The antifungal activity of salivary histatin 5 towards C albicans and C glabrata
El Hachem, Lea
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Candida ablicans is an opportunistic fungus colonizing the oral cavity. Histatin 5, a basic histidine-rich protein, exerts fungistatic as well as fungicidal activities towards C. albicans. Another Candida species, C. glabrata, has been shown to be much less sensitive to histatin 5. We hypothesized that the cell wall plays a determining role in fungal sensitivity to histatin 5. The aim of this study was to compare C. albicans and C. grabrata blastoconidia and spheroplast cell sensitivities to histatin 5 in growth inhibition and killing assays. C. ablicans and C. grabrata cells were cultured in 20% Sabouraud Dextrose Broth at 30 degree C. After 24h the cells were harvested, dilluted in the same broth and exposed to a dilution series of histatin 5. After 24h incubation, growth inhibition was monitored spectrophotometrically at 620 nm, and the IC superscript 50 values were determined. For the cell killing assay, the cultured cells were aliquoted into two portions. The first half was diluted in 10mM phosphate buffer containing 1.4 M sorbitol. The second half was first converted into spheroplasts using zymolyase enzyme. Blastoconidia and spheroplasts were each mixed with a dilution series of histatin 5, and after 1.5 h incubation cells were diluted and plated on agar, followed by colony counting and LC subscript 50 value determination. In the growth inhibition assays, histatin 5 showed IC subscript 50 values of 9.75[plus or minus]0.25 and [more than] 225 [mega]/ml towards C. albicans and C. glabrata blastoconidia, respectively. In the killing assays, histatin 5 showed LC superscript 50 values of 3.2[plus or minus]0.2 and [more than]200[mega] /mI towards C. albicans and C. grabrata blastoconidia, respectively. Spheroplast formation of C. albicans and C. grabrafa was successful, but due to the low inherent viability of the preparations on agar plates, LC subscript 50 values could not be established. Alternatively, spheroplast viability following killing assays was assessed by microscopic analysis using trypan blue stain (4%). However, microscopically, cell lysis and clustering was observed at high levels of histatin 5 and quantitative data could not be obtained. Spheroplast viability was also assessed with the fluorescent dye Presto Blue. Fluorescence was measured at an excitation wave length of 560 nm and an emission wavelength of 595 nm. The dye accurately distinguished between life and death of fungal cells but was not suitable for use in buffers employed in spheroplast formation. Our data confirm a high susceptibility of C. ablicans in contrast to a reduced sensitivity of C. grabrata in both growth inhibition and killing assays. Fungal infections are important oral manifestations of immune suppression and naturally occurring salivary proteins are excellent candidates for controling such infections. The most prevalent fungus in such infections, C. ablicans, is succeptible to histatin 5, but infections caused by C. grabrata might need alternative antifungal agents for treatment. Further investigations are needed in order to determine the mechanisms causing the sensitivity of C. ablicans and the resistance of C. grabrata to histatin 5.
PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact firstname.lastname@example.org.Dissertation (MSD) --Boston University, Henry M. Goldman School of Dental Medicine, 2014 (Department of Periodontology and Oral Biology).Includes bibliographic references: leaves 54-59.
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