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.

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.

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.

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.

Human domain antibodies against virulence traits of Candida albicans inhibit fungus adherence to vaginal epithelium and protect against experimental vaginal candidiasis.

Antibody variable domains (domain antibodies [DAbs]) are genetically engineered antibody fragments that include individual heavy-chain (VH) or kappa-chain (Vkappa) variable domains and lack the Fc region. Human DAbs against the 65-kDa mannoprotein (MP65) or the secretory aspartyl proteinase (SAP)-2 of Candida albicans (monospecific DAbs) or against both fungal antigens (heterodimeric, bispecific DAbs) were generated from phage expression libraries. Both monospecific and bispecific DAbs inhibited fungus adherence to the epithelial cells of rat vagina and accelerated the clearance of vaginal infection with the fungus. When heterodimeric DAbs were used, the clearance of infection was at least equivalent to treatment with fluconazole. The in vivo protective effects of DAbs were demonstrated by both pre- and postchallenge schedules of DAb administration and with both fluconazole-susceptible and fluconazole-resistant strains of C. albicans. This is the first demonstration that human DAbs lacking the Fc constituent can efficiently control an infection and can act largely by inhibiting adherence.

In vivo activity of terpinen-4-ol, the main bioactive component of Melaleuca alternifolia Cheel (tea tree) oil against azole-susceptible and -resistant human pathogenic Candida species.

BACKGROUND: Recent investigations on the antifungal properties of essential oil of Melaleuca alternifolia Cheel (Tea Tree Oil, TTO) have been performed with reference to the treatment of vaginal candidiasis. However, there is a lack of in vivo data supporting in vitro results, especially regarding the antifungal properties of TTO constituents. Thus, the aim of our study was to investigate the in vitro and the in vivo anti-Candida activity of two critical bioactive constituents of TTO, terpinen-4-ol and 1,8-cineole. METHODS: Oophorectomized, pseudoestrus rats under estrogen treatment were used for experimental vaginal infection with azole (fluconazole, itraconazole) -susceptible or -resistant strains of C. albicans. All these strains were preliminarily tested for in vitro susceptibility to TTO, terpinen-4-ol and 1,8-cineole for their antifungal properties, using a modification of the CLSI (formerly NCCLS) reference M27-A2 broth micro-dilution method. RESULTS: In vitro minimal inhibitory concentrations (MIC90) values were 0.06% (volume/volume) for terpinen-4-ol and 4% (volume/volume) for 1,8-cineole, regardless of susceptibility or resistance of the strains to fluconazole and itraconazole. Fungicidal concentrations of terpinen-4-ol were equivalent to the candidastatic activity. In the rat vaginal infection model, terpinen-4-ol was as active as TTO in accelerating clearance from the vagina of all Candida strains examined. CONCLUSION: Our data suggest that terpinen-4-ol is a likely mediator of the in vitro and in vivo activity of TTO. This is the first in vivo demonstration that terpinen-4-ol could control C. albicans vaginal infections. The purified compound holds promise for the treatment of vaginal candidiasis, and particularly the azole-resistant forms.

Phenotypic and functional characterization of vaginal dendritic cells in a rat model of Candida albicans vaginitis.

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.

Lack of circulating Candida mannoprotein antigen in patients with focal hepatosplenic candidiasis.

The significance of Candida mannoprotein serum detection in 15 patients with haematological malignancies and proven (six cases) or probable (nine cases) hepatosplenic candidiasis was retrospectively evaluated. Circulating mannoprotein antigen was detected in three of six and in one of two serum samples from two patients with probable infection. The antigen was not detected in 38 serum samples of 13 (87%) patients. Thus, in contrast to other deep-seated Candida infections, mannoprotein is infrequently detectable during focal hepatosplenic candidiasis and does not appear to be of diagnostic value.

Therapeutic activity of an engineered synthetic killer antiidiotypic antibody fragment against experimental mucosal and systemic candidiasis.

Peptides derived from the sequence of a single-chain, recombinant, antiidiotypic antibody (IdAb; KT-scFv) acting as a functional internal image of a microbicidal, wide-spectrum yeast killer toxin (KT) were synthesized and studied for their antimicrobial activity by using the KT-susceptible Candida albicans as model organism. A decapeptide containing the first three amino acids (SAS) of the light chain CDR1 was selected and optimized by alanine replacement of a single residue. This peptide exerted a strong candidacidal activity in vitro, with a 50% inhibitory concentration of 0.056 microM, and was therefore designated killer peptide (KP). Its activity was neutralized by laminarin, a beta1-3 glucan molecule, but not by pustulan, a beta1-6 glucan molecule. KP also competed with the binding of a KT-like monoclonal IdAb to germinating cells of the fungus. In a rat model of vaginal candidiasis, local, postchallenge administration of KP was efficacious in rapidly abating infections caused by fluconazole-susceptible or -resistant C. albicans strains. In systemic infection of BALB/c or SCID mice preinfected intravenously with a lethal fungal load, KP caused a highly significant prolongation of the median survival time, with >80% of the animals still surviving after >60 days, whereas >90% of control mice died within 3 to 5 days. KP is therefore the first engineered peptide derived from a recombinant IdAb retaining KT microbicidal activity, probably through the interaction with the beta-glucan KT receptor on target microbial cells.

In vitro and in vivo activity of tea tree oil against azole-susceptible and -resistant human pathogenic yeasts.

A tea tree oil (TTO) preparation of defined chemical composition was studied, using a microbroth method, for its in vitro activity against 115 isolates of Candida albicans, other Candida species and Cryptococcus neoformans. The fungal strains were from HIV-seropositive subjects, or from an established type collection, including reference and quality control strains. Fourteen strains of C. albicans resistant to fluconazole and/or itraconazole were also assessed. The same preparation was also tested in an experimental vaginal infection using fluconazole-itraconazole-susceptible or -resistant strains of C. albicans. TTO was shown to be active in vitro against all tested strains, with MICs ranging from 0.03% (for C. neoformans) to 0.25% (for some strains of C. albicans and other Candida species). Fluconazole- and/or itraconazole-resistant C. albicans isolates had TTO MIC50s and MIC90s of 0.25% and 0.5%, respectively. TTO was highly efficacious in accelerating C. albicans clearance from experimentally infected rat vagina. Three post-challenge doses of TTO (5%) brought about resolution of infection regardless of whether the infecting C. albicans strain was susceptible or resistant to fluconazole. Overall, the use of a reliable animal model of infection has confirmed and extended our data on the therapeutic effectiveness of TTO against fungi, in particular against C. albicans.

Detection of human P-glycoprotein-like molecule in azole-resistant Candida albicans from HIV+ patients.

Azole resistance in Candida albicans may be due to several mechanisms. It has been demonstrated that C. albicans possesses sequences with a high degree of homology with the human MDR-1 gene coding for P-glycoprotein (P-gp), belonging to the ATP-binding cassette transporter (ABC) superfamily and responsible for the multidrug resistance (MDR) in tumor cells. On this basis, the expression and intracellular localization of human P-gp-like molecule in C. albicans strains showing different sensitivity to fluconazole were investigated by flow cytometry and immunoelectron microscopy. Post-embedding immunolabeling revealed that monoclonal antibody (mAb) MM4.17, which recognizes an external epitope of human P-gp, reacted with both fluconazole-sensitive (3153 and CO 23-1) and fluconazole-resistant (AIDS 68 and CO 23-2, isolated from AIDS patient and in vitro drug-selected, respectively) strains of C. albicans. However, the resistant strains displayed a number of MM4.17-reactive epitopes much higher than the drug-sensitive ones. The C. krusei ATCC 6458 strain, whose resistance is not mediated by the presence of ABC transporters, was not reactive at all with mAb MM4.17. The specificity of the immunolabeling was confirmed by a competitive inhibition assay performed by using phage clone particles capable of mimicking the MM4.17-reactive epitope. The flow cytometric analysis confirmed a higher level of intracytoplasmic P-gp expression in azole-resistant strains of C. albicans. Both cyclosporin A and verapamil, which are well-known MDR inhibitors, strongly reduced the MICs for fluconazole and itraconazole of the tested azole-resistant AIDS 68 strain, while they did not influence the MICs of either the sensitive 3153 strain of C. albicans or the ATCC 6458 strain of C. krusei. Overall, our data suggest the existence of a P-gp-like drug efflux pump in C. albicans that may participate in the mechanisms of azole-resistance of this fungus.

Immune cell-mediated protection against vaginal candidiasis: evidence for a major role of vaginal CD4(+) T cells and possible participation of other local lymphocyte effectors.

The protective roles of different lymphocyte subsets were investigated in a rat vaginal candidiasis model by adoptive transfer of vaginal lymphocytes (VL) or sorted, purified CD3(+) T cells, CD4(+) or CD8(+) T cells, or CD3(-) CD5(+) B cells from the vaginas of naïve or immune rats following three rounds of Candida albicans infection. The adoptive transfer of total VL from nonimmune animals did not alter the course of vaginal candidiasis of the recipient rats. In contrast, the animals receiving total VL or CD3(+) T cells from immune rats showed a highly significant acceleration of fungus clearance compared with animals which received nonimmune VL. The animals with vaginal CD3(-) CD5(+) B cells transferred from immune rats also had fewer Candida CFU than the controls, but fungal clearance was significantly retarded with respect to the animals administered immune T cells. Sorted, purified CD4(+) and CD8(+) vaginal T cells from immune rats were also adoptively transferred to naïve animals. Although both populations were seen to accelerate the clearance of the fungus from the vagina, CD4(+) T cells were much more effective than CD8(+) T cells. Overall, there was no difference between the antifungal effects of immune vaginal CD4(+) T cells and those achievable with the transfer of whole, immune VL. Histological observations of the vaginal tissues of rats with adoptively transferred immune T cells demonstrated a remarkable accumulation of lymphocytes in the subepithelial lamina propria and also infiltrating the mucosal epithelium. These results strongly suggest that distinct vaginal lymphocyte subsets participate in the adaptive anti-Candida immunity at the vaginal level, with the vaginal CD4(+) T cells probably playing a major role.

Deletion of the two-component histidine kinase gene (CHK1) of Candida albicans contributes to enhanced growth inhibition and killing by human neutrophils in vitro.

We have observed that human neutrophils (polymorphonuclear leukocytes [PMNs]) have an increased growth-inhibitory and killing effect on a strain of Candida albicans with a deletion of CHK1, a gene encoding a putative histidine kinase. The PMN effect was not due to increased phagocytosis of the null strain. This observation may partially explain the reduced virulence in a hematogenously disseminated murine model of candidiasis.