Quantitative expression of the Candida albicans secreted aspartyl proteinase gene family in human oral and vaginal candidiasis

Abstract

A quantitative real-time RT-PCR system was established to identify which secreted aspartyl proteinase (SAP) genes are most highly expressed and potentially contribute to Candida albicans infection of human epithelium in vitro and in vivo. C. albicans SC5314 SAP1-10 gene expression was monitored in organotypic reconstituted human epithelium (RHE) models, monolayers of oral epithelial cells, and patients with oral (n=17) or vaginal (n=17) candidiasis. SAP gene expression was also analysed in Deltasap1-3, Deltasap4-6, Deltaefg1 and Deltaefg1/cph1 mutants to determine whether compensatory SAP gene regulation occurs in the absence of distinct proteinase gene subfamilies. In monolayers, RHE models and patient samples SAP9 was consistently the most highly expressed gene in wild-type cells. SAP5 was the only gene significantly upregulated as infection progressed in both RHE models and was also highly expressed in patient samples. Interestingly, the SAP4-6 subfamily was generally more highly expressed in oral monolayers than in RHE models. SAP1 and SAP2 expression was largely unchanged in all model systems, and SAP3, SAP7 and SAP8 were expressed at low levels throughout. In Deltasap1-3, expression was compensated for by increased expression of SAP5, and in Deltasap4-6, expression was compensated for by SAP2: both were observed only in the oral RHE. Both Deltasap1-3 and Deltasap4-6 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 Deltaefg1/cph1 and Deltaefg1, but SAP5 expression was only dramatically reduced in Deltaefg1/cph1 despite the absence of hyphal growth in both mutants. This indicates that hypha formation is the predominant cause of tissue damage, and that SAP5 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 SAP gene expression levels during human mucosal infections; the results suggest that SAP5 and SAP9 are the most highly expressed proteinase genes in vivo. 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.

Fig. 1

Quantitative gene expression changes of SAP2 and the SAP4–6 subfamily in culture medium. (a) SAP2 upregulation in YCB/1 % BSA over 30 h; (b) SAP4–6 upregulation in PBS/10 % serum over 24 h, normalized to ACT1.

Fig. 2

Histological light micrographs of SC5314, Δsap1–3, Δsap4–6, Δefg1 and Δefg1/Δcph1 infecting oral RHE for up to 24 h. Little difference in surface biofilm development or invasion was evident over 24 h with SC5314, Δsap1–3 or Δsap4–6. In contrast, the Δefg1 and Δefg1/Δcph1 mutants were not invasive and surface biofilms did not develop. Δefg1 cells were clearly present at 12 h and 24 h but Δefg1/Δcph1 cells were difficult to detect.

Fig. 3

Compensatory upregulation of SAP1–10 in hypha-deficient mutants at 12 h and 24 h in the oral RHE model, relative to YPD-grown preculture cells. SAP5 was markedly reduced in expression in Δefg1 and particularly in Δefg1/Δcph1 at 12 h and 24 h. SAP1, SAP3, SAP7 and SAP8 appeared to be moderately upregulated in both Δefg1 and Δefg1/Δcph1 at 12 h, but this diminished by 24 h.

Fig. 4

Quantitative expression of SAP1–10 mRNA expression in patient samples. Samples from individual patients are represented by different coloured bars. mRNA transcripts (arbitrary units) of SAP1–10 in (a) oral and (b) vaginal (n=17) samples, normalized against the housekeeping control gene CEF3 (10⁴ mRNA transcripts). SAP9 was the most highly expressed gene in vivo. SAP5 was also relatively highly expressed, and together with SAP10 was expressed in all patient samples (n=17/17).