Zinc Binding by Histatin 5 Promotes Fungicidal Membrane Disruption in C

Journal of Fungi

Article Zinc Binding by Histatin 5 Promotes Fungicidal Membrane Disruption in C. albicans and C. glabrata

Hannah L. Norris, Rohitashw Kumar , Chih Yean Ong, Ding Xu and Mira Edgerton *

Department of Oral Biology, School of Dental Medicine, University at Buffalo, Foster Hall Buffalo, NY 14214, USA; hlnorris@buffalo.edu (H.L.N.); rohitash@buffalo.edu (R.K.); chihyean@buffalo.edu (C.Y.O.); dingxu@buffalo.edu (D.X.) * Correspondence: edgerto@buffalo.edu

Received: 29 June 2020; Accepted: 29 July 2020; Published: 31 July 2020

Abstract: Histatin 5 (Hst 5) is an antimicrobial peptide produced in human saliva with antifungal activity for opportunistic pathogen Candida albicans. Hst 5 binds to multiple cations including dimerization-inducing zinc (Zn2+), although the function of this capability is incompletely understood. Hst 5 is taken up by C. albicans and acts on intracellular targets under metal-free conditions; however, Zn2+ is abundant in saliva and may functionally affect Hst 5. We hypothesized that Zn2+ binding would induce membrane-disrupting pores through dimerization. Through the use of Hst 5 and two derivatives, P113 (AA 4-15 of Hst 5) and Hst 5∆MB (AA 1-3 and 15-19 mutated to Glu), we determined that Zn2+ significantly increases killing activity of Hst 5 and P113 for both C. albicans and Candida glabrata. Cell association assays determined that Zn2+ did not impact initial surface binding by the peptides, but Zn2+ did decrease cell association due to active peptide uptake. ATP efflux assays with Zn2+ suggested rapid membrane permeabilization by Hst 5 and P113 and that Zn2+ affinity correlates to higher membrane disruption ability. High-performance liquid chromatography (HPLC) showed that the higher relative Zn2+ affinity of Hst 5 likely promotes dimerization. Together, these results suggest peptide assembly into fungicidal pore structures in the presence of Zn2+, representing a novel mechanism of action that has exciting potential to expand the list of Hst 5-susceptible pathogens.

Keywords: Candida albicans; Candida glabrata; Histatin 5; P113; zinc; membrane disruption

1. Introduction Histatin 5 (Hst 5) is one of multiple Histatin peptides found in the oral environment of humans and higher primates [1,2]. Hst 5 is notable for its high antimicrobial activity against the oral opportunistic pathogen Candida albicans [2] and other Candida species [3,4]. The full mechanism of action remains unclear, but multiple cellular effects are known, including efflux of small molecules like potassium, magnesium [5], ATP [6], ROS generation [7], and cell cycle arrest [8]. At the same time, it was discovered that Hst 5 binds to multiple divalent cations including copper and zinc [9], potentially with physiological relevance [10], though the function of this capability is still poorly understood [11–16]. The effects of Zn2+ binding on the conformation, oligomerization state, and membrane interactions of the Hst 5 peptide have been described [11,14,15], but this has also raised questions about the practical application of Zn2+ to alter or improve fungicidal activity. Though there are studies on the effect of both copper [12] and iron [16] on Hst 5 killing activity, comparatively little is known about the direct effect of Zn2+ on Hst 5 efficacy or the mechanism involved. Hst 5 kills fungal cells through multiple intracellular mechanisms under established in vitro experimental conditions in which there is a requirement for Hst 5 to be actively taken up by fungal polyamine transporters Dur3 and Dur31 [17]. Exogenous expression of these transporters in the naturally Hst 5-resistant Candida glabrata

J. Fungi 2020, 6, 124; doi:10.3390/jof6030124 www.mdpi.com/journal/jof J. Fungi 2020, 6, 124 2 of 16 results in significantly increased killing activity, which has further substantiated Hst 5 as having an intracellular mechanism of action against fungi [18]. This mechanism is unusual because other cationic antimicrobial peptides (AMPs) that are biochemically similar to Hst 5 function predominantly by pore formation and act directly upon microbial membranes [19]. In fact, although active uptake of Hst 5 is required to achieve efficient antifungal activity under in vitro conditions, Hst 5 binds as well to model membranes as true membrane translocating peptides [20] and causes visible, though mild membrane defects in C. albicans [21]. Despite the ability of Hst 5 to interact with fungal membranes, the peptide is only capable of lethally disrupting a small proportion (10%) of C. albicans membranes at high concentrations [22] or when remaining on the cell surface over time [23]. Thus, the ability of Hst 5 to associate with membranes is almost entirely separate from membrane disruption. Although Hst 5 appears to have little ability to disrupt membranes in low salt buffers (i.e. 10 mM sodium phosphate buffer (NaPB)), introduction of other divalent ions such as Zn2+ may alter the functional properties of Hst 5 in the oral environment. Saliva is the functional environment for Hst 5 and contains measurable levels of numerous metal cations [24]. We found that Zn2+ is the most abundant metal in the saliva of healthy adults among other salivary metals, including Cu, Fe, Ni, and Mn [24]. Zinc concentrations in healthy adult saliva range from 0–3.77 µM[24], though this does not include fluctuations that may occur from diet or Zn2+ supplementation [25,26]. Concentrations of Hst 5 in whole saliva range from 0.7–5.6 µM[27]. Based upon these studies, an average physiological ratio of salivary Zn2+: Hst 5 is less than one Zn2+ per peptide. However, very little work has been done to study how Hst 5 activity is modulated at this physiological ratio [10,13,16]. A review by Łoboda et al. (2018) [28] highlights the diversity of bactericidal and fungicidal membrane binding and pore forming peptides and proteins potentiated by metal binding [28]. Zinc in particular has been implicated in enhancing Hst 5 bacterial and model membrane interactions [13,15], and there are numerous other examples of Zn2+-enhanced protein- and peptide-membrane interactions in eukaryotic and prokaryotic cells [29–31]. Besides this, multiple factors highlight the possibility that addition of Zn2+ might increase membrane disruption capabilities compared to Hst 5 alone. Hst 5 is capable of catalyzing vesicle fusion in the presence of Zn2+ [10], indicating direct binding with membrane phospholipids and the ability to affect membrane structure. Importantly, Zn2+ binding stabilizes Hst 5 in bioactive conformations [11] and also promotes dimerization [14]. Peptide dimerization and aggregation have a complex relationship with AMP activity [32,33] but are highly potentiating for some pore-forming peptides [34–36], and pre-assembly of oligomeric peptide structures can speed up pore formation [34,37,38]. Furthermore, Zn2+ increases the surface adsorption capabilities of Hst 5 at a broad pH range [15], which may lead to general membrane disruption by increasing the number of molecules at the membrane and carpeting the cell surface with high charges [39]. Though our previous work has established that Hst 5 functions by disrupting intracellular ion balance, these studies were carried out without Zn2+ and at relatively high doses of added Hst 5. In this study, we examine Hst 5 at a much lower dose per fungal cell, with added Zn2+ at a physiological ratio. To understand the role of peptide length and sequence in the effects of Zn2+ binding, we utilized Hst 5 and two derivative peptides, including a Hst 5 proteolytic product P113 and a metal-binding mutant Hst 5∆MB, which lack canonical ATCUN and HExxH metal-binding motifs. P113 is often referred to as the “active” fragment of Hst 5 because it retains all of the candidacidal activity of the full-length Hst 5 peptide [40]. A direct comparison of Zn2+ affinity between Hst 5 and P113 has never been carried out, but we expected P113 to have lower Zn2+ affinity than Hst 5 due to the lower number of Zn2+-binding residues. Through a comparison of these three peptides, we show the importance of Zn2+ affinity and dimerization to a novel Zn2+-mediated membrane-disruption mechanism of fungicidal activity for Hst 5. This is the first study to show that Zn2+ binding significantly potentiates Hst 5 and P113 fungicidal activity, and furthermore, it is the first study linking Zn2+ binding to in vitro evidence of membrane disruption in C. albicans. J. Fungi 2020, 6, 124 3 of 16

2. Materials and Methods

2.1. Yeast Strain, Media, and Reagent Preparation Experiments were carried out with Candida albicans strain SC5314 [41] or Candida glabrata strain Cg931010 [42]. Liquid and solid pre-mixed yeast-peptone-dextrose media was used to grow yeast cells (YPD and YPD-Agar, respectively) (Fisher Scientific, Waltham, MA, USA). All media was prepared in deionized water with 50 µg/ mL supplemented uridine (Sigma Aldrich, St. Louis, MO, USA), and sterilized by autoclave. Metal salt solutions of ZnSO 7H O (Fisher Scientific, Waltham, MA, USA) 4· 2 were made in High-performance liquid chromatography (HPLC)-grade water (JT Baker, Phillipsburg, NJ, USA) and used within one day of preparation. Here, 10 mM sodium phosphate buffer, pH 7.4 (NaPB) (Fisher Scientific, Waltham, MA, USA) was prepared directly in HPLC water containers (JT Baker, Phillipsburg, NJ, USA) to prevent metal ion contamination, and filter sterilized. Zincon monosodium salt dye (Sigma Aldrich, St. Louis, MO, USA) for metal titrations was prepared in 100% methanol (Fisher Scientific, Waltham, MA, USA). Briefly, cell culture conditions were as follows for C. albicans and C. glabrata in all experiments: Liquid cell cultures were inoculated from a single colony and grown overnight (30 ◦C, 220 rpm). Cultures were then diluted to OD600 of 0.3–0.4 and re-cultured to OD600 = 0.9–1.0. Cells were then washed twice in NaPB and diluted for use in experiments. For candidacidal assays, a cell concentration of 3.4 107 cells/ mL was used for a dosage of 0.44 fmol × peptide/cell, and a cell concentration of 1.0 106 cells/ mL was used for all other dosages. Uptake assays × and ATP assays were both performed with a cell concentration of 3.4 107 cells/ mL. × 2.2. Peptides Three peptides—Histatin 5, P113, and Histatin 5∆MB—were used in this study. Hst 5 is 24 amino acids long (DSHAKRHHGYKRKFHEKHHSHRGY), while the proteolytic product P113 is only 12 AA (AKRHHGYKRKFH) but retains similar killing efficacy. As a control, we designed a metal binding mutant of Hst 5 (Hst 5 ∆MB, QQQAKRHHGYKRKFQQQQQSHRGY) that replaces both the N-terminal ATCUN motif (AA 1-3) and the canonical HExxH motif (AA 15-19) of Hst 5 with glutamines. Though we were primarily interested in the effects of Zn2+ rather than copper in this study, we chose to mutate both metal binding sites for the purpose of comparing and contrasting activity and function of Hst 5∆MB and P113. All three peptides were synthetically manufactured by Genemed Synthesis, INC. (San Antonio, Texas, USA) and dissolved in RNase and DNase free water (Corning, Corning, NY, USA). Master stocks for long term storage were maintained at 80 C, while diluted working stock − ◦ solutions were stored at 20 C, which were thawed as needed and kept at 4 C to minimize peptide − ◦ ◦ precipitation from repeated freeze-thaw cycles. Peptide solution concentrations were confirmed by NanoDrop (ThermoFisher Scientific, Waltham, MA, USA).

2.3. Peptide Solubility Assay Aggregation was quantified by measuring peptide precipitation from NaPB. Molar ratios of [0:0], [0:1], [1:2], [1:1], [2:1], [3:1], and [4:1] ZnSO4 to 15 µM peptide were used and samples were incubated for 30 min at room temperature (20 C) before centrifugation. Samples were spun at 20000 g for 10 min ◦ × at room temperature and the protein concentrations of the supernatants were subsequently measured using microvolume spectrophotometry (NanoDrop One, Thermofisher Scientific, Waltham, MA, USA) Data were cleaned using robust regression and outlier removal (ROUT) with Q set to 1%. Cleaned data were baseline corrected by subtracting peptide- and metal- free NaPB (0:0). Total moles of peptide per sample (1:2, 1:1, 2:1, 3:1, 4:1) were subtracted from the corresponding Zn2+-free controls (0:1) to provide moles lost value. Percent aggregation out of total peptide added was calculated. Curves were fit using a second order polynomial. Statistical differences between the peptides at each metal salt titration were calculated using two-way ANOVA with Tukey’s multiple comparison test. J. Fungi 2020, 6, 124 4 of 16

2.4. Zinc-Binding Competition Assay Binding competition assays were carried out utilizing the colorimetric copper and Zn2+ binding dye Zincon. Peptides (20 µM) were mixed with Zincon at a 1:1 ratio and titrated with increasing equivalents of ZnSO4. Absorbance at 621 nm was measured at each addition of metal salt using a Lambda 25 UV/Vis spectrophotometer (Perkin Elmer, Waltham, MA, USA). Data were curve ﬁt using a one site competition equation.

2.5. High-Performance Liquid Chromatography To determine the oligomerization state of Hst 5, Hst 5∆MB, and P113, experiments were performed using an Aglient 1260 Infinity modular HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) and a Zenix SEC-80 column (Sepax Technologies, Inc., Newark, DE, USA). The mobile phase used was 10 mM NaPB, pH 7.4, with or without 164.67 µM ZnSO4. Hst 5, Hst 5∆MB, or P113 at 329.34 µM were prepared in 10 mM NaPB and allowed to come to room temperature before loading. Samples were run at a flow rate of 1 mL/min for 15 min, and protein peaks were measured using absorbance at 210 nm. Analysis of HPLC traces were performed using OpenLab software (Agilent Technologies Inc., Santa Clara, CA, USA). Peak identification and peak baselines were determined manually using visual comparison to known standards. Subsequently, statistical differences in the percentage of monomer, dimer, and higher-order peaks of peptide alone and added Zn2+ traces were compared in GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA) using two-way ANOVA and Tukey’s multiple comparisons test. Representative traces were subjected to second-order smoothing.

2.6. Candidacidal Assays Cells (C. albicans or C. glabrata) were cultured as previously described [43]. Briefly, cells were cultured in YPD to OD600 = 1.0 (30 ◦C, 220 rpm), then washed in NaPB, and added to peptides alone or peptides pre-incubated with Zn2+ in NaPB at a dosage of 0.44, 3.75, 7.50, 15, or 30 fmol peptide/cell. After incubation (30 ◦C, 220 rpm), cells were then diluted, plated, and grown for at least 18 h on YPD-agar to determine viability. Experiments were repeated on at least three different days. Significance of differences in killing between Zn2+ and Zn2+-free samples were calculated using one-way ANOVA with Sidak’s multiple comparisons test.

2.7. Peptide Uptake Assay

2+ C. albicans cells were added to pre-incubated peptides (30 min, 20 ◦C) alone or at a 1 Zn :2 peptide ratio, and additionally to Hst 5 at a 3 Zn2+:2 peptide ratio, using a dose of 0.44 fmol peptide/cell. After cells were added, samples were vortexed briefly and immediately centrifuged for 2 min at 6000 g at 20 C, after which 10 µL supernatant was removed to new tubes and stored at 4 C for × ◦ ◦ quantification. Pelleted cells were resuspended with vortexing and incubated at 30 ◦C with shaking at 220 rpm. Samples were removed from 30 ◦C after intervals of 10, 15, and 20 min total incubation time for centrifugation and supernatant removal as described above. Protein concentration in supernatant samples was measured by NanoDrop. Experiments were repeated on at least three different days. The initial time point was taken as a measure of surface binding, and was subtracted from 10, 15, and 20-min time points to determine uptake. Linear regressions were performed on uptake values. Significance of differences in uptake rate and binding between zinc added and zinc free samples were calculated using two-way ANOVA with Sidak’s multiple comparisons test.

2.8. Extracellular ATP Quantiﬁcation Candida albicans (SC5314) or Candida glabrata (Cg931010) cells were prepared as for peptide uptake assays, with the exception that cells were added to samples at a dosage of 0.88 fmol peptide/cell in a 1:2 ratio of ZnSO4:peptide. After samples were mixed, each was immediately centrifuged for 2 min at 6000 g at room temperature, after which 10 µL supernatant was removed to new tubes and added × J. Fungi 2020, 6, 124 5 of 16

J. Fungi 2020, 6, x FOR PEER REVIEW 8 of 16 to 84 µL boiling TE buffer (50 mM Tris pH 7.5, 2 mM EDTA pH 8) (1-min time point). Pelleted cells werenew resuspended tubes and added with to vortexing. 84 µL boiling After TE this, buffer samples (50 mM were Tris incubated pH 7.5, 2 atmM 30 ◦EDTAC (220 pH rpm) 8) (1-min and removed time frompoint). incubation Pelleted for cells a 10-minwere resuspended time point. with Samples vortexing. in TE buAfterffer this, were samples boiled forwere two incubated more minutes at 30 °C and stored(220 rpm) on ice and for removed quantitation. from Then,incubation 25µL for of a each 10-mi samplen time werepoint. added Samples to 10in TEµL buffer Staybrite were adenosine boiled triphosphatefor two more (ATP) minutes assay and mix stored and 65 onµ Lice assay for quanti buffertation. (Biovision, Then, Milpitas,25µL of each CA, sample USA), and were luminescence added to 10 µL Staybrite adenosine triphosphate (ATP) assay mix and 65 µL assay buffer (Biovision, Milpitas, was immediately measured using a FlexStation 3 plate reader (Molecular Devices, San Jose, CA, USA). CA, USA), and luminescence was immediately measured using a FlexStation 3 plate reader Experiments were carried out on at least three separate days. Data were interpolated using a standard (Molecular Devices, San Jose, CA, USA). Experiments were carried out on at least three separate days. curve to determine moles of ATP in each sample. Significant differences between Zn2+ added and Data were interpolated using a standard curve to determine moles of ATP in each sample. Significant Zn2+-free samples at each time point were determined using one-way ANOVA and Sidak’s multiple differences between Zn2+ added and Zn2+-free samples at each time point were determined using one- comparisonsway ANOVA test. and Sidak’s multiple comparisons test. 2.9. Statistical Analysis 2.9. Statistical Analysis AllAll calculationscalculations and and statistical statistical analysis analysis were performed were performed using GraphPad using GraphPadPrism 7.04 (GraphPad Prism 7.04 (GraphPadSoftware, Software,San Diego, San CA, Diego, USA). CA, USA). 3. Results 3. Results 3.1. Zinc Binding Characteristics of Hst 5, P113, and Hst 5∆MB 3.1. Zinc Binding Characteristics of Hst 5, P113, and Hst 5ΔMB Hst 5 is comprised of 24 amino acid residues with multiple overlapping metal binding motifs Hst 5 is comprised of 24 amino acid residues with multiple overlapping metal binding motifs 2+ 2+ includingincluding a Cua Cubinding2+ binding ATCUN ATCUN motif motif [12] and[12] twoand discretetwo discrete Zn bindingZn2+ binding motifs motifs including including a canonical a 2+ HExxHcanonical motif HExxH and another motif and Zn another-affinity Zn sequence2+-affinity closersequence to thecloser N-terminus, to the N-terminus, (H)AKRHH (H)AKRHH [14]. (Figure [14]. 1). The(Figure P113 1). proteolytic The P113 productproteolytic of product Hst 5 is of 12 Hst amino 5 is 12 acids amino in acids length in andlength spans and aminospans amino acids acids 4–15 of 2+ Hst4–15 5 [ 40of]. Hst P113 5 [40]. also P113 has somealso has metal some binding metal binding ability dueability to adue lower to a alowerffinity affinity Zn binding Zn2+ binding site despite site 2+ lackingdespite the lacking HExxH the motif HExxH [44 ]motif and most[44] and of themost Cu of thebinding Cu2+ binding ATCUN ATCUN motif [motif45]. Our [45]. designed Our designed peptide Hstpeptide 5∆MB Hst is the5ΔMB same is the length same as length Hst 5 as but Hst we 5 but expected we expected that the that mutant the mutant peptide peptide and P113and P113 would bothwould have both decreased have decreased Zn2+ binding Zn2+ binding compared compared to Hst to 5 asHst both 5 as containboth contain only oneonly Znone2+ Znbinding2+ binding motif (AKRHH).motif (AKRHH).

FigureFigure 1. 1.A A comparison comparison ofof thethe lengthlength and metal binding binding regions regions of of amino amino acid acid sequences sequences for forHst Hst 5, 5, 2+ 2+ HstHst 5∆ 5MB,ΔMB, and and P113. P113. Three Three metal metal bindingbinding motifsmotifs are labeled—ATCUN (Cu (Cu2+, orange),, orange), HExxH HExxH (Zn (Zn2+, , blue),blue), and and (H)AKRHH (H)AKRHH (Zn (Zn2+2+,, lightlight blue). P113 P113 is is truncated truncated to to amino amino acids acids 4-15 4-15 of ofHst Hst 5. 5.The The ATCUN ATCUN (AA(AA 1-3) 1-3) and and HExxH HExxH (AA (AA 15-19) 15-19) motifs motifs ofof HstHst 55∆ΔMB are mutated mutated to to glutamines. glutamines.

2+ SinceSince Zn Zn2+binding binding stronglystrongly affects aﬀects Hst 5 5 solubility solubility [46], [46], we we compared compared solubility solubility of ofthese these peptides peptides inin 10 10 mM mM NaPB NaPB by by varying varying the the molar molar ratioratio ofof ZnZn22++ addedadded to to Hst Hst 5 5(red), (red), Hst Hst 5Δ 5MB∆MB (blue), (blue), or orP113 P113 (green)(green) at at Zn Zn2+2+to to peptide peptide ratiosratios fromfrom 0:1 to to 4:1 4:1 (Figure (Figure 2A).2A). All All three three peptides peptides were were completely completely soluble soluble withwith one one Zn Zn2+2+to to two two peptides. peptides. However, a a 1:1 1:1 ratio ratio resulted resulted in in 3% 3% precipitation precipitation of ofHst Hst 5, 5,14% 14% of ofHst Hst 5∆5MB,ΔMB, and and 21% 21% precipitation precipitation of of P113,P113, andand a 4:1 ratio further further caused caused 68%, 68%, 25%, 25%, and and 51% 51% precipitation precipitation ofof Hst Hst 5, 5, Hst Hst 5 ∆5ΔMB,MB, andand P113,P113, respectively. Therefore, Therefore, a aratio ratio of of1 Zn 1 Zn2+ to2+ 2to peptides 2 peptides was chosen was chosen to

J. Fungi 2020, 6, 124 6 of 16 J. Fungi 2020, 6, x FOR PEER REVIEW 8 of 16 tomaintain maintain full full solubility solubility of of all all peptides in subsequentsubsequent experiments.experiments. Also,Also, thisthis ratioratio mostmost closely closely representsrepresents physiological physiological ratios ratios of of Hsts Hsts with with Zn Zn2+2+levels levels in in saliva saliva [ 24[24,27].,27].

Figure 2. The three Hsts are all soluble at a physiological ratio of Zn2+ to peptide but differ in relative ZnFigure2+ affi nity.2. The (A three) Percent Hsts solubilityare all soluble of 15 µatM a Hstphysiological 5 (red), Hst ratio 5∆MB of Zn (blue),2+ to peptide and P113 but (green) differ in in 10 relative mM sodiumZn2+ affinity. phosphate (A) Percent buffer solubility pH 7.4 with of 15 increasing µM Hst 5 ratio (red), of Hst Zn 52+ΔMBincubated (blue), and at room P113 temperature(green) in 10 formM 30sodium min. Peptide phosphate solubility buffer was pH measured 7.4 with increasing via NanoDrop ratio A205 of Zn after2+ incubated removal at of room insoluble temperature aggregates for by 30 centrifugation.min. Peptide solubility (B) Binding was competition measured via assay NanoDrop with Zincon A205 and after peptides removal at of 20 insolubleµM in 10 aggregates mM sodium by phosphatecentrifugation. buffer ( pHB) Binding 7.4 to establish competition relative assay affi nitywith of Zincon Hst 5 (red),and peptides Hst 5∆MB at 20 (blue), µM andin 10 P113 mM (green)sodium 2+ forphosphate Zn at increasingbuffer pH 7.4 molar to establish ratios. Absorbance relative affinity of Zincon of Hst 5 at (red), 621 nmHst was5ΔMB measured (blue), and as P113 an inverse (green) indicatorfor Zn2+ ofat relative increasing peptide molar affi nity,ratios. as Absorbance Zincon hasa of blue Zincon color at when 621 boundnm was to zinc.measured Experiments as an inverse were performedindicator inof triplicaterelative peptide and averaged. affinity, as Zincon has a blue color when bound to zinc. Experiments were performed in triplicate and averaged. The relative binding affinity of each peptide for Zn2+ was measured by its ability to compete for 2+ 2+ Zn withThe relative indicator binding dye Zincon, affinity which of each has peptide a high absorbance for Zn2+ was value measured at 621 nmby its when ability Zn to boundcompete and for 2+ lowZn2+ absorbance with indicator when dye not Zincon, Zn bound which (Figure has a2 B).high Zincon absorbance+ Hst 5value∆MB at (blue 621 line)nm when was not Zn significantly2+ bound and 2+ dilowfferent absorbance from Zincon when alone not Zn (black2+ bound line), (Figure confirming 2B). Zincon that Hst + Hst 5∆ 5MBΔMB had (blue the line) lowest was a ffinotnity significantly for Zn 2+ amongdifferent the from tested Zincon peptides. alone In(black contrast, line), Hstconfirming 5 (red line) that competedHst 5ΔMB Zn had theaway lowest from affinity Zincon for up Zn to2+ 2+ 2+ aamong ratio of the two tested Zn peptides.to one peptide In contrast, before Hst binding 5 (red line) sites competed were saturated Zn2+ away and from excess Zincon Zn upremained to a ratio 2+ inof solution two Zn2+ (indicated to one peptide by a largebefore increase binding in sites absorbance were saturated at three and Zn excessto one Zn peptide).2+ remained This in solution agrees 2+ with previous findings that Hst 5 contains two higher-affinity2+ Zn binding sites [47]. It is interesting (indicated by a large increase in absorbance at three Zn to one peptide). This agrees with previous tofindings note that that the Hst majority 5 contains of Hst two 5 higher-affinity precipitation occurredZn2+ binding after sites both [47]. high It is a ffiinterestingnity zinc to binding note that sites the weremajority filled, of and Hst coincided 5 precipitation with an occurred increase after in Zincon both absorbance,high affinity indicating zinc binding that sites fully were saturated filled, zinc and 2+ bindingcoincided decreased with an peptide increase solubility. in Zincon P113 absorbance (green line), hadindicating intermediate that fully Zn saturatedaffinity relative zinc binding to Hst 2+ 5decreased and Hst 5 peptide∆MB, and solubility. P113 competed P113 (green Zn line)away had fromintermediate Zincon onlyZn2+ affinity at a molar relative ratio to of Hst 1:1 5 orand less. Hst 2+ These5ΔMB, results and P113 indicate competed that Hst Zn 52+ has away two from Zn Zinconbinding only sites at anda molar P113 ratio has one,of 1:1 while or less. Hst These 5∆MB results has 2+ 2+ onlyindicate low athatffinity Hst for5 has Zn twodespite Zn2+ binding containing sites and one P113 Zn hasbinding one, while motif. Hst This 5ΔMB also has indicates only low that affinity the 2+ 2+ Znfor Znbinding2+ despite motif containing present in one Hst Zn 5∆2+MB binding and P113 motif. has This variable also aindicatesffinity for that Zn thedependent Zn2+ binding upon motif the surroundingpresent in Hst amino 5ΔMB acids. and P113 has variable affinity for Zn2+ dependent upon the surrounding amino acids. 3.2. Hst 5∆MB C. albicans Killing is Reduced Accompanying Mutation of one Zn2+ Binding Site While Hst 5 2+ Killing3.2. Hst Is Increased5ΔMB C. inalbicans the Presence Killing of is Zn Reduced Accompanying Mutation of one Zn2+ Binding Site While Hst 5 KillingWe first is Increased tested the in fungicidalthe Presence activity of Zn2+ of Hst 5, P113 and Hst 5∆MB in standard buffer (10 mM NaPBWe free first of trace tested metals) the fungicidal to determine activity the of importance Hst 5, P113 of and the intactHst 5Δ ATCUNMB in standard and HExxH buffer motifs (10 mM in killingNaPB (Figurefree of 3trace). We metals) tested to candidacidal determine the activity importance of the three of the peptides intact ATCUN over a rangeand HExxH of doses motifs (0.44, in 3.75,killing 7.5, (Figure 15 and 303). fmolWe /testedcell) following candidacidal 1 h incubation activity of (Figurethe three3A). peptides Hst 5 and over P113 a range had similar of doses killing (0.44, activity3.75, 7.5, over 15 and the entire30 fmol/ range cell) offollowing doses tested 1 h incubation achieving (Figure 90% killing 3A). Hst at 305 and fmol P113/cell. had Hst similar 5∆MB killing had activity over the entire range of doses tested achieving 90% killing at 30 fmol/cell. Hst 5ΔMB had

J.J. Fungi Fungi 20202020,, 66,, x 124 FOR PEER REVIEW 87 of of 16 16 decreased killing activity compared to Hst 5 and P113 at most doses, up to 80% killing at a dose of 30 fmol/decreased cell. killing activity compared to Hst 5 and P113 at most doses, up to 80% killing at a dose of 30 fmolNext/ cell. we examined how the presence of zinc altered killing activity (Figure 3B). To determine the importanceNext we examined of Zn2+ binding how the sites presence for functional of zinc altered activity killing of the activity peptides, (Figure Zn2+ 3wasB). Toadded determine to the 2+ 2+ bufferthe importance at a ratio of 1 Zn Zn2+binding to 2 peptides sitesfor at the functional 0.44 and activity 7.5 fmol/cell of the doses. peptides, Strikingly, Zn was the added presence to the of 2+ Znbu2+ff ersignificantly at a ratio of increased 1 Zn to killing 2 peptides of Hst at 5 the from 0.44 10 andto 70% 7.5 at fmol 0.44/cell fmol/cell doses. and Strikingly, P113 from the 10% presence to 50% of 2+ butZn didsignificantly not significantly increased alter killingthe killing of Hst activity 5 from of Hst 10 to 5Δ 70%MB. atA 0.44similar fmol elevation/cell and of P113 killing from with 10% Zn to2+ was50% found but did for not Hst significantly 5 at 7.5 fmol/cell, alter the but killing the activity relative of increase Hst 5∆MB. was A less similar due elevationto higher ofbasal killing killing. with 2+ TheseZn was results found correlate for Hst with 5 at the 7.5 relative fmol/cell, Zn but2+ binding the relative affinity increase among was the less three due peptides. to higher The basal relatively killing. 2+ lowThese Zn results2+ binding correlate affinity with of the Hst relative 5ΔMB Zn wasbinding associated affinity with among no theincrease three peptides.in killing Theactivity, relatively the 2+ moderatelow Zn bindingrelative aaffinityffinity of of Hst P113 5∆ MBfor wasZn2+ associatedwith a significant with noincrease increase in in killing killing activity, activity, the while moderate the 2+ highestrelative affinity affinity ofZn P1132+ binding for Zn by withHst 5 a was significant associated increase with in even killing greater activity, killing while activity the highest with aaddedffinity 2+ 2+ ZnZn2+. binding by Hst 5 was associated with even greater killing activity with added Zn .

Figure 3. Hst 5∆MB has less killing activity than Hst 5 or P113 and does not have increased killing Figureactivity 3. with Hst added5ΔMB Znhas2 +less.(A killing) Candidacidal activity than assays Hst of 5C. or albicans P113 andSC5314 does cellsnot have with increased 1 h incubation killing at 2+ activity30 ◦C of with Hst 5added (red), HstZn 5. ∆(AMB) Candidacidal (blue), and P113 assays (green) of C. at albicans dosages SC5314 of 0.44, cells 3.75, 7.5,with 15, 1 h and incubation 30 fmol/cell at 30in˚C 10 of mM Hst sodium5 (red), Hst phosphate 5ΔMB (blue), buﬀer and pH P113 7.4. (green) Data represent at dosages at of least 0.44, two 3.75, replicates 7.5, 15, and for 30 each fmol/cell dose.

in(B )10 Candidacidal mM sodium assays phosphate of C. albicansbuffer pHSC5314 7.4. cellsDatawith represent 1 h incubation at least two at 30 replicates◦C of Hst for 5 (red), each Hstdose. 5∆ MB(B) Candidacidal(blue), and P113 assays (green) of C. albicans with and SC5314 without cells a with 1:2 ratio1 h incubation of Zn2+: at peptide 30 °C of (hatched) Hst 5 (red), at dosagesHst 5ΔMB of (blue),0.44 fmol and/cell P113 (left) (green) and 7.5 with fmol and/cell without (right) ina 1:2 10 mMratio sodium of Zn2+: phosphate peptide (hatched) buffer pH at 7.4.dosages Squares of 0.44 and fmol/celltriangles indicate(left) and individual 7.5 fmol/cell replicates. (right) Significancein 10 mM sodium of differences phosphate in killing buffer activity pH 7.4. were Squares calculated and trianglesusing one indicate way ANOVA individual with replicates. Sidak’s multipleSignificance comparisons of differences test. in * killing indicates activityp 0.05, were ** calculated indicates ≤ usingp 0.01, one **** way indicates ANOVAp with0.0001. Sidak’s multiple comparisons test. * indicates p ≤ 0.05, ** indicates p ≤ ≤ ≤ 0.01, *** indicates p ≤ 0.001, **** indicates p ≤ 0.0001. Since Hst 5 killing is most highly associated with its intracellular uptake [17], we hypothesized 2+ that introductionSince Hst 5 killing of Zn is mostwould highly alter theassociated Hst 5 secondary with its intracellular or tertiary structure uptake [17], to permit we hypothesized more rapid thatuptake introduction by C. albicans of Zncells.2+ would We therefore alter the performed Hst 5 secondary Hst-cell-association or tertiary structure assays to to determine permit more the relative rapid 2+ uptakesurface by binding C. albicans and uptake cells. ofWe the therefore three peptides performed with Hst-cell or without-association Zn (Figure assays4). Ato pre-incubationdetermine the relativetime point surface (1 min) binding was taken and touptake determine of the initial three surface peptides binding with ofor thewithout three peptides,Zn2+ (Figure which 4). rangedA pre- incubationfrom 600 to time 1100 point pmol (1 out min) of 3000 was pmol taken total to determ added,ine but initial differences surface were binding not significant of the three and peptides, addition which ranged from 600 to 1100 pmol out of 3000 pmol total added, but differences were not significant

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2+ ofand Zn additiondid not of changeZn2+ did the not relative change initial the relative binding initial of peptide binding to theof peptide cell (Figure to the4A). cell Unexpectedly, (Figure 4A). 2+ additionUnexpectedly, of Zn additionto Hst 5of at Zn a 1:22+ to ratio Hst (dashed5 at a 1:2 red ratio line) (dashed did not red signiﬁcantly line) did not alter significantly total uptake alter or total rate ofuptake uptake or comparedrate of uptake to Hst compared 5 alone (solid to Hst red 5 alon line)e (Figure (solid 4redB). line) Hst 5(Figure was the 4B). only Hst peptide 5 was tothe retain only 2+ 2+ relativelypeptide to high retain uptake relatively with ahigh 1:2 ratiouptake of Znwith toa 1:2 peptide, ratio of so Zn we2+ also to peptide, tested a so higher we also molar tested ratio a of higher Zn 2+ tomolar ascertain ratio ifof ZnZn2+ tobinding ascertain at a if second Zn2+ binding site decreased at a second Hst 5site uptake. decreased The additionHst 5 uptake. of higher The levelsaddition of 2+ Znof higherat a 3:2levels ratio of (dotted Zn2+ at reda 3:2 line) ratio reduced (dotted the red rate line) and reduced total uptake the rate of and Hst total 5 to 470 uptake pmol. of AsHst expected 5 to 470 frompmol. its As low expected killing from activity, its low Hst killing 5∆MB activity, (solid blue Hst line) 5ΔMB had (solid a total blue uptake line) ofhad only a total 370 uptake pmols, of which only 2+ was370 pmols, further which decreased was to further 90 pmol decreased (dashed to blue 90 line)pmol by (dashed the addition blue line) of Zn by .the The addition total uptake of Zn of2+. P113 The alonetotal uptake (green of line) P113 reached alone (green 1570 pmol line) whichreached was 1570 signiﬁcantly pmol which (p was0.001) significantly reduced (p to ≤ 4200.001) pmol reduced with 2+ ≤ theto 420 addition pmol ofwith Zn the(dashed addition green of Zn line).2+ (dashed Thus, green the increased line). Thus, killing the activityincreased of Hstkilling 5 and activity P113 of in Hst the 2+ presence5 and P113 of in Zn thewas presence not a of result Zn2+ of was increased not a result peptide of increased binding or peptide uptake. binding or uptake.

FigureFigure 4. ZincZinc decreases decreases uptake uptake of of P113 P113 at at a aone one Zn Zn2+ to2+ twoto two peptides peptides ratio ratio and andHst Hst5 at 5a atthree a three Zn2+ 2+ Znto twoto peptides two peptides ratio. ratio.Cell association Cell association assays assays were wereperformed performed at 30 ˚ atC 30with◦C 0.44 with fmol/cell. 0.44 fmol Hst/cell. 5 Hst(red), 5 (red),Hst 5Δ HstMB 5 (blue),∆MB (blue), and P113 and (green) P113 (green) in 10 mM in 10 sodium mM sodium phosphate phosphate buffer bupHff er7.4 pH with 7.4 or with without or without added 2+ 2+ 2+ addedZn2+ atZn a oneat Zn a one2+ toZn two peptidesto two peptides ratio (hatched) ratio (hatched) or a three or a Zn three2+ to Zn two topeptides two peptides ratio (dots). ratio (dots). Time Timepoints points were weretaken taken at 1, 10, at 1, 15, 10, and 15, 20 and m. 20 (A m.) One (A) minute One minute time timepoints points were were taken taken as initial as initial binding binding and and(B) cell (B) association cell association attributed attributed to active to active uptake uptake was ca waslculated calculated by subtracting by subtracting the initial the initial time point time point from fromthe 10, the 15, 10, and 15, and20 m 20 time m time points. points. Linear Linear regression regressionss were were performed performed to to dete determinermine rate rate of of uptake. ExperimentsExperiments were repeatedrepeated onon atat leastleast threethree separateseparate daysdays andand averaged.averaged. SignificanceSignificance of didifferencesfferences inin initialinitial bindingbinding werewere calculatedcalculated usingusing one-wayone-way ANOVA with Sidak’s multiple comparisons test. SignificanceSignificance of didifferencesfferences inin raterate ofof uptakeuptake werewere calculatedcalculated usingusing one-wayone-way ANOVAANOVA withwith Sidak’sSidak’s multiple comparisons test. **** indicates p 0.0001 multiple comparisons test. **** indicates p ≤ 0.0001 We next assessed whether the increase in Zn2+-mediated killing activity of Hst 5 and P113 was We next assessed whether the increase in Zn2+-mediated killing activity of Hst 5 and P113 was associated with ATP efflux, a classic indicator of membrane disruption by AMPs [21]. ATP efflux was associated with ATP efflux, a classic indicator of membrane disruption by AMPs [21]. ATP efflux was measured for each peptide with or without Zn2+ after 1 min and 10 min of incubation with C. albicans measured for each peptide with or without Zn2+ after 1 min and 10 min of incubation with C. albicans (Figure(Figure5 5).). We We found found that that Hst Hst 5 5 (red) (red) induced induced a a total total ATP ATP e effluxfflux ofof 317317 pmolpmol atat 1 min, which increased to 732 pmol after a 10 min incubation. Addition of Zn2+ (red hatched) significantly increased ATP to 732 pmol after a 10 min incubation. Addition of Zn2+ (red hatched) significantly increased ATP releaserelease toto 11801180 pmolpmol at at 1 1 min min and and 1513 1513 pmol pmol at at 10 10 min, min, representing representing a 3.7-folda 3.7-fold increase increase in in ATP ATP effl effluxux at 1at min1 min and and 2.1-fold 2.1-fold at at 10 10 min. min. Hst Hst 5 ∆5ΔMBMB alone alone (blue) (blue) initiated initiated ATPATP eeffluxfflux ofof 8585 andand 257257 pmolspmols atat 1 and 10 min that did not significantly increase with added Zn2+ (blue hatched). For P113 alone (green), and 10 min that did not significantly increase with added Zn2+ (blue hatched). For P113 alone (green), ATP efflux was 304 and 582 pmols at 1 and 10 min, while the addition of Zn2+ (green hatched) did ATP efflux was 304 and 582 pmols at 1 and 10 min, while the addition of Zn2+ (green hatched) did not notincrease increase release release at 1 at min 1 min but but significantly significantly increased increased ATP ATP release release to to 1471 1471 pmol pmol (2.5-fold) (2.5-fold) at at 10 10 min. TheseThese resultsresults showshow thatthat Hst Hst 5 5 or or P113 P113 alone alone induce induce some some release release of of ATP ATP from from cells, cells, but but the the addition addition of Zn2+ significantly elevated ATP efflux suggesting membrane permeabilization of C. albicans cells. of Zn2+ significantly elevated ATP efflux suggesting membrane permeabilization of C. albicans cells.

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Figure 5. Zinc induces almost immediate ATP efflux. C. albicans SC5314 bioluminescent ATP efflux assay. Figure 5. Zinc induces almost immediate ATP efflux. C. albicans SC5314 bioluminescent ATP efflux Cells were treated at 30 C with 0.88 fmol/ cell. Hst 5 (red), Hst 5∆MB (blue), or P113 (green) in 10 mM assay. Cells were treated◦ at 30 °C with 0.88 fmol/ cell. Hst 5 (red), Hst 5ΔMB (blue), or P113 (green) in sodium phosphate buffer pH 7.4 with or without added Zn2+ at a one to two peptides ratio (hatched). 10 mM sodium phosphate buffer pH 7.4 with or without added Zn2+ at a one to two peptides ratio Cells were removed from samples via centrifugation and aliquots of supernatant were taken at 1 min (hatched). Cells were removed from samples via centrifugation and aliquots of supernatant were and 10 min time points to measure extracellular ATP by luminescence. Experiments were repeated on taken at 1 min and 10 min time points to measure extracellular ATP by luminescence. Experiments at least three separate days and averaged. Significance of differences in ATP efflux calculated using one were repeated on at least three separate days and averaged. Significance of differences in ATP efflux way ANOVA with Sidak’s multiple comparison test. ** indicates p 0.01, *** indicates p 0.001. calculated using one way ANOVA with Sidak’s multiple comparison≤ test. ** indicates≤ p ≤ 0.01, *** Sinceindicates Zn2+ pappeared ≤ 0.001. to initiate fungicidal activity by membrane disruption, we examined whether Zn2+ could function in a similar manner against C. glabrata that does not transport Hst 5 intracellularly Since Zn2+ appeared to initiate fungicidal activity by membrane disruption, we examined and consequently is insensitive to Hst 5. We therefore performed killing (Figure6A) and ATP whether Zn2+ could function in a similar manner against C. glabrata that does not transport Hst 5 efflux assays (Figure6B) in C. glabrata with peptides with and without added Zn2+. We found that a intracellularly and consequently is insensitive to Hst 5. We therefore performed killing (Figure 6A) 7.5 fmol/cell dose of Hst 5 (red) induced 30% killing, which is 40% less than in C. albicans under the and ATP efflux assays (Figure 6B) in C. glabrata with peptides with and without added Zn2+. We found same conditions, while the addition of Zn2+ (red hatched) increased killing to nearly 100%. Hst 5∆MB that a 7.5 fmol/ cell dose of Hst 5 (red) induced 30% killing, which is 40% less than in C. albicans under (blue) only produced about 20% killing with or without added Zn2+, but P113 (green) increased from the same conditions, while the addition of Zn2+ (red hatched) increased killing to nearly 100%. Hst about 40% to 80% killing with the addition of Zn2+. As suggested by the low killing activity of Hst 5ΔMB (blue) only produced about 20% killing with or without added Zn2+, but P113 (green) increased 5, ATP efflux was minimal in C. glabrata when treated with peptide alone. Hst 5 induced 10 pmol from about 40% to 80% killing with the addition of Zn2+. As suggested by the low killing activity of ATP efflux total at 1 min which increased to 40 pmol after 10 min. However, when Zn2+ was added, Hst 5, ATP efflux was minimal in C. glabrata when treated with peptide alone. Hst 5 induced 10 pmol ATP efflux increased to 270 pmol at one min, which did not further increase after 10 min, indicating that ATP efflux total at 1 min which increased to 40 pmol after 10 min. However, when Zn2+ was added, maximal ATP efflux occurred almost immediately. Interestingly, although ATP efflux due to Hst 5∆MB ATP efflux increased to 270 pmol at one min, which did not further increase after 10 min, indicating alone never exceeded 30 pmol total, when Zn2+ was added, a significant increase to over 100 pmol that maximal ATP efflux occurred almost immediately. Interestingly, although ATP efflux due to Hst occurred by 10 min. This indicates that Hst 5∆MB is also minimally capable of membrane disruption, 5ΔMB alone never exceeded 30 pmol total, when Zn2+ was added, a significant increase to over 100 though to a lesser extent than Hst 5. P113 also had very low ATP efflux like Hst 5 or Hst 5∆MB, but pmol occurred by 10 min. This indicates that Hst 5ΔMB is also minimally capable of membrane when Zn2+ was added, an immediate increase in ATP efflux was apparent, reaching 200 pmol at 1 min disruption, though to a lesser extent than Hst 5. P113 also had very low ATP efflux like Hst 5 or Hst and almost 260 pmol at 10 min. 5ΔMB, but when Zn2+ was added, an immediate increase in ATP efflux was apparent, reaching 200 pmol at 1 min and almost 260 pmol at 10 min.

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Figure 6. Zinc greatly increases Hst 5 and P113 killing of C. glabrata, and induces almost immediate Figure 6. Zinc greatly increases Hst 5 and P113 killing of C. glabrata, and induces almost immediate ATP efflux. C. glabrata Cg931010 cells were used in candidacidal assays and ATP efflux assays. (A) For ATP efflux. C. glabrata Cg931010 cells were used in candidacidal assays and ATP efflux assays. (A) candidacidal assays, cells were incubated for 1 h at 30 C with Hst 5 (red), Hst 5∆MB (blue), and For candidacidal assays, cells were incubated for 1 h at 30◦ ˚C with Hst 5 (red), Hst 5ΔMB (blue), and P113 (green) with and without a 1:2 ratio of Zn2+ to peptides (hatched) at 7.5 fmol peptides/cell in P113 (green) with and without a 1:2 ratio of Zn2+ to peptides (hatched) at 7.5 fmol peptides/cell in 10 10 mM sodium phosphate buffer pH 7.4. Squares and triangles indicate individual replicates. (B) In mM sodium phosphate buffer pH 7.4. Squares and triangles indicate individual replicates. (B) In bioluminescent ATP efflux assays, cells were treated with 0.88 fmol/cell Hst 5 (red), Hst 5∆MB (blue), bioluminescent ATP efflux assays, cells were treated with 0.88 fmol/cell Hst 5 (red), Hst 5ΔMB (blue), or P113 (green) in 10 mM sodium phosphate buffer pH 7.4 with or without added Zn2+ at a one to or P113 (green) in 10 mM sodium phosphate buffer pH 7.4 with or without added Zn2+ at a one to two two peptides ratio (hatched). Cells were removed from samples via centrifugation and aliquots of peptides ratio (hatched). Cells were removed from samples via centrifugation and aliquots of supernatant were taken at 1 and 10 min time points to measure extracellular ATP by luminescence. supernatant were taken at 1 and 10 min time points to measure extracellular ATP by luminescence. Both experiments were repeated on at least three separate days and averaged. Significance of differences Both experiments were repeated on at least three separate days and averaged. Significance of in killing and ATP efflux were calculated using one way ANOVA with Sidak’s multiple comparison differences in killing and ATP efflux were calculated using one way ANOVA with Sidak’s multiple test. * indicates p 0.05, **** indicates p 0.0001 comparison test. *≤ indicates p ≤ 0.05, ****≤ indicates p ≤ 0.0001 We hypothesized that peptide-membrane-disruption effects induced by Zn2+ may be partly a 2+ resultWe of hypothesized Zn2+-induced that peptide peptide-membra dimerization,ne-disruption since dimerization effects isinduced dependent by Zn on themay number be partly and a 2+ locationresult of ofZn histidines-induced [ 12peptide] and hasdimerization, previously since been correlateddimerization to fusogenicis dependent activity on the [10 number]. Therefore, and peptidelocation dimerizationof histidines induced[12] and byhas Zn previously2+ was examined been correlated using size-exclusion to fusogenic HPLCactivity (Figure [10]. Therefore,7). Hst 5 peptide dimerization induced by Zn2+ was examined using size-exclusion HPLC (Figure 7). Hst 5 (red (red trace) eluted in two peaks—one at 8 min that we interpreted as dimer formation and a second trace) eluted in two peaks—one at 8 min that we interpreted as dimer formation and a second larger larger peak at 9 min—that represented the majority of the peptide and was assigned as the monomeric peak at 9 min—that represented the majority of the peptide and was assigned as the monomeric fraction. The addition of Zn2+ to the mobile phase resulted in a distinguishable change of the relative fraction. The addition of Zn2+ to the mobile phase resulted in a distinguishable change of the relative ratio between the monomeric and dimeric peaks. Analysis of peak areas found that addition of Zn2+ ratio between the monomeric and dimeric peaks. Analysis of peak areas found that addition of Zn2+ increased the percentage of peptide in dimer form from 4% to 24%. For Hst 5∆MB (blue trace), increased the percentage of peptide in dimer form from 4% to 24%. For Hst 5ΔMB (blue trace), the the majority of peptide eluted as a monomer peak at 8 min, and the addition of Zn2+ to the mobile majority of peptide eluted as a monomer peak at 8 min, and the addition of Zn2+ to the mobile phase phase did not change the elution profile. P113 (green trace) eluted as three peaks—the smallest at 8 min, did not change the elution profile. P113 (green trace) eluted as three peaks—the smallest at 8 min, a a larger peak at 9 min, and another larger peak at 11.5 min—corresponding to tetrameric, dimeric, and larger peak at 9 min, and another larger peak at 11.5 min—corresponding to tetrameric, dimeric, and monomeric fractions by molecular weight. Interestingly, this shows that a large proportion (about 50%) monomeric fractions by molecular weight. Interestingly, this shows that a large proportion (about of P113 naturally forms dimers in 10 mM NaPB (pH 7.4). The addition of Zn2+ to the mobile phase 50%) of P113 naturally forms dimers in 10 mM NaPB (pH 7.4). The addition of Zn2+ to the mobile slightly increased the percentage of dimeric and oligomeric species of P113. Thus, Zn2+ induces dimer phase slightly increased the percentage of dimeric and oligomeric species of P113. Thus, Zn2+ induces formation in Hst 5, but not Hst 5∆MB, and somewhat increases dimeric and higher order structures dimer formation in Hst 5, but not Hst 5ΔMB, and somewhat increases dimeric and higher order in P113. structures in P113.

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Figure 7. Zinc binding by Hst 5 promotes zinc-induced soluble dimers. Size-exclusion high-performance liquidFigure chromatography 7. Zinc binding (HPLC) by Hst was 5 performed promotes with zinc-induced protein detection soluble at A210dimers. to determine Size-exclusion dimerization high- stateperformance of (top, red)liquid Hst chromatography 5, (middle, blue) (HPLC) Hst 5∆ MB,was andperformed (bottom, with green) protein P113 detection at a concentration at A210 ofto 329.34determineµM dimerization in mobile phase state composed of (top, red) of Hst either 5, (middle, zinc-free blue) 10 mM Hst sodium 5ΔMB, phosphateand (bottom, bu green)ffer pH P113 7.4, orat bua concentrationffer with added of zinc329.34 at µM a concentration in mobile phas of 164.67e composedµM. Each of either condition zinc-free was run10 mM in triplicate. sodium Peakphosphate identification buffer pH was 7.4, made or buffer based uponwith added comparison zinc at to a known concentration standards of and164.67 area µM. under Each the condition curve of peakswas run was in usedtriplicate. to calculate Peak identification the relative percentage was made of based peptide upon in comparison monomer (M), to known dimer (D), standards and higher and orderarea under oligomer the curve (T) form of peaks (circle was graphs). used Representativeto calculate the tracesrelative of percentage individual of runs peptide were in subjected monomer to second(M), dimer order (D), smoothing and higher for order visualization. oligomer (T) form (circle graphs). Representative traces of individual runs were subjected to second order smoothing for visualization. 4. Discussion 4. DiscussionWe found that Zn2+ binding to Hst 5 and P113 significantly increased their ability to kill C. albicans concurrentWe found with that rapid Zn2+ ATP binding release to suggestingHst 5 and P113 membrane significan disruption.tly increased In step their with ability this to finding, kill C. 2+ wealbicans discovered concurrent that Zn withbinding rapid potentiatesATP release the suggesting candidacidal membrane activityof disruption. Hst 5 and P113In step for C.with glabrata this byfinding, a similar we discovered mechanism, that which Zn2+ is binding of exciting potentiates clinical relevancethe candidacidal because activityC. glabrata of Hstis highly 5 and resistantP113 for toC. Hstglabrata 5 [18 by] and a similar other mechanism, AMPs [48]. Thiswhich work is of is exciting also of clinical significance relevance in understanding because C. glabrata the role is highly of Hst 2+ 2+ 5resistant in the oral to Hst environment, 5 [18] and other as we AMPs found [48]. an This average work physiological is also of significance ratio of Hstin understanding 5 and Zn (1 the Zn role:2 peptides)of Hst 5 in potentiates the oral environment, the candidacidal as we activity foun ofd an Hst average 5 against physiological these two Candida ratio ofspp, Hst suggesting 5 and Zn that2+ (1 2+ 2+ Zn 2+:2binding peptides) is relevant potentiates to the the function candidacidal of Hst 5 inactivity typical of levels Hst of5 Znagainstfound these in salivatwo Candida [24]. spp, suggestingOf the threethat Zn classic2+ binding models is ofrelevant membrane to the disruption function of by Hst antimicrobial 5 in typical peptides—carpet, levels of Zn2+ found barrel in saliva stave, and[24]. toroidal as reviewed in Łoboda et al (2018) [28]—our data suggests that Hst 5 most likely functions in theOf toroidal the three model. classic Hst models 5 binding of membrane to C. albicans disruptionwas not by significantly antimicrobial changed peptides—carpet, by the addition barrel of 2+ Znstave,, suggestingand toroidal that as reviewed Hst 5 does in not Łoboda function et al through(2018) [28]—our non-specific data carpetsuggests disruption. that Hst 5 Barrel-stave most likely pore-formingfunctions in the peptides toroidal require model. a hydrophobicHst 5 binding character to C. albicans to insert was into not asignificantly membrane. changed Hst 5 is highlyby the chargedaddition andof Zn lacks2+, suggesting the hydrophobicity that Hst necessary5 does not to function insert directly through into non-specific a membrane carpet to form disruption. a typical barrel-staveBarrel-stave pore-forming pore [19]. Toroidal peptides pores require are formeda hydrophobic by more character polar peptides to insert thatinto binda membrane. to lipid Hst head 5 groupsis highly and charged induce and curvature lacks the until hydrophobicity pores develop, necessary consistent to withinsert Hst directly 5 membrane into a membrane disruption to having form a toroidaltypical barrel-stave mechanism. pore [19]. Toroidal pores are formed by more polar peptides that bind to lipid headWe groups also showedand induce that curvature Hst 5 dimerizes until pores at a low deve 1:2lop, ratio consistent of Zn2+ withto peptides. Hst 5 membrane This agrees disruption with the historicalhaving a toroidal view that mechanism. the canonical Zn2+-binding HExxH motif facilitates Zn2+-bridged dimerization interactionsWe also [10 showed] and recent that Hst work 5 dimerizes showing dimerizationat a low 1:2 ratio experimentally of Zn2+ to peptides. [14]. Dimerization This agrees of with Hst 5the in solutionhistorical could view be that an importantthe canonical precursor Zn2+-binding to membrane HExxH disruption, motif facilitates as this is aZn mechanism2+-bridged that dimerization can speed interactions [10] and recent work showing dimerization experimentally [14]. Dimerization of Hst 5 in solution could be an important precursor to membrane disruption, as this is a mechanism that can

J. Fungi 2020, 6, x FOR PEER REVIEW 8 of 16 J. Fungi 2020, 6, 124 12 of 16 speed up pore formation after membrane binding [32,34]. However, Hst 5ΔMB does not dimerize in 2+ 2+ upsolution pore formationwith added after Zn membrane but does binding induce [ 32ATP,34]. efflux However, in C. Hst glabrata 5∆MB, suggesting does not dimerize that Zn in solutionbinding withwithout added soluble Zn2 +dimerizationbut does induce can slightly ATP effl increaseux in C. glabratamembrane, suggesting stress. Additionally, that Zn2+ binding P113 naturally without 2+ solubleforms dimers dimerization but did can not slightly show any increase indication membrane of membrane stress. Additionally, disruption without P113 naturally the addition forms of dimers Zn , butsuggesting did not showthat pore-forming any indication peptide–peptide of membrane disruption interactions without at the additioncell membrane of Zn2+ ,are suggesting specifically that 2+ 2+ pore-formingmediated by Zn peptide–peptide binding. A model interactions of Zn -induced at the cell peptide membrane self-assembly are specifically at the membrane mediated accounts by Zn2+ binding.for the both A modeldelayed of membrane Zn2+-induced disruption peptide by self-assembly P113 seen in at our the ATP membrane assays in accounts C. albicans, for theand both the 2+ delayedslight increase membrane in ATP disruption efflux in by C. P113 glabrata seen indue our toATP Hst assays5ΔMB inwithC. albicans,added Znand.the First, slight pore-forming increase in ATPpeptides efflux have in C. length glabrata requirementsdue to Hst 5 and∆MB must with span added the Zn width2+. First, of a pore-forming membrane in peptides order to have generate length a 2+ requirementspore [49]. P113 and binds must Zn span with the less width relative of a affinity membrane and inis orderhalf the to length generate of aHst pore 5, so [49 it]. may P113 be binds less 2+ Znprone2+ with to pore less assembly relative a ffiatnity the andmembrane. is half the In lengthour proposed of Hst 5,model, so it may P113 be must less pronego through to pore more assembly Zn - atinduced the membrane. aggregation In oursteps proposed than Hst model, 5 in order P113 to must form go athrough pore because more Znit is2+ -inducedtoo short aggregationto span the stepsmembrane than Hstand 5therefore in order requires to form atwice pore the because number it is of too molecules short to to span form the a membranepore the same and size therefore as Hst requires5 (Figure twice8). In thecontrast, number we ofpropose molecules that toHst form 5ΔMB a pore undergoes the same slow size and as Hstreduced 5 (Figure aggregation8). In contrast, due to 2+ welower propose Zn affinity that Hst and 5∆ MBminimal undergoes soluble slow dimerization and reduced so that aggregation membrane due effects to lower are Zn much2+ a ffilessnity lethal and minimalto cells. soluble dimerization so that membrane effects are much less lethal to cells.

Figure 8. A model of ZnZn22++-induced-induced pore pore formation formation by by Hst Hst 5 an andd P113. Zinc binding induces soluble dimers of Hst 5 thatthat bindbind toto thethe cellcell surfacesurface ofof C.C. albicansalbicans andand C.C. glabrataglabrata.. InteractionInteraction withwith fungalfungal membranes induces dimer units to assemble into higher order structures, which induces membrane curvature andand toroidaltoroidal pores.pores. P113 functionsfunctions similarlysimilarly butbut isis dimericdimeric beforebefore ZnZn22++ bindingbinding and requires more assembly steps to form a pore structure of the same size as Hst 5. Hst 5Δ∆MB binds to zinc but does not form dimers or assemble into higher order structuresstructures whenwhen membranemembrane bound.bound.

Cytosolic transporttransport isis an an important important determinant determinant of ofC. C. albicans albicanssusceptibility susceptibility to killing to killing by Hst by 5Hst [17 5]. 2+ We[17]. found We found that the that molar the molar binding binding ratio of ratio Zn ofwith Zn2+ Hst with 5 profoundlyHst 5 profoundly affected affected activepeptide active peptide uptake 2+ (Figureuptake 4(Figure), suggesting 4), suggesting that dual that Zn dualbinding Zn2+ binding plays a roleplays in a fine role tuning in fine the tuning effect the and effect targets and of targets Hst 5. Weof Hst previously 5. We previously established established that P113 that requires P113 a requ specificires aminoa specific acid amino sequence acid forsequence cytosolic for transportcytosolic bytransportC. albicans by C.cells albicans [50], andcells later [50], this and was later proposed this was toproposed be three to amino be three acids, amino Lys-Phe-His acids, Lys-Phe-His (KFH) that

J. Fungi 2020, 6, 124 13 of 16 make up AA 10–12 for P113 or 13–15 for Hst 5 [51]. The Zn2+ coordinating site of P113 is known and includes part of the KFH sequence [44], which suggests that Zn2+ binding could interfere with intracellular transport. Indeed, we found that uptake of P113 was lost with the addition of a 1:2 molar ratio of Zn2+:peptide. Similarly, Hst 5∆MB, in which KFH has been mutated to KFQ, has low uptake regardless of the addition of Zn2+. In light of this, it is significant that Hst 5 does not have decreased uptake at a molar ratio of 1 Zn2+ to 2 peptides, but uptake was decreased when three Zn2+ per two peptides was used. This suggests that Zn2+ does not first bind at the KFH sequence, but the second bound Zn2+ blocks KFH to reduce uptake. This effect is not related to the dimerization of Hst 5 or P113, since P113 is 50% dimer with or without added Zn2+, and Hst 5 has good uptake at a ratio of Zn2+ with significant dimer formation. We speculate that transport of Zn2+-bound Hst 5 into the cytosol is a functional mechanism to maximize killing activity. While it is generally accepted that salivary Hst 5 functions in innate immunity for oral opportunistic pathogens [52], not all fungal species have susceptibility to intracellularly targeted Hst 5. Increasing the molar binding ratio of Zn2+ to peptide modulates the target of Hst 5 from intracellular to the membrane, but there is an ideal ratio with maximal solubility, killing activity, and dimer formation where both targets can be acted on simultaneously. This is important because differences in ATP efflux and killing activity indicate that C. albicans and C. glabrata are not equally susceptible to membrane disruption by Hst 5, which is likely due to variability in cell wall or membrane components. Dual Zn2+ binding allows Hst 5 to act on the separate fungicidal targets of two oral pathogens with differing susceptibilities under the same conditions, suggesting that Hst 5 could also be involved in the control of other fungal and bacterial pathogens regardless of uptake ability. Further work is needed to elucidate the full scope of the role of Hst 5 in oral health in combination with salivary Zn2+.

5. Conclusions In this study, we determined a new mode of action for Hst 5 that is modulated by zinc binding at a physiological molar ratio of Zn2+:peptide. This ﬁnding could have important implications in our understanding of the innate immune mechanisms in the oral environment, and furthermore provides valuable information to clinicians about the eﬀect of Zn2+ on Hst 5 and derivatives like P113 that might have clinical relevance in treatment of oral fungal infections. This work highlights that Zn2+ ions have a functional antifungal role in the complex oral environment.

Author Contributions: Conceptualization, M.E. and H.L.N.; methodology, M.E. and H.L.N.; validation, H.L.N.; formal analysis, H.L.N.; investigation, H.L.N. and C.Y.O.; resources M.E., D.X., R.K., and H.L.N.; data curation, H.L.N.; writing—original draft preparation, M.E. and H.L.N.; writing—review and editing, M.E., H.L.N., R.K., D.X., and C.Y.O.; visualization, M.E. and H.L.N; supervision, M.E., R.K., and D.X.; project administration, M.E.; funding acquisition, M.E. and H.L.N. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by National Institute of Health NIDCR grants R01DE010641 to M.E., F31DE028458 to H.L.N., and NIAMS grant R01AR070179 to D.X. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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