The secreted aspartyl proteinases Sap1 and Sap2 cause tissue damage in an in vitro model of vaginal candidiasis based on reconstituted human vaginal epithelium

Abstract

Secreted aspartyl proteinases (Saps) contribute to the ability of Candida albicans to cause mucosal and disseminated infections. A model of vaginal candidiasis based on reconstituted human vaginal epithelium (RHVE) was used to study the expression and role of these C. albicans proteinases during infection and tissue damage of vaginal epithelium. Colonization of the RHVE by C. albicans SC5314 did not cause any visible epithelial damage 6 h after inoculation, although expression of SAP2, SAP9, and SAP10 was detected by reverse transcriptase PCR. However, significant epithelial damage was observed after 12 h, concomitant with the additional expression of SAP1, SAP4, and SAP5. Additional transcripts of SAP6 and SAP7 were detected at a later stage of the artificial infection (24 h). Similar SAP expression profiles were observed in three samples isolated from human patients with vaginal candidiasis. In experimental infection, secretion of antigens Sap1 to Sap6 by C. albicans was confirmed at the ultrastructural level by using polyclonal antisera raised against Sap1 to Sap6. Addition of the aspartyl proteinase inhibitors pepstatin A and the human immunodeficiency virus proteinase inhibitors ritonavir and amprenavir strongly reduced the tissue damage of the vaginal epithelia by C. albicans cells. Furthermore, SAP null mutants lacking either SAP1 or SAP2 had a drastically reduced potential to cause tissue damage even though SAP3, SAP4, and SAP7 were up-regulated in these mutants. In contrast the vaginopathic potential of mutants lacking SAP3 or SAP4 to SAP6 was not reduced compared to wild-type cells. These data provide further evidence for a crucial role of Sap1 and Sap2 in C. albicans vaginal infections.

FIG. 1.

Light micrographs of RHVE and SAP expression pattern as obtained by RT-PCR 6, 12, and 24 h after infection with C. albicans SC5314. (A) Colonization of RHVE without marked morphological alteration and expression of SAP2, SAP9, and SAP10 were observed 6 h after infection with SC5314. (B) Invasion of C. albicans with vacuolization and edema at 12 h was accompanied by additional expression of SAP1, SAP4, and SAP5. (C) Later stage of infection at 24 h with extensive edema, vacuolization, and additional SAP6 and SAP7 transcripts. (D) A 564-bp fragment size obtained by amplification with EFB1 primers demonstrates the cDNA origin of the templates used for panels A to C, whereas the same set of primers amplified an 928-bp intron-containing fragment when genomic (g) DNA was used as a template. In lanes 1 the molecular mass marker (M) XIII (PEQLAB, Erlangen, Germany) was used, giving fragments that were 1,353, 1,078, 872, and 603 bp long.

FIG. 2.

Appearance of RHVE 24 h after infection with C. albicans SC5314 (A), SC5314 in the presence of pepstatin A (15 μM) (B), sap1 (C), and sap2 (D) mutant cells. Severe vacuolization, edema, and detachment of all epithelial layers are only seen in SC5314 infection without the addition of pepstatin A (A). In contrast, a reduced-virulence phenotype was observed in the presence of 15 μM pepstatin A (B) and after infection with the sap1 (C) and the sap2 (D) mutants.

FIG. 3.

Appearance of RHVE 24 h after infection with C. albicans SC5314 (A), sap3 (B), and sap4-to-sap6 (C) mutant cells. Tissue damage caused by sap3 (B) and sap4-to-sap6 (C) mutants is not attenuated compared with SC5314 (A) infection. Histological damage after infection with the sap4-to-sap6 triple mutant (C) seemed to be increased compared with the parental strain (A).

FIG. 4.

Appearance of RHVE 12 h after infection with C. albicans SC5314 (A and D), sap1 mutant (sap1::hisG/sap1::hisG::URA3::hisG) (B), SAP1 revertant (sap1::hisG/sap1::hisG/SAP1) (C), sap2 mutant (sap2::hisG/sap2::hisG::URA3::hisG) (E), and SAP2 revertant (sap2::hisG/sap2::hisG/SAP2) (F) cells. A similar degree of tissue damage was seen after infection with SC5314 (A and D) and the SAP1 (C) and SAP2 (F) reconstituted strains, while reduced tissue damage was observed for sap1 (B) and sap2 mutants (E).

FIG. 5.

Electron microscopy with postembedding immunogold labeling, by use of 10-nm gold particles, in a sample of RHVE taken 12 h after infection with SC5314. Increased amounts of Sap1-to-Sap3 (A) compared to Sap4-to-Sap6 (B) antigen were detected within the cell wall of C. albicans yeast cells. The scale bar represents 0.5 μm.

FIG. 6.

Analysis of the in vivo expression of the SAP gene family in three patients with vaginal candidiasis (A) indicating the number of positive signals detected for each SAP gene. RT-PCR products of RNA (lanes 2 to 11) from patient 3 (B). Molecular mass marker pBR322 DNA/MvaI (M) (MBI Fermentas, St. Leon-Rot, Germany) with fragments that were 1,857, 1,058, 929, and 383 bp long (lane 1).