The effect of histatin 5, adsorbed on PMMA and hydroxyapatite, on Candida albicans colonization

Abstract

The limited number of treatments for oral candidiasis resulted in the emergence of azole-resistant Candida albicans strains, thus enforcing the need for novel antifungal treatments. Although histatin 5 (H5) demonstrates antifungal activity, its inhibitory effect when adhered to hydroxyapatite and Polymetylmethacrylate (PMMA) surfaces, resembling conditions of the in vivo pellicle, remains unexplored. The objective of this in vitro study was to determine whether surface-adhered H5 inhibits the colonization of C. albicans on hydroxyapatite and/or PMMA. The C. albicans assay involved developing a mono-protein pellicle (either H5 or albumin) on hydroxyapatite and PMMA discs, introducing C. albicans and counting the number of adhered cells, throughout time, using scanning electron microscopy. A negative binomial statistical model and the Tukey-Kramer test were used for statistical analysis, with p < 0.01 indicating significance. H5-coated PMMA had significantly reduced number of cells compared to albumin-coated PMMA at 30, 90 and 1440 min (p < 0.0001), with the number of cells decreasing significantly in 90 and 1440 min (p < 0.0001). Similarly, H5-coated hydroxyapatite had significantly fewer cells compared to the albumin-coated surface at 90 and 1440 min (p < 0.0001), with the number of cells decreasing significantly at 30, 90 and 1440 min (p < 0.0001). In conclusion, C. albicans colonization was most inhibited by PMMA and hydroxyapatite-adhered H5 after 1440 min, illustrating the time-dependent effect of H5. In addition, yeast cells colonized albumin-coated PMMA, while dense hyphal networks formed on albumincoated hydroxyapatite, suggesting that C. albicans morphology is influenced by the surface available for albumin adhesion.

Miniaturized Digestion and Extraction of Surface Proteins fromCandida albicansfollowing Treatment with Histatin 5 for Mass Spectrometry Analysis

“…To begin, we reproduced the adhesion of C. albicans on HSA-coated glass surfaces as previously reported [5], and then the cells were treated with varying concentrations of HTN5. Given the physiological concentration of HTN5 in the oral cavity, 10–30 μ M [31], we performed HTN5 treatments at 20, 25, and 30  μ g/mL (Figures 2(b) and 2(c)).…”

“…During an experiment, each sample spot was covered with an inverted glass vial containing a wet cotton, which maintained a high humidity environment and minimized evaporation of the sample droplet (Figure 1(c)). To prepare for cell adhesion, the glass surface within the hydrophobic circles was treated with 25  μ L of 0.10 mg/mL human serum albumin (HSA) solution (Sigma Aldrich) for 2 hours at room temperature (RT), based on a protocol previously reported by our group [5]. The HSA-coated surface was then washed three times with water.…”

“…For infections to begin, adhesion to oral surfaces of protein films is the initial step [5, 6]. From these proteins, salivary proteins such as histatins, statherins, and acidic proline-rich ones have exhibited antifungal properties [7, 8].…”

“…From these proteins, salivary proteins such as histatins, statherins, and acidic proline-rich ones have exhibited antifungal properties [7, 8]. The family known as histatins (HTN), comprised of HTN1, HTN3, and HTN5, have been of increasing interest due to its clear ability to control and kill C. albicans [5, 7, 9, 10]. Studies targeting the effects of HTN5 on cellular respiration at the mitochondrial level have been performed; however, the exact mechanism remains unknown [9, 11, 12].…”

Nanoscale Adhesion Forces between Enamel Pellicle Proteins and Hydroxyapatite

“…Furthermore, many studies indicate that H5 exhibits potent antifungal activity against the pathogenic Candida albicans, which is the primary pathogen responsible for initiating oral candidiasis (Gyurko et al, 2000;Pusateri et al, 2009). Relevant to the adhesion of H5 is a recent discovery showing that this protein retains its antifungal effect against C. albicans after adsorption to HA (Vukosavljevic et al, 2012). Histatins and some other salivary proteins are well-known for their antibacterial functions.…”