The human salivary peptide histatin 5 exerts its antifungal activity through the formation of reactive oxygen species

Abstract

Previous studies have shown that the human salivary antifungal peptide histatin 5 is taken up by Candida albicans cells and associates intracellularly with mitochondria. The purpose of the present study was to investigate the biological consequence of this specific subcellular targeting. Histatin 5 inhibited respiration of isolated C. albicans mitochondria as well as the respiration of intact blastoconidia in a dose and time-dependent manner. A nearly perfect correlation was observed between histatin-induced inhibition of respiration and cell killing with either logarithmic-or stationary-phase cells, but stationary-phase cells were less sensitive. Because nonrespiring yeast cells are insensitive to histatin 5, the potential mechanistic relationship between histatin 5 interference with the respiratory apparatus and cell killing was explored by using an oxygen radical sensitive probe (dihydroethidium). Fluorimetric measurements showed that histatin 5 induced the formation of reactive oxygen species (ROS) in C. albicans cells as well as in isolated mitochondria and that ROS levels were highly correlated with cell death. In the presence of an oxygen scavenger (L-cysteine), cell killing and ROS formation were prevented. In addition, the membrane-permeant superoxide dismutase mimetic 2,2,6,6-tetramethylpiperidine-N-oxyl, abolished histatin-induced ROS formation in isolated mitochondria. In contrast to histatin 5, the conventional inhibitors of the respiratory chain, sodium cyanide or sodium azide, neither induced ROS nor killed yeast cells. These data provide strong evidence for a comprehensive mechanistic model of histatin-5-provoked yeast cell death in which oxygen radical formation is the ultimate and essential step.

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

“…Our data regarding the respiratory activity of histatin-treated isolated mammalian mitochondria are in agreement with previous studies that reported a dosedependent inhibition of the respiratory activity and a large dissipation of the mitochondrial membrane potential (DW) in C. albicans cells, following histatin 5 exposure [17,19]. First, Helmerhorst et al suggested that histatin-5, after its entrance in the yeast cell, reaches the mitochondria and inhibits the complexes I and/or III of the respiratory chain.…”

“…It is interesting to note that the peptide at a concentration of 100 lM inhibits mitochondrial respiration only in the presence of glutamate-malate as substrates, implying that inhibition of respiration was occurring probably at the level of the complex I of the respiratory chain. It should be noted that, although the concentration of histatin-5 used in these experiments would not be achievable under physiological conditions, in earlier in vitro studies concerning the toxic effects of histatin-5 on cells, this peptide was used in similar concentration [11,17,19,22].…”

“…Whether dissipation of the transmembrane potential (DW) is a primary effect of histatin-5 on the mitochondria or a consequence of cytochrome c release needs probably further work, although our data favour the first possibility. Helmerhorst et al suggested that a large accumulation of unstable reactive species within the mitochondria of yeast cells after treatment with histatin-5 might likely represent an inevitable consequence of impairment of the mitochondrial respiratory enzymes induced by histatin-5 [19]. Similarly, we suggest that a slight membrane damage induced by the oxygen reactive species generated within the mammalian mitochondria as a consequence of the histatin-5-dependent inhibition of the complex I of the respiratory chain might account for the slight dissipation of the transmembrane potential (DW), we observed, under our experimental conditions, in isolated mammalian mitochondria subsequent to histatin-5 exposure.…”

“…Recently, Helmerhorst et al have demonstrated that histatin-5 after its internalisation within the yeast cell reaches the mitochondria, dissipating the mitochondrial membrane potential (DW). Mitochondria thus seem to represent the main target for histatin within the yeast cell [18,19] and in particular Helemehorst et al have recently reported that histatin-5 is able to induce the formation of reactive oxygen species (ROS) in C. albicans cells as well as in isolated mitochondria suggesting that this is the ultimate and essential step for the histatin-5-provoked yeast cell death.…”

Antimicrobial peptides: mode of action and perspectives of practical application

“…Так, в работе [84] было высказано предположение, что токсичность антимикробного пептида хистатина-5 в отношении C. albicans, C. neoformans и некоторых других грибков обусловлена образованием активных форм кислорода (АФК) в их клетках под действием этого пептида. Авторы [84] выдвинули гипотезу о том, что после попадания в клетки микроскопических грибов хистатин-5 проникает в митохондрии и ингибирует цикл кофермента Q, что приводит к накоплению в клетке АФК, и, как следствие, гибели клетки вследствие окисления под действием АФК внутриклеточных субстратов. С другой стороны, в статье [85] высказана другая точка зрения заключающаяся в том, что хистатин-5 не приводит к повышению уровня АФК в клетках грибов, а фунгицидное действие этого пептида обусловлено вызванной им утечкой АТР из грибковых клеток.…”