Susceptibility of Candida dubliniensis to salivary histatin 3

Abstract

Candida dubliniensis is a recently described Candida species associated with oral candidiasis in human immunodeficiency virus (HIV)-infected patients and patients with AIDS. The majority of C. dubliniensis clinical isolates tested to date are susceptible to the commonly used antifungal drugs, including fluconazole, ketoconazole, itraconazole, and amphotericin B. However, the appearance of fluconazole-resistant C. dubliniensis strains in this patient group is increasing. Histatins are a family of basic histidine-rich proteins present in human saliva which have therapeutic potential in the treatment of oral candidiasis. The mechanism of action of histatin is distinct from that of commonly used azole and polyene drugs. Characterization of the antifungal activity of histatin has mainly been carried out using C. albicans but it is also effective in killing C. glabrata and C. krusei. Here we report that C. dubliniensis is also susceptible to killing by histatin 3. The concentration of histatin 3 giving 50% killing (the IC(50) value) ranged from 0.043 to 0.196 mg/ml among different strains of C. dubliniensis. The least-susceptible C. dubliniensis strain, P9224, was found to internalize histatin at a lower rate than the C. albicans reference strain CA132A. The dissociation constant (K(d)) for the least-susceptible strain (C. dubliniensis 9224) was ninefold higher than that for the C. albicans reference strain. These results suggest that histatin 3 may have potential as an effective antifungal agent, particularly in the treatment of oral candidiasis in HIV-infected patients and patients with AIDS in which resistance to the commonly used antifungal drug fluconazole has emerged.

FIG. 1.

Candidacidal activity of histatin 3 against various C. dubliniensis strains and C. albicans CA132A. Results are means ± standard deviations (error bars) of three independent assays. Where error bars are not visible, they are smaller than the data symbol.

FIG. 2.

Binding of ¹⁴C-labeled histatin to CA132A (□), P0508 (⧫), P9224 (○). A total of 10⁷ cells/ml were incubated for 15 min with 0 to 16 μM [¹⁴C]histatin 3. Assays were performed in 10 mM potassium phosphate buffer, pH 7.4. Data points shown are the means ± standard deviations (error bars) of duplicate determinations from a representative binding assay.

FIG. 3.

Binding and internalization of fluorescently labeled histatin to C. albicans and C. dubliniensis strains. Cells from C. albicans CA132A and C. dubliniensis P0508 and P9224 were incubated with 12 μM fluorescently labeled histatin at 37°C for the periods indicated. The number of cells exposed in each case was 3.4 × 10⁶ cells/ml. Micrographs are for a film exposure time of 5 s. Scale bars, 3 μm.