Expression of Penicillium marneffei glucan synthase genes in response to cell-wall stressors

Abstract

Penicillium marneffei is an opportunistic, dimorphic fungal pathogen that causes penicilliosis in AIDS patients. In an effort to genetically characterize this organism, Kummasook et al. developed an Agrobacterium tumefaciens-mediated transformation system in 2010. This system allowed for the production of thousands of P. marneffei mutant strains that could be further assessed in order to assess gene-to-function relationships. In 2014, Suwunnakorn et al., characterized one of these mutants, I231, and determined the presence of an interrupted yakA gene which codes for a dual-specificity tyrosine phosphorylation-regulated protein kinase. They determined that the yakA mutant produced fewer conidia, possessed increased chitin content, and exhibited unique cell-wall stress responses when compared to wild-type P. marneffei. The present study seeks to investigate these stress responses in the yakA mutant, a F4 wild-type strain, and the yakA complement strain CY21 at a genetic level. Using qRT-PCR, expression of three glucan synthase genes (fksP, ags2, and kre6) was quantified using the benA beta-tubulin gene as a reference gene for normalization. The three strains were subjected to cell-wall stressors including the anionic dye Congo red, the detergent sodium dodecyl sulfate, and the glucan synthase-specific antifungal drug caspofungun and assessed for glucan synthase gene expression. The resulting gene expressions indicated that the yakA mutant actually possessed relatively larger stress responses than the wild-type, indicating that the yakA gene and its resulting protein may be responsible for the regulation of glucan synthase gene expression in response to cell-wall stress. This reasoning is reinforced by the presence of increased chitin in the yakA mutant, a characteristic common to fungal mutants lacking proper glucan regulation in their cell-walls.