Figure 4.

C. neoformans Cho1 is a PS synthase, and its expression is regulated by inositol. A, expression of Cn CHO1 gene fully complements S. cerevisiae (Sc) cho1Δ mutant auxotrophic for ethanolamine (Etn). WT S. cerevisiae can grow well in the absence of ethanolamine unlike the Sc cho1Δ mutant transformed with pTH19 (empty vector), which is ethanolamine auxotrophic. Sc cho1Δ expressing Cn CHO1 restores growth in the absence of ethanolamine, indicating that C. neoformans CHO1 gene can fully complement Sc CHO1 gene function. B, enzymatic analysis of Saccharomyces strains for PS synthase activity. Cho1 enzyme activity in WT S. cerevisiae was absent in Sc cho1Δ strain. This activity was successfully restored when the Sc cho1Δ strain was transformed with Cn CHO1 gene indicating that it is a PS synthase. The assay was performed in duplicate and repeated twice. C, Cn CHO1 gene expression is regulated by inositol. CHO1 gene expression is suppressed in the presence of 1% inositol as compared with its expression in the presence of 0.1% inositol indicating that phospholipid precursor inositol negatively regulates CHO1 gene expression. The analysis of CHO1 gene expression was performed in triplicate, and the two-tail t test was used for statistical analysis (* indicates p < 0.001).