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Vulvovaginal candidiasis is a common mucosal infection affecting a large proportion of women with some of them affected by recurrent often intractable forms of the disease. Thus, there is an increasing interest in understanding the pathogenesis of this disease. The aim of our work was to characterize, in animal models of vaginal candidiasis, the components of the host-fungus interaction at the mucosal level.The evidence of an immune response in the vaginal compartment was very encouraging to identify the proper targets for new strategies for vaccination or immunotherapy of vaginal candidiasis. Aspartyl-proteinase (Sap2), which is an important immunodominant antigens and virulence factors of C.albicans acting in mucosal infections, was assembled with virosomes and a vaccine PEV7 was obtained. The results obtained in the mouse model and in the clinical trial conducted by Pevion on women have evidenced that the vaccine PEV7, intravaginally administered, has an encouraging therapeutic potential for the treatment of recurrent vulvovaginal candidiasis. This opens the way to a modality for anti-Candida protection at mucosal level.

ScOpi1p is a well-characterized transcriptional repressor and master regulator of inositol and phospholipid biosynthetic genes in the baker's yeast Saccharomyces cerevisiae. An ortholog has been shown to perform a similar function in the pathogenic fungus Candida glabrata, but with the distinction that CgOpi1p is essential for growth in this organism. However, in the more distantly related yeast Yarrowia lipolytica, the OPI1 homolog was not found to regulate inositol biosynthesis, but alkane oxidation. In Candida albicans, the most common cause of human candidiasis, its Opi1p homolog, CaOpi1p, has been shown to complement a S. cerevisiae opi1∆ mutant for inositol biosynthesis regulation when heterologously expressed, suggesting it might serve a similar role in this pathogen. This was tested in the pathogen directly in this report by disrupting the OPI1 homolog and examining its phenotypes. It was discovered that the OPI1 homolog does not regulate INO1 expression in C. albicans, but it does control SAP2 expression in response to bovine serum albumin containing media. Meanwhile, we found that CaOpi1 represses filamentous growth at lower temperatures (30 °C) on agar, but not in liquid media. Although, the mutant does not affect virulence in a mouse model of systemic infection, it does affect virulence in a rat model of vaginitis. This may be because Opi1p regulates expression of the SAP2 protease, which is required for rat vaginal infections.

The in vitro screening of stereoisomeric bicyclic peptidomimetics towards SAP2 of Candida albicans revealed a constrained chemotype as aspartic protease inhibitor in the micromolar to nanomolar range. The results indicated that the acetal bridge may serve as a transition-state isostere, and that the right match between interactions with subsites and the orientation by hydrogen bonding with Gly85 is the main requisite for inhibitory activity. Molecular docking calculations suggested the bicyclic acetal scaffold to be capable of interacting with the two catalytic aspartic acids, thus resulting in good inhibitory activity with only two hydrophobic groups addressing the enzyme catalytic subsites., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A novel vaccine (PEV7) consisting of a truncated, recombinant aspartyl proteinase-2 of Candida albicans incorporated into influenza virosomes was studied. This vaccine candidate generated a potent serum antibody response in mouse and rat following intramuscular immunization. Anti-Sap2 IgG and IgA were also detected in the vaginal fluid of rats following intravaginal or intramuscular plus intravaginal administration. In a rat model of candidal vaginitis, PEV7 induced significant, long-lasting, likely antibody-mediated, protection following intravaginal route of immunization. PEV7 was also found to be safe in a repeated-dose toxicological study in rats. Overall, these data provide a sound basis to envisage the clinical development of this new candidate vaccine against candidal vaginitis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Sap2 (secreted aspartyl proteinase2) is a member of the Sap family of Candida albicans, a human opportunistic pathogen, which acts as a virulence factor in experimental animal models of mucosal candidiasis. The C. albicans SAP2 was subcloned into vector pDS56-RBSII-6xhis, under the control of an inducible promoter to produce a truncated 6xhis-tagged, enzymatically inactive Sap2, lacking the N-terminus 76 amino acids (rSap2t). This recombinant protein was purified to homogeneity by one-step nickel-chelate affinity chromatography and used to immunize intravaginally oophorectomized estradiol-treated rats. These animals raised local anti-rSap2t immunoglobulin G (IgG) and IgA antibodies and were protected from the challenge of a highly vaginopathic strain of the fungus. Protection was possibly due to the specific antibodies as suggested by the passive transfer of immune vaginal fluid and the protective effects of passive vaccination with anti-rSap2t IgM and IgG monoclonal antibodies. Hence, this new recombinant proteinase constitutes a novel tool to investigate mechanisms of anti-Candida protection at the vaginal level and as vaccination against vaginal candidiasis, a common, frequently recurrent and sometimes antimycotic-refractory infection in women., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The screening of a library of small molecule peptidomimetics toward secreted aspartic proteinase-2 (SAP2) of Candida albicans allowed us to identify two compounds that showed in vitro inhibitory potency comparable to pepstatin A. In an experimental model of vaginal candidiasis, the two candidate compounds were as active as a therapeutic dose of fluconazole. Importantly, this activity was fully preserved when the challenger was a fluconazole-resistant strain of the fungus. Altogether, our data demonstrate SAP2 as a valid C. albicans target for the development of new drugs against this important human pathogen.

The Ssk1p response regulator of Candida albicans is required for oxidant adaptation, survival in human neutrophils, and virulence in a disseminated murine model of candidiasis. We have previously shown that the amino acid residues D556 and D513 of the Ssk1p receiver domain are critical to the Ssk1p in oxidant stress adaptation and morphogenesis. Herein, transcriptional profiling is used to explain the oxidant sensitivity and morphogenesis defect of two point mutants (D556N and D513K, respectively) compared with a WT strain. In the D556N mutant, during oxidative stress (5 mM H(2)O(2)), a downregulation of genes associated with redox homeostasis and oxidative stress occurred, which accounted for about 5% of all gene changes, including among others, SOD1 (superoxide dismutase), CAP1 (required for some types of oxidant stress), and three genes encoding glutathione biosynthesis proteins (GLR1, GSH1, and GSH2). Mutant D513K was not sensitive to peroxide but was impaired in its yeast $/to hyphal transition. We noted downregulation of genes associated with morphogenesis and cell elongation. Virulence of each mutant was also evaluated in a rat vaginitis model of candidiasis. Clearance of an SSK1 null and the D556N mutants from the vaginal canal was significantly greater than wild type or the D513K mutant, indicating that a change in a single amino acid of the Ssk1p alters the ability of this strain to colonize the rat vaginal mucosa.

Mannoproteins are fungal cell wall components which play a main role in host-parasite relationship. Camp65p is a putative beta-glucanase mannoprotein of 65 kDa which has been characterized as a main target of human immune response against Candida albicans. However, nothing is known about its specific contribution to the biology and virulence of this fungus. We constructed CAMP65 knock-out mutants including null camp65/camp65 and CAMP65/camp65 heterozygous strains. The null strains had the same growth rate and morphology under yeast form as the wild-type strain but they were severely affected in hyphal morphogenesis both in vitro and in vivo. Hyphae formation was restored in revertant strains. The null mutants lost adherence to the plastic, and this was in keeping with the strong inhibition of fungal cell adherence to plastic exerted by anti-Camp65p antibodies. The null mutants were also significantly less virulent than the parental strains, and this loss of virulence was observed both in systemic and in mucosal C. albicans infection models. Nonetheless, the virulence in both infectious models was regained by the CAMP65 revertants. Thus, CAMP65 of C. albicans encodes a putative beta-glucanase, mannoprotein adhesin, which has a dual role (hyphal cell wall construction and virulence), accounting for the particular relevance of host immune response against this mannoprotein.

Virulence of Candida albicans strains with targeted disruption of secretory aspartyl proteinase genes (SAP1 to SAP6) was assessed in an estrogen-dependent rat vaginitis model. Null sap1 to sap3 but not sap4 to sap6 mutants lost most of the virulence of their parental strain SC5314. In particular, the sap2 mutant was almost avirulent in this model. Reinsertion of the SAP2 gene into this latter mutant led to the to recovery of the vaginopathic potential. The vaginal fluids of the animals infected by the wild type strain or by the sap1 or sap3 mutants expressed a pepstatin-sensitive proteinase activity in vitro. No traces of this activity were found in the vaginal fluid of rats challenged by the sap2 mutant. All strains were capable of developing true hyphae during infection. Thus, members of SAP family, in particular SAP2, play a clear pathogenic role in vaginitis and may constitute a novel target for chemoimmunotherapy of this infection.

Little is known of the biological attributes conferring pathogenicity on the opportunistic fungal pathogen Candida albicans. Infection by this pathogen, as for bacterial pathogens, may rely upon environmental signals within the host niche to regulate the expression of virulence determinants. To determine if C. albicans responds to the pH of the host niche, we tested the virulence of strains with mutations in either of two pH-regulated genes, PHR1 and PHR2. In vitro, PHR1 is expressed when the ambient pH is at 5.5 or higher and deletion of the gene results in growth and morphological defects at neutral to alkaline pHs. Conversely, PHR2 is expressed at an ambient pH below 5.5, and the growth and morphology of the null mutant is compromised below this pH. A PHR1 null mutant was avirulent in a mouse model of systemic infection but uncompromised in its ability to cause vaginal infection in rats. Since systemic pH is near neutrality and vaginal pH is around 4.5, the virulence phenotype paralleled the pH dependence of the in vitro phenotypes. The virulence phenotype of a PHR2 null mutant was the inverse. The mutant was virulent in a systemic-infection model but avirulent in a vaginal-infection model. Heterozygous mutants exhibited partial reductions in their pathogenic potential, suggesting a gene dosage effect. Unexpectedly, deletion of PHR2 did not prevent hyphal development in vaginal tissue, suggesting that it is not essential for hyphal development in this host niche. The results suggest that the pH of the infection site regulates the expression of genes essential to survival within that niche. This implies that the study of environmentally regulated genes may provide a rationale for understanding the pathobiology of C. albicans.

The expression of the Candida albicans complement-binding C3d protein (MP60) was investigated both in vitro and in vivo by immunogold labelling and electron microscopy. In vivo expression was determined in a rat vaginitis model. Reactivity of in vitro-grown cells to an anti-MP60 rabbit serum was associated with both cytoplasmic and cell wall sites. Immunostaining in the cell wall of both yeast and hyphae was most concentrated in the inner, electron-lucid layer. Immunogold stained preparations of C. albicans from vaginal smears of infected animals also showed intense localization of the MP60 in the inner cell wall, plasma membrane. However, immunogold label was also intense at the cell surface in these samples, mostly in the area of close adherence with the keratinocytes of the vaginal epithelia. These observations indicate that MP60 is expressed both in vitro and in vivo, but to a different degree in the different cell wall layers.

Several strains of Candida albicans were compared for their ability to cause vaginal infection in a rat model, and their vaginopathic potentials were correlated with the expression of two aspartyl proteinases genes (SAP1 and SAP2) and adherence in vivo to the vaginal epithelium. Dot blot reactions and Northern blot analysis with RNA extracted from the vaginal fluid of rats infected with the highly vaginopathic strains H12 and 10261 demonstrated the expression of both SAP1 and SAP2 during the first week of infection. In contrast, neither gene was expressed during infection by a nonvaginopathic strain (N), even though the organism could be recovered during the first 24 h postinfection. A moderately vaginopathic strain (P) also expressed both genes, but the level of SAP1 mRNA appeared to decrease prior to that of SAP2. Neither gene was expressed, even by the highly vaginopathic strains, after the first week of infection, concomitant with a decrease in the number of organisms recovered from the vaginas. Analysis of in vivo adherence showed that the nonvaginopathic strain (N) adhered to vaginal epithelial cells less readily than the highly vaginopathic strain (H12) and moderately vaginopathic strain (P). Thus, in addition to its inability to express SAP1 and SAP2 in vivo, the nonvaginopathic strain does not colonize host cells to the same extent as the other strains tested. Our results demonstrate the early in vivo expression of two aspartyl proteinase gene during candidal vaginitis and suggest its association with the establishment of a vaginal infection.

Experimental infections were produced in guinea pigs, rabbits and dogs with lesions similar to those seen in human seborrheic dermatitis and otitis externa by cutaneous application of cultures of Malassezia furfur and M. pachydermatis. Infected animals were treated by topical application of a concentrated yeast killer toxin (Hansenula anomala UCSC 25F). Clinical recovery as well as negative mycological test cultures of infected animals proved to the clearly associated with the treatment by the killer toxin.