Iron binding modulates candidacidal properties of salivary histatin 5

Abstract

Salivary protein histatin 5 (Hst 5) is fungicidal toward Candida albicans, the causative agent of oropharyngeal candidiasis. However, its activity in saliva is compromised by salivary protease-mediated degradation and interaction with salivary salts. Hst 5 has also been shown to bind various metals in saliva-namely, Zn, Cu, and Ni. Surprisingly, interactions of Hst 5 with Fe have not been studied, although iron is one of the most abundant metals present in saliva. Using circular dichroism, we show that Hst 5 can bind up to 10 equivalents of iron as measured by loss of its alpha-helical secondary structure that is normally observed for it in trifluoroethylene. A significant decrease in the candidacidal ability of Hst 5 was observed upon iron binding, with increasing iron concentrations being inversely proportional to Hst 5 killing activity. Binding assays showed that the decrease in killing was likely a result of reduced binding (10-fold reduction) of Fe-Hst 5 to C. albicans cells. Protease stability analysis showed that Fe-Hst 5 was completely resistant to trypsin digestion. In contrast, zinc binding had limited effects on Hst 5 fungicidal activity or protease susceptibility. RNA sequencing results identified changes in iron uptake genes in Hst 5-treated C. albicans cells. Our findings thus suggest that consequences of Hst 5 binding iron not only affect candidacidal ability and proteolyic stability of Hst 5, but may also contribute to a novel killing mechanism involving interference with cellular iron metabolism.

Keywords: Candida albicans; antimicrobial peptides; proteolysis; saliva; transition elements; zinc.

Figure 1.

Circular dichroism (CD) spectra of histatin 5 titrated with Fe³⁺ or Zn²⁺ showed changes in secondary structure upon metal binding. Hst 5 (250 μL of 60 μM) in 95% trifluoroethylene was titrated with 7.5-mM solution of ferric chloride or zinc chloride equivalents in increments of 2 μL to achieve a Hst 5:metal ratio ranging from 1:1 to 1:10; spectra was recorded between 190 and 280 nm. (A) Hst 5 can bind up to 10 equivalents of iron, resulting in complete loss of secondary structure at 10 equivalents (solid, dashed, dotted, and dot-dashed black and gray lines represent 0-3 and 4-7 equivalents, respectively; solid, dashed, and dotted light gray lines represent 8-10 equivalents of iron). (B) Hst 5 shows saturation in changes in secondary structure upon binding 4 equivalents of zinc, with negligible additional change at 10 equivalents (solid black, dotted black, solid gray, and dotted light gray lines represent 0, 2, 4, and 10 equivalents of Zn, respectively).

Figure 2.

Histatin 5 (Hst 5) killing and cellular binding are affected upon metal binding. Hst 5 (30 μM) with or without metals was used to perform killing assays via a microdilution plate method. (A) Killing abilities of Hst 5 are significantly reduced upon binding with iron, starting at 2 equivalents of iron, with incremental decrease upon addition of further equivalents (white bar, Hst 5 alone; light gray bars, Fe:Hst 5; and dark gray bar, 300 μM iron alone). (B) Killing abilities of Hst 5 (30 μM) are significantly reduced upon binding with 2 equivalents of Zn; however, Zn alone had toxicity (white bar, Hst 5 alone; light gray bars, Zn:Hst 5; and dark gray bars, zinc alone). (C) Cellular binding of Hst 5 is affected upon its binding to iron and zinc. X is the number of times that the concentration is of Hst 5 alone (30 μM).

Figure 3.

Presence of iron but not zinc in whole saliva (WS) significantly affects the killing ability of native or exogenously added Hst 5. WS and phosphate buffered saline were mixed (1:1 by volume), and respective metals and/or exogenous histatin 5 (Hst 5; 30 μM) were added to the mixture before the killing assay was performed. (A) Addition of 300 μM iron (10 times the expected concentration of Hst 5 in WS) significantly reduced the candidacidal activity of WS, as well as activity of exogenously added Hst 5 in WS. (B) Addition of up to 300 μM zinc (10 times the expected concentration of Hst 5 in WS) did not affect the candidacidal ability of native or exogenously added Hst 5 in WS. White bars represent the killing ability of Hst 5 alone in vitro, while light and dark gray bars represent the killing abilities of native or exogenously added Hst 5, respectively, in WS. X is the number of times that the concentration is of Hst 5 alone (30 μM).

Figure 4.

Mass spectrometry of trypsin-digested Hst 5 shows proteolytic stability in the presence of iron. Solutions of Hst 5 (6.2 mM) and respective metals (12.4 mM) were mixed in a total volume of 100 μL to achieve a Hst 5:metal ratio of 1:10, to which 6.3 μg of trypsin in 100 μL volume was added such that Hst 5:trypsin equaled 30:1. Hst 5 alone had a characteristic mass/charge (m/z) peak of 760, while Hst 5 treated with trypsin showed signature masses for the degradation products based on potential trypsin cleavage sites and disappearance of the 760 peak. The profile of Hst 5 in the presence of iron and trypsin was similar to that of Hst 5 alone showing iron binding–mediated proteolytic stability. The profile of Hst 5 in the presence of zinc and trypsin was similar to that of trypsin alone, showing that zinc binding does not offer proteolytic stability to Hst 5.