1887

Abstract

A quantitative real-time RT-PCR system was established to identify which secreted aspartyl proteinase () genes are most highly expressed and potentially contribute to infection of human epithelium and . SC5314 gene expression was monitored in organotypic reconstituted human epithelium (RHE) models, monolayers of oral epithelial cells, and patients with oral (=17) or vaginal (=17) candidiasis. gene expression was also analysed in Δ, Δ, Δ and Δ mutants to determine whether compensatory gene regulation occurs in the absence of distinct proteinase gene subfamilies. In monolayers, RHE models and patient samples was consistently the most highly expressed gene in wild-type cells. was the only gene significantly upregulated as infection progressed in both RHE models and was also highly expressed in patient samples. Interestingly, the subfamily was generally more highly expressed in oral monolayers than in RHE models. and expression was largely unchanged in all model systems, and , and were expressed at low levels throughout. In Δ, expression was compensated for by increased expression of , and in Δ, expression was compensated for by : both were observed only in the oral RHE. Both Δ and Δ mutants caused RHE tissue damage comparable to the wild-type. However, addition of pepstatin A reduced tissue damage, indicating a role for the Sap family as a whole in inducing epithelial damage. With the hypha-deficient mutants, RHE tissue damage was significantly reduced in both Δ and Δ, but expression was only dramatically reduced in Δ despite the absence of hyphal growth in both mutants. This indicates that hypha formation is the predominant cause of tissue damage, and that expression can be hypha-independent and is not solely controlled by the Efg1 pathway but also by the Cph1 pathway. This is believed to be the first study to fully quantify gene expression levels during human mucosal infections; the results suggest that and are the most highly expressed proteinase genes . However, the overall contribution of the Sap1–3 and Sap4–6 subfamilies individually in inducing epithelial damage in the RHE models appears to be low.

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2008-11-01
2019-09-18
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