxidative burst. The cDNA and genomic DNA of SOD1 from Cn yeast were recently cloned by functional complementation of SOD1delta mutant of Saccharomyces cerevisiae and by Southern hybridization of cosmid library using Cn SOD1 cDNA as a probe. The deduced 154 amino acid sequence of Cn SOD1 cDNA showed approximately 62 percent identity with other fungal SODs. This preliminary data will be the basis for the proposed study which aims at (i) construction of SOD1 knockout and revertant mutants to define the phenotypic and biochemical consequences of CuZn-SOD deficiency and its role in animal virulence (ii) determination of protective mechanisms of CuZn-SOD against oxidative damage produced by human phagocytes in vitro, (iii) regulation of SOD] transcripts and proteins during oxidative and other stress conditions to define the role of this oxidant in Cn biology. These studies are potentially significant because the results are expected to (a) define the precise antioxidant role of CuZn-SOD, (b) characterize the mechanism used by Cn to survive under oxidative stress, (c) expand understanding of host-pathogen interactions in cryptococcosis, which may aid in improved design of drugs and or vaccines, and (d) provide methodological basis for study of other fungal antioxidants.
