Genetically engineered human salivary histatin genes are functional in Candida albicans: development of a new system for studying histatin candidacidal activity

Abstract

Histatins are a structurally related family of salivary proteins known as histidine-rich proteins that are produced and secreted by the human major salivary glands. In vitro, histatins are potent cytotoxic proteins with selectivity for pathogenic yeasts including Candida albicans. Studies that investigate the mechanism of action of histatin proteins upon this important human pathogen have used a candidacidal assay in which the histatin is applied extracellularly. In order to develop a model system to study the mechanism of histatin action independently from binding and translocation events, the authors constructed C. albicans strains that contain chromosomally encoded human salivary histatin genes under the control of a regulated promoter. Intracellular expression of either histatin 5 or histatin 3 induced cell killing and ATP release in parallel. Since histatin killing can be initiated solely from intracellular sites, extracellular binding and internalization are preceding transport events. Thus the mechanism of histatin-induced ATP release does not require extracellular binding, and intracellular targets alone can activate ATP release. By employing a codon-optimization strategy it was shown that expression of heterologous sequences in C. albicans can be a useful tool for functional studies.

Respiratory inhibition of isolated mammalian mitochondria by salivary antifungal peptide histatin-5

“…This finding supports the hypothesis that binding of histatin-5 to fungal cell membrane and its internalisation and transport represent critical events in the entire killing process [38,39] mediated by histatin-5.…”

Candida albicansCell Wall Proteins

“…The ATP itself has a cytotoxic role because the removal of the released ATP can inhibit the killing. When histatin 5 was directly introduced into the fungal cell, bypassing the entry step, it was still capable of inducing the release of ATP and killing C. albicans (11). This observation suggested that the signal for killing did not come externally.…”

How Does It Kill?: Understanding the Candidacidal Mechanism of Salivary Histatin 5

“…Also, it was shown that in spite of the cellular loss of ATP upon Hst 5 exposure, cells continued to maintain their membrane potential and remained alive for up to 80 min after ATP efflux (23). ATP release and killing of C. albicans cells from endogenously expressed chromosomally encoded Hst 5 provided further strong evidence that Hst 5 targets are intracellular (27).…”