Differential expression of secreted aspartyl proteinases in a model of human oral candidosis and in patient samples from the oral cavity

Abstract

SummaryCandida albicans, an opportunistic pathogen in humans, secretes secretory aspartyl proteinases (Saps), which have been correlated with virulence. We examined the temporal regulation of the mRNA expression of seven known members of the SAP gene family by reverse transcription polymerase chain reaction (RT-PCR) in (i) an in vitro model of oral candidosis based on reconstituted human epithelium (RHE); and (ii) clinical samples from patients with oral candidosis. SAP1 and SAP3 transcripts were first detected 42 h after inoculation of RHE, while at the same time, slight morphological alterations in the epithelium were documented by light microscopy. SAP6 expression occurred 6 h later concomitantly with germ tube formation of some infecting Candida cells and severe lesions of the epithelial tissue. SAP2 and SAP8 RT-PCR products were first detected 60 h after infection, while SAP4 and SAP5 transcripts were never discovered. Thus, a temporal progression of SAP expression in the order SAP1 and SAP3 > SAP6 > SAP2 and SAP8 was observed at the same time as increasing RHE damage occurred. At the protein level, Sap antigen was found within the C. albicans yeast cells and the epithelial cells by immunoelectron microscopy using an anti-Sap murine monoclonal antibody directed against the gene products Sap1-3. Expression of SAP1-3 and 6 was also detected by RT-PCR in samples from patients suffering from oral candidosis. Our results suggest that the pathogenesis of experimental and clinical oral candidosis is associated with the differential and temporal regulation of SAP gene expression.

Reduced expression of the hyphal-independent Candida albicans proteinase genes SAP1 and SAP3 in the efg1 mutant is associated with attenuated virulence during infection of oral epithelium

“…For this study, the clinical C. albicans wild-type isolate designated SC5314 (Gillum et al, 1984), the single mutants cph1 (Lo et al, 1997) and efg1 (Stoldt et al, 1997), the double mutant cph1/efg1 (Lo et al, 1997) and an EFG1 reconstituted strain (Lo et al, 1997) were used. For the incubation of C. albicans with PMNs and for the infection of the reconstituted epithelium, inocula were prepared as reported previously (Schaller et al, 1998(Schaller et al, , 1999.…”

“…The medium was changed every 24 h. Three replicate infection experiments were performed with the C. albicans wild-type SC5314, the single mutants cph1 and efg1, the double mutant cph1/efg1 and the EFG1 revertant. Epithelia were infected with 2310 6 C. albicans yeast cells of each strain in 50 ìl PBS for 12, 36, 46 and 52 h as described previously (Schaller et al, 1998(Schaller et al, , 1999. Histological lesions of the samples caused by C. albicans were investigated by light microscopy.…”

“…Light microscopical studies were performed as previously described (Schaller et al, 1998(Schaller et al, , 1999 to evaluate histological changes during infection. A part of each specimen was fixed, postfixed and embedded in glycide ether.…”

“…Post-embedding immunogold labelling was carried out as previously described (Schaller et al, 1998(Schaller et al, , 1999 for intracellular detection of Sap antigen in epithelial samples infected with SC5314, cph1, efg1, cph1/efg1 and the EFG1 revertant taken after 12 and 36 h. After fixation, specimens were embedded in LR-White. Sections, 80-100 nm thick, were mounted on nickel grids.…”

“…We therefore investigated the interaction of the cph1, efg1 and cph1/efg1 mutants, their parental wild-type strain SC5314 and an EFG1 revertant with reconstituted human oral epithelium (RHE) and with PMNs. Previously we have shown that certain secreted aspartyl proteinases (Saps) contribute to virulence in this model (Schaller et al, 1998(Schaller et al, , 1999 and that the mucosal immune response is modulated differentially by different Candida species depending on their virulence potential (Schaller et al, 2002). Recently it has been shown that Efg1 is involved in the regulation of both hyphal formation and expression of hyphal-associated SAP genes (Schröppel et al, 2000;Staib et al, 2002;Felk et al, 2002).…”