Anti-candidal activity of genetically engineered histatin variants with multiple functional domains.

The human bodily defense system includes a wide variety of innate antimicrobial proteins. Histatins are small molecular weight proteins produced by the human salivary glands that exhibit antifungal and antibacterial activities. While evolutionarily old salivary proteins such as mucins and proline-rich proteins contain large regions of tandem repeats, relatively young proteins like histatins do not contain such repeated domains. Anticipating that domain duplications have a functional advantage, we genetically engineered variants of histatin 3 with one, two, three, or four copies of the functional domain by PCR and splice overlap. The resulting proteins, designated reHst3 1-mer, reHist3 2-mer, reHis3 3-mer and reHist3 4-mer, exhibited molecular weights of 4,062, 5,919, 7,777, and 9,634 Da, respectively. The biological activities of these constructs were evaluated in fungicidal assays toward Candida albicans blastoconidia and germinated cells. The antifungal activities per mole of protein increased concomitantly with the number of functional domains present. This increase, however, was higher than could be anticipated from the molar concentration of functional domains present in the constructs. The demonstrated increase in antifungal activity may provide an evolutionary explanation why such domain multiplication is a frequent event in human salivary proteins

Amino acid sequences of recombinant histatin 3 multimers¹.

1<p>The fungicidal domain sequences are underlined.</p>2<p>The bolded R residue in the ReHst3 2-mer indicates an unintentional amino acid substitution introduced by the DNA polymerase.</p

PCR primers used in this study.

1<p>Bolded nucleotide regions represent the functional domain; underlined nucleotide regions represent the BamHI cleavage site.</p

Killing activity of recombinant histatin 3 mers.

<p>A, <i>C. albicans</i> blastoconidia killing. Cells were grown to exponential phase in ¼ strength Sabouraud dextrose broth; B, Germ tube killing. Formation of germ tubes was induced by incubating blastoconidia in RPMI-1640 for 3 hr at 37°C. Killing was determined for peptide concentrations between 0 and 50 µM after an incubation time of 60 min at 37°C. Black bars, reHst3 1-mer; crosshatched bars, reHst3 2-mer; white bars, reHst3 3-mer; dotted bars, reHst3 4-mer. The experiments were performed in triplicate. The average and standard deviation (SD) of a representative experiment is shown.</p