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.

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Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans , Candida glabrata , Cryptococcus neoformans , and Malassezia furfur , including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro . An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.

BackgroundThe MP65 gene of Candida albicans (orf19.1779) encodes a putative β-glucanase mannoprotein of 65 kDa, which plays a main role in a host-fungus relationship, morphogenesis and pathogenicity. In this study, we performed an extensive analysis of a mp65Δ mutant to assess the role of this protein in cell wall integrity, adherence to epithelial cells and biofilm formation.ResultsThe mp65Δ mutant showed a high sensitivity to a range of cell wall-perturbing and degrading agents, especially Congo red, which induced morphological changes such as swelling, clumping and formation of hyphae. The mp65Δ mutant showed an activation of two MAPKs (Mkc1p and Cek1p), a high level of expression of two stress-related genes (DDR48 and SOD5), and a modulated expression of β-glucan epitopes, but no gross changes in cell wall polysaccharide composition. Interestingly, the mp65Δ mutant displayed a marked reduction in adhesion to BEC and Caco-2 cells and severe defects in biofilm formation when compared to the wild type. All of the mentioned properties were totally or partially recovered in a revertant strain, demonstrating the specificity of gene deletion.ConclusionsWe demonstrate that the MP65 gene of Candida albicans plays a significant role in maintaining cell wall integrity, as well as in adherence to epithelia and biofilm formation, which are major virulence attributes of this fungus.

A 65 kDa-constituent (MP65) of a whole-cell mannoprotein (MP) fraction of Candida albicans was purified by immunoaffinity chromatography with monoclonal antibodies directed against periodate-insensitive, protease-sensitive MP epitopes, putatively polypeptide in nature. These antibodies were obtained by immunization of mice with concanavalin A bead-coupled, low-glycosylated MP from hyphal cells of C. albicans grown in the presence of a subinhibitory dose of tunicamycin. The immunoaffinity-purified MP65 molecule had a pI of 4.1 and a protein/polysaccharide ratio of 1.8:1. It was resistant to hydrolysis by endoglycosidase H, endoglycosidase F, or N-glycanases but still reactive with concanavalin A. The polysaccharide moiety of MP65 was composed exclusively of mannose and glucose at a ratio of 12.7 to 1. The protein moiety showed numerous potential O-glycosidic linkage sites as suggested by the high proportion of serine and threonine (together accounting for more than 20% of the total amino acid composition) and susceptibility to diluted alkali. This treatment and digestion with ␣-mannosidase caused a reduction in the MP65 molecular mass to around 54 kDa. The N-terminal sequence of MP65 protein moiety was rich in alanine and valine (7 of 13 amino acids) and did not show any significant homology with deposited sequences in data banks. Purified MP65, at doses of a few nanograms, induced extensive T-cell proliferation of human peripheral blood mononuclear cells. This proliferation was specifically inhibited, in a dose-response fashion, by the antigen-binding fragment of the monoclonal antibody used for immunoaffinity purification. Overall, these results highlight biochemical and molecular details of MP65, a main target of human T-cell response to C. albicans.

This study analyzes the phenotype of vaginal dendritic cells (VDCs), their antigenic presentation and activation of T-cell cytokine secretion, and their protective role in a rat model of Candida vaginitis. Histological observation demonstrated a significant accumulation of OX62؉ VDCs in the mucosal epithelium of Candida albicans-infected rats at the third round of infection. We identified two subsets of OX62 ؉ VDCs differing in the expression of CD4 molecule in both noninfected and Candida-infected rats. The OX62 ؉ CD4 ؉ subset of VDCs displayed a lymphoid cell-like morphology and expressed the T-cell antigen CD5, whereas the OX62 ؉ CD4؊ VDC subset exhibited a myeloid morphology and was CD5 negative. Candida infection resulted in VDC maturation with enhanced expression of CD80 and CD134L on both CD4 ؉ and CD4 ؊ VDC subsets at 2 and 6 weeks after Candida infection. CD5 ؊ CD4 ؊ CD86 ؊ CD80 ؊ CD134L ؉ VDCs from infected, but not noninfected, rats spontaneously released large amounts of interleukin-12 (IL-12) and tumor necrosis factor alpha, whereas all VDC subsets released comparable levels of IL-10 and IL-2 cytokines. Furthermore, OX62؉ VDCs from infected rats primed naïve CD4 ؉ T-cell proliferation and release of cytokines, including gamma interferon, IL-2, IL-6, and IL-10, in response to staphylococcal enterotoxin B stimulation in vitro. Adoptive transfer of highly purified OX62 ؉ VDCs from infected rats induced a significant acceleration of fungal clearance compared with that in rats receiving naive VDCs, suggesting a protective role of VDCs in the anti-Candida mucosal immunity. Finally, VDC-mediated protection was associated with their ability to rapidly migrate to the vaginal mucosa and lymph nodes, as assessed by adoptive transfer of OX62 ؉ VDCs labeled with 5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester.Vulvovaginal candidiasis, mostly caused by Candida albicans, is a common mucosal infection affecting a large proportion of women of child-bearing age (44). There is broad agreement that local rather than systemic immunity plays a critical role (18,19). This aspect has been previously reported (13) where the accelerated clearing of the infection occurred following immunization in an oophorectomized rat model of Candida albicans infection. This was characterized by the increased number of activated CD4 ϩ CD25 ϩ T cells and CD5 ϩ B cells; in vitro proliferation of vaginal lymphocytes in response to Candida antigenic stimulation; and the presence of cytokines, namely, interleukin-12 (IL-12), gamma interferon (IFN-␥), and IL-2, and protective antibodies (Abs) against mannan and aspartyl proteinase antigens in the vaginal fluids (13). More importantly, adoptive transfer of vaginal lymphocytes, and in particular purified CD4 ϩ T cells, can protect normal rats from Candida infection (42).Dendritic cells (DCs) are key inducers of both innate and adaptive immunity (2, 4). These cells patrol the tissues, phagocytosing pathogens, and also infected or dying cells. Once they are exposed to inflammatory mediato...

Highly active anti-retroviral therapies (HAART) with human immunodeficiency virus (HIV) protease inhibitors (PIs) or nonnucleoside reverse-transcriptase inhibitors (NNRTI) were compared for their effect on prevalence, aspartyl proteinase (Sap) production and the biotypes and anti-mycotic sequential susceptibility of Candida spp. isolates from the oral cavity in a longitudinal prospective study. HAART-PI, but not HAART-NNRTI strongly inhibited Sap expression in the oral cavity without exerting any consistent effect on the role of Candida spp. isolation or selection of low virulence or anti-mycotic resistant fungus biotype. More importantly, the sequential isolates of Candida albicans from HAART-PI, but not those from suspended HAART-NNRTI, showed an increased Sap production in vitro. While further demonstrating that HIV-PI inhibit Sap expressions, our results do not support the view that the mentioned inhibition could eliminate Candida or its selection of the oral cavity.

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.