Role of CpALS4790 and CpALS0660 in Candida parapsilosis Virulence: Evidence from a Murine Model of Vaginal Candidiasis.

The Candida parapsilosis genome encodes for five agglutinin-like sequence (Als) cell-wall glycoproteins involved in adhesion to biotic and abiotic surfaces. The work presented here is aimed at analyzing the role of the two still uncharacterized ALS genes in C. parapsilosis, CpALS4790 and CpALS0660, by the generation and characterization of CpALS4790 and CpALS066 single mutant strains. Phenotypic characterization showed that both mutant strains behaved as the parental wild type strain regarding growth rate in liquid/solid media supplemented with cell-wall perturbing agents, and in the ability to produce pseudohyphae. Interestingly, the ability of the CpALS0660 null mutant to adhere to human buccal epithelial cells (HBECs) was not altered when compared with the wild-type strain, whereas deletion of CpALS4790 led to a significant loss of the adhesion capability. RT-qPCR analysis performed on the mutant strains in co-incubation with HBECs did not highlight significant changes in the expression levels of others ALS genes. In vivo experiments in a murine model of vaginal candidiasis indicated a significant reduction in CFUs recovered from BALB/C mice infected with each mutant strain in comparison to those infected with the wild type strain, confirming the involvement of CpAls4790 and CpAls5600 proteins in C. parapsilosis vaginal candidiasis in mice.

CpALS4770 and CpALS4780 contribution to the virulence of Candida parapsilosis.

The ability of yeast to adhere to biotic and abiotic surfaces represents an essential trait during the early stages of infection. Agglutinin-like sequence (Als) cell-wall proteins play a key role in adhesion of Candida species. Candida parapsilosis genome encompasses 5 ALS members, of which only the role of CPAR2_404800 has been elucidated. The present project was aimed at investigating the contribution of C. parapsilosis Als proteins by generating edited strains lacking functional Als proteins. CPAR2_404770 and CPAR2_404780, further indicated as CpALS4770 and CpALS4780, were selected for the generation of single and double edited strains using an episomal CRISPR/Cas9 technology. Phenotypic characterization of mutant strains revealed that editing of both genes had no impact on the in vitro growth of C. parapsilosis or on morphogenesis. Notably, CpALS4770-edited strain showed a reduction of biofilm formation and adhesive properties to human buccal cells (HBECs). Conversely, single CpALS4780-edited strain did not show any difference compared to the wild-type strain in all the assays performed, while the double CpALS4770-CpALS4780 mutant revealed an increased ability to produce biofilm, a hyper-adhesive phenotype to HBECs, and a marked tendency to form cellular aggregates. Murine vaginal infection experiments indicated a significant reduction in CFUs recovered from BALC/c mice infected with single and double edited strains, compared to those infected with the wild-type strain. These finding clearly indicate that CpAls4770 plays a role in adhesion to biotic and abiotic surfaces, while both CpALS4770 and CpALS4780 genes are required for C. parapsilosis ability to colonize and persist in the vaginal mucosa.

Candida vaginitis: virulence, host response and vaccine prospects.

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.

Use of SCW4 gene primers in PCR methods for the identification of six medically important Aspergillus species.

Aspergillus species are the cause of invasive mold infections in immunocompromised patients: Aspergillus fumigatus, A. flavus and A. terreus account for most cases of invasive aspergillosis (IA). As certain species are associated with higher mortality and vary in their resistance to antifungal therapy, diagnosis requires increasingly rapid molecular methods that enable sensitive detection and species discrimination. We have developed PCR and Multiplex PCR assays for the detection of six medically important Aspergillus spp. species DNA in bronchoalveolar lavage (BAL) specimens from hematology and intensive care unit (ICU) patients at risk of IA, using different species and genus-specific PCR primers, selected within the SCW4 gene, encoding a cell wall glucanase of A. fumigatus, similar to mannoprotein Mp65 of Candida albicans. The genus-specific PCR primers were able to amplify only Aspergillus DNAs but not that belonging to other fungal genera tested. The species-specific PCR primers allowed differentiation of each Aspergillus species by the amplicon length produced. The methods described in this study are rapid (less than 4 h), reproducible, simple and specific and demonstrate potential application in the clinical laboratory.

Studies of Immune Responses in Candida vaginitis.

The widespread occurrence of vaginal candidiasis and the development of resistance against anti-fungal agents has stimulated interest in understanding the pathogenesis of this disease. The aim of our work was to characterize, in an animal model of vaginal candidiasis, the mechanisms that play a role in the induction of mucosal immunity against C. albicans and the interaction between innate and adaptive immunity. Our studies evidenced the elicitation of cell-mediated immunity (CMIs) and antibody (Abs)-mediated immunity with a Th1 protective immunity. An immune response of this magnitude in the vagina was very encouraging to identify the proper targets for new strategies for vaccination or immunotherapy of vaginal candidiasis. Overall, our data provide clear evidence that it is possible to prevent C. albicans vaginal infection by active intravaginal immunization with aspartyl proteinase expressed as recombinant protein. This opens the way to a modality for anti-Candida protection at the mucosa. The recombinant protein Sap2 was assembled with virosomes, and a vaccine PEVION7 (PEV7) was obtained. The results have given evidence that the vaccine, constituted of virosomes and Secretory aspartyl proteinase 2 (Sap2) (PEV7), has an encouraging therapeutic potential for the treatment of recurrent vulvovaginal candidiasis.

Candida albicans OPI1 regulates filamentous growth and virulence in vaginal infections, but not inositol biosynthesis.

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.

AIDS-related mycoses: the way forward.

The contribution of fungal infections to the morbidity and mortality of HIV-infected individuals is largely unrecognized. A recent meeting highlighted several priorities that need to be urgently addressed, including improved epidemiological surveillance, increased availability of existing diagnostics and drugs, more training in the field of medical mycology, and better funding for research and provision of treatment, particularly in developing countries.

Evaluation of efficacy, pharmacokinetics and tolerability of peptidomimetic aspartic proteinase inhibitors as cream formulation in experimental vaginal candidiasis.

OBJECTIVES: It has been previously shown that the treatment with the two protease inhibitors APG12 and APG19 confers protection in a rat model of mucosal candidiasis; in this study, we examined whether these peptidomimetic inhibitors are also effective as a cream formulation in reducing Candida albicans vaginal infection. METHODS: These efficacy studies were performed in a rat model of estrogen-dependent rat vaginitis by C. albicans on both azole-susceptible and azole-resistant C. albicans, and on both caspofungin-susceptible and caspofungin-resistant C. albicans strains. In vivo studies were also conducted in female albino rats and rabbits to obtain information about the safety, local tolerability and principal pharmacokinetics parameters of the two compounds. KEY FINDINGS AND CONCLUSIONS: Both hit compounds showed remarkable results within the 48-h range as effective inhibitors of the infection, particularly causing rapid decay of vaginal C. albicans burden. Importantly, the two compounds showed marked acceleration of fungus clearance in the rats challenged with the fluconazole-resistant as well as with the capsofungin-resistant strain of C. albicans. Both compounds showed fast elimination rates when given by the intravenous route, and poor systemic absorption after intravaginal cream administration. Test drugs were also well tolerated in 7-day local tolerability experiments in the rabbit.

IL-22 and IDO1 affect immunity and tolerance to murine and human vaginal candidiasis.

The ability to tolerate Candida albicans, a human commensal of the gastrointestinal tract and vagina, implicates that host defense mechanisms of resistance and tolerance cooperate to limit fungal burden and inflammation at the different body sites. We evaluated resistance and tolerance to the fungus in experimental and human vulvovaginal candidiasis (VVC) as well as in recurrent VVC (RVVC). Resistance and tolerance mechanisms were both activated in murine VVC, involving IL-22 and IL-10-producing regulatory T cells, respectively, with a major contribution by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 was responsible for the production of tolerogenic kynurenines, such that replacement therapy with kynurenines restored immunoprotection to VVC. In humans, two functional genetic variants in IL22 and IDO1 genes were found to be associated with heightened resistance to RVVC, and they correlated with increased local expression of IL-22, IDO1 and kynurenines. Thus, IL-22 and IDO1 are crucial in balancing resistance with tolerance to Candida, their deficiencies are risk factors for RVVC, and targeting tolerance via therapeutic kynurenines may benefit patients with RVVC.

Comparison of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis adhesive properties and pathogenicity.

Retrospective studies indicate that Candida metapsilosis and Candida orthopsilosis each represents 1-10% of the infections/colonisations attributed to C. parapsilosis by conventional biochemical tests. Little is known on the virulence properties of these fungi and on their role in the establishment/progression of the infection. In this study, the adhesive properties of clinical isolates belonging to the 'psilosis' species were assessed in an in vitro model of co-incubation with human buccal epithelial cells (HBECs). Ectophosphatase activity was also measured for all isolates, since the activity of this enzyme has previously been linked to adhesion properties in C. parapsilosis. The results indicate that whilst C. parapsilosis and C. orthopsilosis strains showed similar adhesion abilities, C. metapsilosis isolates displayed a significantly lower ability to adhere to HBECs (P<0.05). No evidence of a correlation between ectophosphatase activity and adhesion was observed, and this finding was also confirmed by phosphatase inhibition experiments. Experimental vaginal candidiasis induced in oestrogen-treated mice with representative isolates of the 3 species indicated that mice infected with C. metapsilosis displayed a reduced vaginal fungal burden, especially in the early stages of the infection. The overall findings confirm that C. orthopsilosis has a comparable behaviour to C. parapsilosis, whilst C. metapsilosis seems to possess a reduced virulence potential.

Bicyclic peptidomimetics targeting secreted aspartic protease 2 (SAP2) from Candida albicans reveal a constrained inhibitory chemotype.

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.

A virosomal vaccine against candidal vaginitis: immunogenicity, efficacy and safety profile in animal models.

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.

Peptides of the constant region of antibodies display fungicidal activity.

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.

A highly immunogenic recombinant and truncated protein of the secreted aspartic proteases family (rSap2t) of Candida albicans as a mucosal anticandidal vaccine.

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.

The MP65 gene is required for cell wall integrity, adherence to epithelial cells and biofilm formation in Candida albicans.

BACKGROUND: The MP65 gene of Candida albicans (orf19.1779) encodes a putative ß-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. RESULTS: The 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 ß-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. CONCLUSIONS: We 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.

Rapid, simple, and low-cost identification of Candida species using high-resolution melting analysis.

The feasibility of using high-resolution melting analysis (HRMA) was examined for its rapid and simple detection of 5 clinically relevant Candida species (C. albicans, C. glabrata, C. kefyr, C. parapsilosis, and C. guilliermondii). HRMA was able to differentiate clinical Candida species and resulted in being sensitive, reproducible, and inexpensive.

Loss of mitochondrial functions associated with azole resistance in Candida glabrata results in enhanced virulence in mice.

Mitochondrial dysfunction is one of the possible mechanisms by which azole resistance can occur in Candida glabrata. Cells with mitochondrial DNA deficiency (so-called "petite mutants") upregulate ATP binding cassette (ABC) transporter genes and thus display increased resistance to azoles. Isolation of such C. glabrata mutants from patients receiving antifungal therapy or prophylaxis has been rarely reported. In this study, we characterized two sequential and related C. glabrata isolates recovered from the same patient undergoing azole therapy. The first isolate (BPY40) was azole susceptible (fluconazole MIC, 4 µg/ml), and the second (BPY41) was azole resistant (fluconazole MIC, >256 µg/ml). BPY41 exhibited mitochondrial dysfunction and upregulation of the ABC transporter genes C. glabrata CDR1 (CgCDR1), CgCDR2, and CgSNQ2. We next assessed whether mitochondrial dysfunction conferred a selective advantage during host infection by testing the virulence of BPY40 and BPY41 in mice. Surprisingly, even with in vitro growth deficiency compared to BPY40, BPY41 was more virulent (as judged by mortality and fungal tissue burden) than BPY40 in both systemic and vaginal murine infection models. The increased virulence of the petite mutant correlated with a drastic gain of fitness in mice compared to that of its parental isolate. To understand this unexpected feature, genome-wide changes in gene expression driven by the petite mutation were analyzed by use of microarrays during in vitro growth. Enrichment of specific biological processes (oxido-reductive metabolism and the stress response) was observed in BPY41, all of which was consistent with mitochondrial dysfunction. Finally, some genes involved in cell wall remodelling were upregulated in BPY41 compared to BPY40, which may partially explain the enhanced virulence of BPY41. In conclusion, this study shows for the first time that mitochondrial dysfunction selected in vivo under azole therapy, even if strongly affecting in vitro growth characteristics, can confer a selective advantage under host conditions, allowing the C. glabrata mutant to be more virulent than wild-type isolates.

Plant production of anti-ß-glucan antibodies for immunotherapy of fungal infections in humans.

There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting ß-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the ß-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially ß1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases.

Protection against rat vaginal candidiasis by adoptive transfer of vaginal B lymphocytes.

Vulvovaginal candidiasis is a mucosal infection affecting many women, but the immune mechanisms operating against Candida albicans at the mucosal level remain unknown. A rat model was employed to further characterize the contribution of B and T cells to anti-Candida vaginal protection. Particularly, the protective role of vaginal B cells was studied by means of adoptive transfer of vaginal CD3(-) CD5(+) IgM(+) cells from Candida-immunized rats to naïve animals. This passive transfer of B cells resulted into a number of vaginal C. albicans CFU approximately 50% lower than their controls. Sorted CD3(-) CD5(+) IgM(+) vaginal B lymphocytes from Candida-infected rats proliferated in response to stimulation with an immunodominant mannoprotein (MP) antigen of the fungus. Importantly, anti-MP antibodies and antibody-secreting B cells were detected in the supernatant and cell cultures, respectively, of vaginal B lymphocytes from infected rats incubated in vitro with vaginal T cells and stimulated with MP. No such specific antibodies were found when using vaginal B cells from uninfected rats. Furthermore, inflammatory and anti-inflammatory cytokines, such as interleukin-2 (IL-2), IL-6 and IL-10, were found in the supernatant of vaginal B cells from infected rats. These data are evidence of a partial anti-Candida protective role of CD3(-) CD5(+) IgM(+) vaginal B lymphocytes in our experimental model.

Outcome of experimental rat vaginitis by Candida albicans isolates with different karyotypes.

Candida albicans isolates with different genomic background, designed as b and c karyotypes, have been previously shown to differentially modulate their response to macrophage candidacidal activity. While b-type isolates were susceptible to intracellular killing, strains with c karyotype survived upon internalization and were able to replicate inside macrophages. Furthermore, it was also shown that c type strains escape microglial cell mediated growth inhibition, suggesting that these strains form a more virulent cluster. In this report, the pathogenicity exerted by C. albicans isolates with b and c karyotypes was analyzed in vivo using a model of experimental rat vaginitis. Although both types induced infection, c-type-infected animals suffered from more persistent vaginitis, confirming the higher virulence potential the c karyotype exerted in vivo. The analysis of fungal cells recovered from vaginal fluids of infected animals indicated that c-type was more prone to undergo morphogenesis and to express SAP2 than b-type; these different traits may account for the differences observed in the outcome of experimental rodent vaginitis induced by the two C. albicans karyotypes.