CANDIDACIDAL ACTIVITY OF HUMAN SALIVARY HISTATIN RECOMBINANT VARIANTSPRODUCED BY SITE-DIRECTED MUTAGENESIS

Histatin 5 (Hst5) is a 24-amino acid (aa) member of the Hst family tha t is found in human salivary secretions and exhibits candidacidal acti vity. Hst5 contains a 13-aa region that alone is capable of killing fu ngal pathogens and is referred to as the functional domain. To investi gate the role of specific aa located within the functional domain, the pRSET bacterial expression system was used to produce recombinant Hst 5 (re-Hst5) and several re-variants that were generated by site-direct ed mutagenesis. The vector pRSETC expresses genes of interest as fusio n proteins attached to the carboxy end of an N-terminal His(6) tag tha t binds to nickel (Ni2+). The re-variants were generated using the pol ymerase chain reaction (PCR) and had Gly substituted for either the Hi s, Glu or Lys/Arg within the functional domain. PCR products that enco ded either the wild-type or variant forms of re-Hst5 were inserted int o pRSETC and produced as fusion proteins which were affinity purified from cell lysates by Ni2+-Sepharose chromatography. Fusion proteins we re digested with CNBr and re-Hsts were purified by reversed-phase high performance liquid chromatography (RP-HPLC). Re-Hsts were tested in b ioassays to measure the ability to kill both Candida albicans (C. albi cans) blastoconidia and spheroplasts which were generated by removal o f the cell wall. In both assays, re-Hst5 displayed dose-dependent cand idacidal activity that was nearly identical to that of native Hst5 pur ified from human salivary secretions. Re-Hst5 variants with either Glu or Lys/Arg substitutions demonstrated significantly lower candidacida l activity in both assays, while the variant with His mutated showed e ssentially no activity at physiological concentrations. These results indicate that acidic and basic aa within the functional domain contrib ute to candidacidal activity and that the His are essential for candid acidal activity. Additionally, since C. albicans spheroplasts were als o susceptible to Hsts, the cell wall is not an essential component in the Hst mechanism of candidacidal action.