The role of the cysteine/glutathione regulatory genes CDO1, GSH1, and GSH2 in yeast-mold dimorphism of the pathogenic fungus Histoplasma capsulatum

The dimorphism of Histoplasma capsulatum (Hc) from a mold to yeast is regulated by many environmental factors such as temperature and thiol concentrations. Histoplasma exists in the soil (or in vitro at 25ºC) as a multicellular saprophytic mold. In the lungs of an infected host (or in vitro at 37ºC), a shift to the unicellular parasitic yeast occurs. Sulfhydryl groups (-SH), especially cysteine, are necessary in the culture medium for the mold to yeast transition. Cysteine is the precursor for the synthesis of glutathione. Enzymes involved in the cysteine metabolism pathway are being studied in order to evaluate their role in the dimorphism.;Cysteine dioxygenase (CDO1), is a cytosolic enzyme that oxidizes cysteine to cysteine sulfinic acid. Glutathione biosynthesis occurs through two enzymes, Gamma-glutamyl cysteine synthetase (GSH1), and glutathione synthetase (GSH2). The isolation of the cDNA of these three genes was accomplished by 5' and 3' RACE (rapid amplification of cDNA ends) PCR. The expression level of each gene in both the yeast and mold morphotypes of four Hc strains was examined by northern blotting and real-time PCR. CDO1, which was previously isolated in our lab, is expressed in both the mold and yeast phases of the organism while GSH1 and GSH2 are expressed in only the yeast phase. Analysis of CDO1 indicated transcript levels and enzyme activity in both the mold and yeast morphotypes. A western blot of CDO1 using a flag tag fusion identified the protein size to be approximately 24 kDa. GSH1 and GSH2 mRNA levels were increased in the yeast morphotypes. GSH1 mRNA levels were increased by 20 fold in the 186 AS strain and 12 fold for GSH2. Northern blot and RT-PCR analyses show GSH1 and GSH2 are weakly expressed in the mold morphotype and strongly upregulated in the yeast morphotype.;Extensive knockout analysis of CDO1, GSH1, and GSH2 was attempted to evaluate the loss of function on dimorphism. No viable transformants were able to be obtained. These results indicate these genes may be essential in order for the organism to survive in the yeast morphotype. Studies in other dimorphic organisms have had similar results.;Because we were unable to create genetic knockouts, we examined the effects of overexpression to attempt to gain more information regarding the function of these genes. The hypothesis that overexpression of GSH1 or GSH2 in the yeast morphotype would inhibit the transition to the mold morphotype at the permissive temperature (25ºC) was therefore tested. Overexpression of GSH1 or GSH2 (driven by the strong Tef1 promoter) resulted in yeast cells that were unable to shift to the mold morphotype at 25ºC. Northern blot analysis of the transformants showed strong expression of GSH1 and GSH2 in the mold morphotype. Growth analysis did not indicate any differences in the rate of growth between the wildtype and transformants in the yeast morphotype.