Secreted aspartic proteinase (Sap) activity contributes to tissue damage in a model of human oral candidosis

Abstract

Secreted aspartic proteinases (Saps) are important virulence factors during Candida albicans mucosal or disseminated infections. A differential expression of individual SAP genes has been shown previously in a model of oral candidosis based on reconstituted human epithelium (RHE), and in the oral cavity of patients. In this study, the ultrastructural localization of distinct groups of Sap isoenzymes expressed during RHE infection was shown by immunoelectron microscopy using specific polyclonal antibodies directed against the gene products of SAP1‐3 and SAP4‐6. Large amounts of Sap1‐3 antigen were found within C. albicans yeast and hyphal cell walls, often predominantly in close contact with epithelial cells, whereas lower quantities of Sap4‐6 were detected in hyphal cells. To elucidate the relevance of the expressed Saps during oral infections, we examined the effect of the aspartic proteinase inhibitor, pepstatin A, during infection of the RHE. The extent of lesions caused by the strain SC5314 was found to be strongly reduced by the inhibitor, indicating that proteinase activity contributes to tissue damage in this model. To clarify which of the SAP genes are important for tissue necrosis, the histology of RHE infection with Δsap1, Δsap2, Δsap3, Δsap4‐6 and three Δsap1/3 double mutants were examined. Although tissue damage was not blocked completely with these mutants, an attenuated phenotype was observed for each of the single sap null mutants, and was more strongly attenuated in the Δsap1/3 double null mutants. In contrast, the lesions caused by the Δsap4‐6 triple mutant were at least as severe as those caused by SC5314. During infection with the mutants, we observed that the SAP gene expression pattern of the Δsap1 and the Δsap1/3 mutants was altered in comparison with the wild‐type strain. Expression of SAP5 was observed only during infection with the Δsap1/3 mutant, whereas upregulation of SAP2 and SAP8 transcripts was observed in the Δsap1 and the Δsap1/3 mutants. These results suggest that Sap1‐3, but not Sap4‐6, contribute to tissue damage in this model. Furthermore, C. albicans may compensate for the deletion of certain SAP genes by upregulation of alternative SAP genes.

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.…”

“…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).…”

Discovery of the gray phenotype and white-gray-opaque tristable phenotypic transitions inCandida dubliniensis

“…These secreted enzymes degrade animal tissues and facilitate fungal growth and penetration during infections. 23,25,32,33 We performed ex vivo tongue infection assays and found that the order of growth rates for the 3 cell types was opaque cells>gray cells>white cells at both 30 C (Fig. S6A) and 37 C (Fig.…”