Candidacidal activity of recombinant human salivary histatin-5 and variants

Abstract

Human salivary histatins possess fungicidal and bactericidal activities. The current investigation evaluates the structure-function relationship of histatins with regard to their candidacidal activity by using recombinant histatin-5 and its variants produced in Escherichia coli. The purified recombinant histatins were examined for their candidacidal activity and secondary structure. The m21 (with Lys-13 replaced by Thr [Lys-133Thr]) and m71 (Lys-133Glu) variants are significantly less effective than recombinant histatin-5 in killing Candida albicans, suggesting that Lys-13 is critical for candidacidal activity. The m68 (Lys-133Glu and Arg-223Gly) variant is significantly less potent than the recombinant histatin-5 as well as m71, indicating that Arg-22 is crucial for the cidal activity. The candidacidal activities of m1 (Arg-123Ile), m2 (Arg-123Ile and Lys-173Asp), m12 (Arg-123Lys and His-213Leu), and m70 (His-193Pro and His-213Arg) variants, however, are comparable to that of recombinant histatin-5, indicating that Arg-12, Lys-17, His-19, and His-21 are not functionally important. The conformational preferences of histatin-5 and variants were determined by circular dichroism. The results indicate that all proteins have a strong tendency to adopt ␣-helical conformation in trifluoroethanol. Previously, we have shown that the ␣-helical conformation is one of the important structural requirements for eliciting appreciable candidacidal activity. Collectively, the data suggest that in addition to the helical conformation, specific residues such as Lys-13 and Arg-22 in the sequence of histatin-5 are, indeed, important for candidacidal activity.

Histatins: antimicrobial peptides with therapeutic potential

“…A number of histatin variants have been assessed for their antifungal activity. Variant M21 (described earlier) was found to be less potent than histatin 5 (Tsai et al 1996), and dhvar 1 and dhvar 2 (multi-site substitutions in the dh-5 region) displayed lower antifungal activity than histatin 5 (Helmerhorst et al 1997). The variants displayed activity against a number of bacteria but their haemolytic activity rendered them unsuitable for future development.…”

“…Variant M21 was created by replacing Lys-13 with Thr, while M71 had Lys-13 replaced by Glu. Both variants demonstrated reduced fungicidal activity, indicating that the Lys-13 region was required for antifungal activity (Tsai et al 1996). A similar strategy was adopted in generating variants with multi-site substitutions in the dh-5 region.…”

Studies of the mechanism of human salivary histatin-5 candidacidal activity with histatin-5 variants and azole-sensitive and -resistant Candida species

“…Construction of the pET-Hsn-5 expression plasmid. For the construction of pET-Hsn-5, Hsn-5 cDNA was recloned from the plasmid pGHsn-5 that we constructed previously (32) into an Escherichia coli pET-30b(ϩ) expression system (18), as follows: the Hsn-5 coding region was PCR amplified from the pGHsn-5 plasmid with primer P1 (5Ј-GCGCCATGGATTCACATGCAAAG AGACATC-3Ј), which annealed to the 5Ј end of the coding region, and primer pGEX#4 (5Ј-TTTCACCGTCATCACCGAAA-3Ј), which annealed to the pGEX-2T sequence about 50 bp downstream of the Hsn-5 stop codon. An NcoI restriction site (CCATGG; underlined in the sequence of primer P1) and a Met codon (ATG; double underlined in the sequence of primer P1) were introduced at the 5Ј end of Hsn-5 for cloning and cyanogen bromide (CNBr) cleavage purposes, respectively.…”

“…The samples were then passed through a DE-52 anion exchanger by using 10 mM Tris buffer (pH 8.0) to remove the neutral and acidic peptides generated by CNBr cleavage of the carrier protein. Final purification of Hsn-5 and the two variants was achieved by reversed-phase high-pressure liquid chromatography (RP-HPLC) as described previously (32).…”

“…Among the three major Hsns (Hsn-1, Hsn-3, and Hsn-5), Hsn-5 is the most potent peptide in killing the blastospore and germinated forms of Candida albicans (36). The level of killing of the blastospore form of C. albicans can reach 80 to 100% at the physiologic concentration (15 M) of Hsn-5 (32).…”