ABSTRACT
In recent years, the incidence and mortality of invasive fungal infections has increased. It is highly desirable to develop novel antifungal agents with new modes of action. Targeting virulence factors represents a new strategy for antifungal drug discovery. Secreted aspartic protease 2 (SAP2), a kind of virulence factor, is an emerging antifungal target. However, discovery of small-molecule SAP2 inhibitors remains a significant challenge. Based on the structure-activity relationship of our previously identified triazine small-molecule SAP2 inhibitor, we were able to identify two potent inhibitors, 8a and 8c, which showed excellent in vivo antifungal activity for the treatment of C. albicans infection. Moreover, compounds 8a and 8b effectively inhibited fungal biofilm. Taken together, triazine SAP2 inhibitors represent promising lead compounds for the discovery of novel antifungal agents.
ABSTRACT
Aims@#The aim of this study was to attempt to evaluate the antifungal activity of carvacrol in combination with fluconazole or amphotericin B against Candida albicans.@*Methodology and results@#Antifungal susceptibilities to carvacrol alone and in combination with fluconazole or amphotericin B were performed using the CLSI standard reference method against clinical isolates of C. albicans isolated from the immuno-compromised patients. Proteinase production assay and the expression of genes associated with secreted aspartyl proteinases synthesis (SAP1 and SAP2) were carried out to evaluate the antifungal activity of carvacrol in combination with fluconazole or amphotericin B against C. albicans. The carvacrol alone and in combination with fluconazole and amphotericin B exhibited strong inhibitory activity. The carvacrol, exhibited significant synergy and bpartial synergy with fluconazole or amphotericin B against the test isolates. The data indicated that combination of carvacrol with fluconazole or amphotericin B exerted antifungal effects through reducing SAP enzyme activity. The expression levels of SAP1 and SAP2 genes were down-regulated by carvacrol, fluconazole and amphotericin B alone. After carvacrol was employed in combination with fluconazole or amphotericin B, the expression levels of SAP1 and SAP2 genes demonstrated the lower expression in comparison to carvacrol alone.@*Conclusion, significance and impact of study@#These results provide proof of concept for the implementation of carvacrol alone or in combination with fluconazole or amphotericin B inhibitors of C. albicans.
ABSTRACT
Background: The secreted aspartyl proteinases 2 (Sap2) of Candida albicans (C. albicans) is a potential marker of candididasis. It is a virulence factor associated with adherence and tissue invasion. Aim: In order to detect Sap2 in clinical sera, we developed an indirect competitive enzyme-linked immunosorbent assay (ELISA). Materials and Methods: Polyclonal antibodies were produced for Sap2 by injecting Sap2 into a New Zealand White inbred rabbit. They could be used at a dilution exceeding 1:1200 in an indirect ELISA, and detected Sap2 concentration up to 1 ng/mL. Results: Of the 286 cancer serum samples tested, 16.8% were found as candidiasis. The test was simple and economical to perform and had a level of sensitivity for detection of low-titer positive sera; thus, it may be proven to be of value in epidemiological studies on candidiasis.
ABSTRACT
Candida albicans is an opportunistic fungal pathogen that causes severe systemic infections in immunosuppressed individuals. C. albicans resistance to antifungal drugs is a severe problem in patients receiving prolonged therapy. Moreover, trailing yeast growth, which is defined as a resistant MIC after 48 h of incubation with triazole antifungal agents but a susceptible MIC after 24 h, has been noted in tests of antifungal susceptibility against some C. albicans isolates. In this context, we recently noticed this phenomenon in our routine susceptibility tests with fluconazole/itraconazole and C. albicans clinical isolates. In the present study, we investigated the production of cell-associated and secreted aspartyl peptidases (Saps) in six trailing clinical isolates of C. albicans, since this class of hydrolytic enzymes is a well-known virulence factor expressed by this fungal pathogen. Sap2, which is the best-studied member of the Sap family, was detected by flow cytometry on the cell surface of yeasts and as a 43-kDa polypeptide in the culture supernatant, as demonstrated by Western blotting assay using an anti-Sap1-3 polyclonal antibody. Released aspartyl peptidase activity was measured with BSA hydrolysis and inhibited by pepstatin A, showing distinct amounts of proteolytic activity ranging from 5.7 (strain 44B) to 133.2 (strain 11) arbitrary units. Taken together, our results showed that trailing clinical isolates of C. albicans produced different amounts of both cellular and secreted aspartyl-type peptidases, suggesting that this phenotypic feature did not generate a regular pattern regarding the expression of Sap.