Histatin 5 variant reduces Candida albicans biofilm viability and inhibits biofilm formation

Abstract

Candida albicans is a commensal organism and opportunistic pathogen that can form biofilms that colonize surfaces of medical devices, such as implants, catheters, and dentures. Compared to planktonic C. albicans cells, cells in biofilms exhibit increased resistance to treatment. Histatin 5 (Hst-5) is an antimicrobial peptide that is natively secreted by human salivary glands and has strong antifungal activity against C. albicans. However, C. albicans produces secreted aspartic proteases (Saps) that can cleave and inactivate Hst-5, limiting its antifungal properties. We previously showed that residue substitutions K11R and K17R within Hst-5 improve its antifungal activity and prevent proteolytic degradation by Saps when treating planktonic C. albicans. Here, we investigated the use of the K11R-K17R peptide as an alternative therapeutic against C. albicans biofilms by assessing its ability to reduce viability of pre-formed biofilms and to inhibit the formation of biofilms and showed that K11R-K17R had improved activity compared to Hst-5. Based on these results, we incorporated K11R-K17R and Hst-5 into polyelectrolyte multilayer (PEM) surface coatings and demonstrated that films functionalized with K11R-K17R reduced the formation of C. albicans biofilms. Our results demonstrate the therapeutic potential of the K11R-K17R Hst-5 variant in preventing and treating biofilms.

Keywords: Antimicrobial peptides; Biofilm; Candida albicans; Histatin 5; Polyelectrolyte multilayer film.

Figure 1.

Antifungal activity of Hst-5 and K11R-K17R against C. albicans. (A) Activity against planktonic cells was evaluated by serially diluting peptides and incubating them with 2.5 × 10⁷ cells/mL for 30 min at 30 °C before quantifying the viability of the cells compared to untreated cells. (B) Activity against biofilms was evaluated by forming biofilms for 24 h, adding serially diluted peptides, incubating the peptides with biofilms for 24 h, and then quantifying the metabolic activity of the treated biofilms compared to untreated biofilms. Error bars indicate the standard error of the mean (N = 6). Asterisks indicate concentrations with substantial differences between the peptides (Q = 0.05).

Figure 2.

Reduction in biofilm formation by C. albicans due to the presence of Hst-5 and K11R-K17R evaluated by (A) metabolic activity and (B) microscopy. Cells were incubated with serially diluted peptides for 24 h to allow biofilm formation. Following incubation, the metabolic activity of the biofilms formed in the presence of peptide was compared with those formed in the absence of peptide, and DIC microscopy images were obtained. In (A), error bars indicate the standard error of the mean (N = 6), and asterisks indicate concentrations with substantial differences between the peptides (Q = 0.05). In (B), peptide concentration was 6 μM, and scale bars are 50 μm.

Figure 3.

Construction of PEMs. (A) Following deposition of base layers on a surface, substrates are dipped in alternating solutions of a polyanion (poly-L-glutamic acid, PGA) and a polycation (poly-L-lysine, PLL), with washing steps after each polymer. The alternating charges allow a film to build on the surface. One bilayer is made up of the coating from dipping in the polyanion and the polycation. (B) A cationic peptide can be incorporated into the film by dipping the substrate into a peptide solution after the polyanion solution. The polyanion, peptide, and polycation deposition forms one trilayer on the surface.

Figure 4.

Thickness of PEMs formed on silicon substrate. After depositing base layers on the substrate, films were fabricated by dipping in alternating solutions of PGA, Hst 5, and PLL, as shown in Figure 3B. The concentration of Hst-5 is indicated, with the 0 mg/mL solution containing no peptide. The film thickness on each substrate was measured by ellipsometry at five points per substrate after depositing 60 trilayers (PGA/Hst-5/PLL), and the experiment was performed three times. Error bars indicate standard error of the mean (N=15). An asterisk indicates a statically significant difference between the indicated means (p≤0.05), while “ns” indicates no significant difference.

Figure 5.

Growth of C. albicans on quartz surfaces coated with PEMs containing Hst-5 and K11R-K17R. Films were fabricated by dipping in alternating solutions of PGA, Hst-5, and PLL, as shown in Figure 3B. The concentration of peptide used in film fabrication is indicated, with the control film utilizing a solution of 0 mg/mL peptide. Films were imaged at 6, 12, and 24 h, and these images are representative of the results from three replicate experiments. The scale bar indicates 100 μm.