Salivary histatin 5 induces non-lytic release of ATP from Candida albicans leading to cell death.

Salivary histatins are potent in vitro antifungal proteins and have promise as therapeutic agents against oral candidiasis. We performed pharmacological studies directed at understanding the biochemical basis of Hst 5 candidacidal activity. Three inhibitors of mitochondrial metabolism: carbonyl cyanide p-chlorophenylhydrazone, dinitrophenol, and azide inhibited Hst 5 killing of Candida albicans, while not inhibiting cellular ATP production. In contrast, Hst 5 caused a drastic reduction of C. albicans intracellular ATP content, which was a result of an efflux of ATP. Carbonyl cyanide p-chlorophenylhydrazone, dinitrophenol, and azide inhibited Hst 5-induced ATP efflux, thus establishing a correlation between ATP release and cell killing. Furthermore, C. albicans cells were respiring and had polarized membranes at least 80 min after ATP release, thus implying a non-lytic exit of cellular ATP in response to Hst 5. Based on evidence that transmembrane ATP efflux can occur in the absence of cytolysis through a channel-like pathway and that released ATP can act as a cytotoxic mediator by binding to membrane purinergic receptors, we evaluated whether extracellular ATP released by Hst 5 may have further functional role in cell killing. Consistent with this hypothesis, purinergic agonists BzATP and adenosine 5'O-(thiotriphosphate) induced loss of C. albicans cell viability and purinergic antagonists prevented Hst 5 killing.     

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

Histatins are salivary cationic peptides that provide the first line of defense against oral candidiasis caused by Candida albicans. This minireview presents a critical evaluation of our knowledge of the candidacidal mechanism of histatin 5 (Hst 5). Hst 5 is the most potent among all histatin family members with regard to its antifungal activity. The mode of action of Hst 5 has been a subject of intense debate. Unlike other classical host innate immune proteins, pore formation or membrane lysis by Hst 5 has largely been disproven, and it is now known that all targets of Hst 5 are intracellular. Hst 5 binds C. albicans cell wall proteins (Ssa1/2) and glycans and is taken up by the cells through fungal polyamine transporters in an energy-dependent manner. Once inside the fungal cells, Hst 5 may affect mitochondrial functions and cause oxidative stress; however, the ultimate cause of cell death is by volume dysregulation and ion imbalance triggered by osmotic stress. Besides these diverse targets, a novel mechanism based on the metal binding abilities of Hst 5 is discussed. Finally, translational approaches for Hst 5, based on peptide design and synergy with other known drugs, are considered a step forward for bench-to-bed application of Hst 5.     

Histatin 5 uptake by Candida albicans utilizes polyamine transporters Dur3 and Dur31 proteins

Histatin 5 (Hst 5) is a salivary gland-secreted cationic peptide with potent fungicidal activity against Candida albicans. Hst 5 kills fungal cells following intracellular translocation, although its selective transport mechanism is unknown. C. albicans cells grown in the presence of polyamines were resistant to Hst 5 due to reduced intracellular uptake, suggesting that this cationic peptide may enter candidal cells through native yeast polyamine transporters. Based upon homology to known Saccharomyces cerevisiae polyamine permeases, we identified six C. albicans Dur polyamine transporter family members and propose a new nomenclature. Gene deletion mutants were constructed for C. albicans polyamine transporters Dur3, Dur31, Dur33, Dur34, and were tested for Hst 5 sensitivity and uptake of spermidine. We found spermidine uptake and Hst 5 mediated killing were decreased significantly in Δdur3, Δdur31, and Δdur3/Δdur31 strains; whereas a DUR3 overexpression strain increased Hst 5 sensitivity and higher spermidine uptake. Treatment of cells with a spermidine synthase inhibitor increased spermidine uptake and Hst 5 killing, whereas protonophores and cold treatment reduced spermidine uptake. Inhibition assays showed that Hst 5 is a competitive analog of spermidine for uptake into C. albicans cells, and that Hst 5 Ki values were increased by 80-fold in Δdur3/Δdur31 cells. Thus, Dur3p and Dur31p are preferential spermidine transporters used by Hst 5 for its entry into candidal cells. Understanding of polyamine transporter-mediated internalization of Hst 5 provides new insights into the uptake mechanism for C. albicans toxicity, and further suggests design for targeted fungal therapeutic agents.     

Cationic peptide-induced remodelling of model membranes: direct visualization by in situ atomic force microscopy

Our understanding of how antimicrobial and cell-penetrating peptides exert their action at cell membranes would benefit greatly from direct visualization of their modes of action and possible targets within the cell membrane. We previously described how the cationic antimicrobial peptide, indolicidin, interacted with mixed zwitterionic planar lipid bilayers as a function of both peptide concentration and lipid composition [Shaw, J.E. et al., 2006. J. Struct. Biol. 154 (1), 42-58]. In the present report, in situ atomic force microscopy was used to characterize the interactions between three families of cationic peptides: (1) tryptophan-rich antimicrobial peptides--indolicidin and two of its analogues, (2) an amphiphilic alpha-helical membranolytic peptide--melittin, and (3) an arginine-rich cell-penetrating peptide--Tat with phase-separated planar bilayers containing 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC)/1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) or DOPC/N-stearoyl-D-erythro-sphingosylphosphorylcholine (SM)/cholesterol. We found that these cationic peptides all induced remodelling of the model membranes in a concentration, and family-dependent manner. At low peptide concentration, these cationic peptides, despite their different biological roles, all appeared to reduce the interfacial line tension at the domain boundary between the liquid-ordered and liquid-disordered domains. Only at high peptide concentration was the membrane remodelling induced by these peptides morphologically distinct among the three families. While the transformation caused by indolicidin and its analogues were structurally similar, the concentration required to initiate the transformation was strongly dependent on the hydrophobicity of the peptide. Our use of lipid compositions with no net charge minimized the electrostatic interactions between the cationic peptides and the model supported bilayers. These results suggest that peptides within the same functional family have a common mechanism of action, and that membrane insertion of short cationic peptides at low peptide concentration may also alter membrane structure through a common mechanism regardless of the peptide's origin.     

Role of membranotropic sequences from herpes simplex virus type I glycoproteins B and H in the fusion process

The entry of enveloped viruses involves attachment followed by close apposition of the viral and plasma membranes. Then, either on the cell surface or in an endocytotic vesicle, the two membranes fuse by an energetically unfavourable process requiring the destabilisation of membrane microenvironment in order to release the viral nucleocapsid into the cytoplasm. The core fusion machinery, conserved throughout the herpesvirus family, involves glycoprotein B (gB) and the non-covalently associated complex of glycoproteins H and L (gH/gL). Both gB and gH possess several hydrophobic domains necessary for efficient induction of fusion, and synthetic peptides corresponding to these regions are able to associate to membranes and induce fusion of artificial liposomes. Here, we describe the first application of surface plasmon resonance (SPR) to the study of the interaction of viral membranotropic peptides with model membranes in order to enhance our molecular understanding of the mechanism of membrane fusion. SPR spectroscopy data are supported by tryptophan fluorescence, circular dichroism and electron spin resonance spectroscopy (ESR). We selected peptides from gB and gH and also analysed the behaviour of HIV gp41 fusion peptide and the cationic antimicrobial peptide melittin. The combined results of SPR and ESR showed a marked difference between the mode of action of the HSV peptides and the HIV fusion peptide compared to melittin, suggesting that viral-derived membrane interacting peptides all act via a similar mechanism, which is substantially different from that of the non-cell selective lytic peptide melittin.     

The antimicrobial peptide histatin-5 causes a spatially restricted disruption on the Candida albicans surface, allowing rapid entry of the peptide into the cytoplasm

Antimicrobial peptides play an important role in host defense against microbial pathogens. Their high cationic charge and strong amphipathic structure allow them to bind to the anionic microbial cell membrane and disrupt the membrane bilayer by forming pores or channels. In contrast to the classical pore-forming peptides, studies on histatin-5 (Hst-5) have suggested that the peptide is transported into the cytoplasm of Candida albicans in a non-lytic manner, and cytoplasmic Hst-5 exerts its candicidal activities on various intracellular targets, consistent with its weak amphipathic structure. To understand how Hst-5 is internalized, we investigated the localization of FITC-conjugated Hst-5. We find that Hst-5 is internalized into the vacuole through receptor-mediated endocytosis at low extracellular Hst-5 concentrations, whereas under higher physiological concentrations, Hst-5 is translocated into the cytoplasm through a mechanism that requires a high cationic charge on Hst-5. At intermediate concentrations, two cell populations with distinct Hst-5 localizations were observed. By cell sorting, we show that cells with vacuolar localization of Hst-5 survived, while none of the cells with cytoplasmic Hst-5 formed colonies. Surprisingly, extracellular Hst-5, upon cell surface binding, induces a perturbation on the cell surface, as visualized by an immediate and rapid internalization of Hst-5 and propidium iodide or rhodamine B into the cytoplasm from the site using time-lapse microscopy, and a concurrent rapid expansion of the vacuole. Thus, the formation of a spatially restricted site in the plasma membrane causes the initial injury to C. albicans and offers a mechanism for its internalization into the cytoplasm. Our study suggests that, unlike classical channel-forming antimicrobial peptides, action of Hst-5 requires an energized membrane and causes localized disruptions on the plasma membrane of the yeast. This mechanism of cell membrane disruption may provide species-specific killing with minimal damage to microflora and the host and may be used by many other antimicrobial peptides.     

Interaction of cationic antimicrobial peptides with model membranes

A series of natural and synthetic cationic antimicrobial peptides from various structural classes, including alpha-helical, beta-sheet, extended, and cyclic, were examined for their ability to interact with model membranes, assessing penetration of phospholipid monolayers and induction of lipid flip-flop, membrane leakiness, and peptide translocation across the bilayer of large unilamellar liposomes, at a range of peptide/lipid ratios. All peptides were able to penetrate into monolayers made with negatively charged phospholipids, but only two interacted weakly with neutral lipids. Peptide-mediated lipid flip-flop generally occurred at peptide concentrations that were 3- to 5-fold lower than those causing leakage of calcein across the membrane, regardless of peptide structure. With the exception of two alpha-helical peptides V681(n) and V25(p,) the extent of peptide-induced calcein release from large unilamellar liposomes was generally low at peptide/lipid molar ratios below 1:50. Peptide translocation across bilayers was found to be higher for the beta-sheet peptide polyphemusin, intermediate for alpha-helical peptides, and low for extended peptides. Overall, whereas all studied cationic antimicrobial peptides interacted with membranes, they were quite heterogeneous in their impact on these membranes.     

Comparative activity and mechanism of action of three types of bovine antimicrobial peptides against pathogenic Prototheca spp.

The yeast‐like algae of the genus Prototheca are ubiquitous saprophytes causing infections in immunocompromised patients and granulomatous mastitis in cattle. Few available therapies and the rapid spread of resistant strains worldwide support the need for novel drugs against protothecosis. Host defence antimicrobial peptides inactivate a wide array of pathogens and are a rich source of leads, with the advantage of being largely unaffected by microbial resistance mechanisms. Three structurally diverse bovine peptides [BMAP‐28, Bac5 and lingual antimicrobial peptide (LAP)] have thus been tested for their capacity to inactivate Prototheca spp. In minimum inhibitory concentration (MIC) assays, they were all effective in the micromolar range against clinical mastitis isolates as well as a Prototheca wickerhamii reference strain. BMAP‐28 sterilized Prototheca cultures within 30–60 min at its MIC, induced cell permeabilization with near 100% release of cellular adenosine triphosphate and resulted in extensive surface blebbing and release of intracellular material as observed by scanning electron microscopy. Bac5 and LAP inactivated Prototheca following 3–6 h incubation at fourfold their MIC and did not result in detectable surface damage despite 70–90% killing, suggesting they act via non‐lytic mechanisms. In circular dichroism studies, the conformation of BMAP‐28, but not that of Bac5 or LAP, was affected by interaction with liposomes mimicking algal membranes. Our results indicate that BMAP‐28, Bac5 and LAP kill Prototheca with distinct potencies, killing kinetics, and modes of action and may be appropriate for protothecal mastitis treatment. In addition, the ability of Bac5 and LAP to act via non‐lytic mechanisms may be exploited for the development of target‐selective drugs. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Gap junction-mimetic peptides do work, but in unexpected ways

Gap junction mimetic peptides containing sequences of the extracellular loops of connexins inhibit the de-novo formation of gap junction channels but do not impair the function of existing cell-cell channels. Recently, a flurry of publications appeared showing that such “GAP” peptides attenuate ATP release and/or surrogate measures of it. Although no direct effect on putative connexin “hemichannels” has ever been shown, the peptide effect has been used as diagnostic tool for demonstrating the existence of such channels. However, testing of the peptides on genuine unapposed membrane channels formed by connexins failed to reveal any inhibitory action of the peptides on channel activity. Instead, membrane channels formed by the unrelated pannexin1 were inhibited in the same concentration range as described for the release of ATP. Consequently, rather than indicating connexin involvement in ATP release, the GAP peptide effects represent supporting evidence for a role of pannexin1 in this process.     

Salivary histatin 5 internalization by translocation,but not endocytosis,is required for fungicidal activity in Candida albicans

Salivary histatin 5 (Hst 5) is a cationic salivary protein with high fungicidal activity against Candida albicans. Binding to the cell wall followed by intracellular translocation is required for killing; however, specific binding components and critical toxic events are not understood. In this study, laminarin (β‐1,3‐glucan) but not sialic acid, mannan or pustulan mediated Hst 5 binding to C. albicans, and was disassociated by 100 mM NaCl. Time‐lapse confocal microscopy revealed a dose‐dependent rate of cytosolic uptake of Hst 5 that invariably preceded propidium iodide (PI) entry, demonstrating that translocation itself does not disrupt membrane integrity. Cell toxicity was manifest by vacuolar expansion followed by PI entrance; however, loss of endocytotic vacuolar trafficking of Hst 5 did not reduce killing. Extracellular NaCl (100 mM), but not sorbitol, prevented vacuolar expansion and PI entry in cells already containing cytosolic Hst 5, thus showing a critical role for ionic balance in Hst 5 toxicity. Hst 5 uptake, but not cell wall binding, was blocked by pretreatment with azide or carbonyl cyanide m‐chlorophenylhydrazone; however, 10% of de‐energized cells had membrane disruption. Thus, Hst 5 is capable of heterogeneous intracellular entry routes, but only direct cytosolic translocation causes cell death as a result of ionic efflux.     

Electrophysiological Studies in Xenopus Oocytes for the Opening of Escherichia coli SecA-Dependent Protein-Conducting Channels

Protein translocation in Escherichia coli requires protein-conducting channels in cytoplasmic membranes to allow precursor peptides to pass through with adenosine triphosphate (ATP) hydrolysis. Here, we report a novel, sensitive method that detects the opening of the SecA-dependent protein-conducting channels at the nanogram level. E. coli inverted membrane vesicles were injected into Xenopus oocytes, and ionic currents were recorded using the two-electrode voltage clamp. Currents were observed only in the presence of E. coli SecA in conjunction with E. coli membranes. Observed currents showed outward rectification in the presence of KCl as permeable ions and were significantly enhanced by coinjection with the precursor protein proOmpA or active LamB signal peptide. Channel activity was blockable with sodium azide or adenylyl 5′-(β,γ-methylene)-diphosphonate, a nonhydrolyzable ATP analogue, both of which are known to inhibit SecA protein activity. Endogenous oocyte precursor proteins also stimulated ion current activity and can be inhibited by puromycin. In the presence of puromycin, exogenous proOmpA or LamB signal peptides continued to enhance ionic currents. Thus, the requirement of signal peptides and ATP hydrolysis for the SecA-dependent currents resembles biochemical protein translocation assay with E. coli membrane vesicles, indicating that the Xenopus oocyte system provides a sensitive assay to study the role of Sec and precursor proteins in the formation of protein-conducting channels using electrophysiological methods.     

Single-molecule investigation of the interactions between reconstituted planar lipid membranes and an analogue of the HP(2-20) antimicrobial peptide

In this study, we employed electrophysiology experiments carried out at the single-molecule level to study the mechanism of action of the HPA3 peptide, an analogue of the linear antimicrobial peptide, HP(2–20), isolated from the N-terminal region of the Helicobacter pylori ribosomal protein. Amplitude analysis of currents fluctuations induced by HPA3 peptide at various potentials in zwitterionic lipid membranes reveal the existence of reproducible conductive states in the stochastic behavior of such events, which directly supports the existence of transmembrane pores induced the peptide. From our data recorded both at the single-pore and macroscopic levels, we propose that the HPA3 pore formation is electrophoretically facilitated by trans-negative transmembrane potentials, and HPA3 peptides translocate into the trans monolayers after forming the pores. We present evidence according to which the decrease in the membrane dipole potential of a reconstituted lipid membranes leads to an augmentation of the membrane activity of HPA3 peptides, and propose that a lower electric dipole field of the interfacial region of the membrane caused by phloretin facilitates the surface-bound HPA3 peptides to break free from one leaflet of the membrane, insert into the membrane and contribute to pore formation spanning the entire thickness of the membrane.     

Constitutive activation of the Saccharomyces cerevislae mating response pathway by a MAP kinase kinase from Candida albicans

The HST7 gene of Candida albicans encodes a protein with structural similarity to MAP kinase kinases. Expression of this gene in Saccharomyces cerevisiae complements disruption of the Ste7 MAP kinase kinase required for both mating in haploid cells and pseudohyphal growth in diploids. However, Hst7 expression does not complement loss of either the Pbs2 (Hog4) MAP kinase kinase required for response to high osmolarity, or loss of the Mkk1 and Mkk2 MAP kinase kinases required for proper cell wall biosynthesis. Intriguingly, HST7 acts as a hyperactive allele of STE7; expression of Hst7 activates the mating pathway even in the absence of upstream signaling components including the Ste7 regulator Ste11, elevates the basal level of the pheromone-inducible FUS1 gene, and amplifies the pseudohyphal growth response in diploid cells. Thus Hst7 appears to be at least partially independent of upstream activators or regulators, but selective in its activity on downstream target MAP kinases. Creation of Hst7/Ste7 hybrid proteins revealed that the C-terminal two-thirds of Hst7, which contains the protein kinase domain, is sufficient to confer this partial independence of upstream activators.Communicated by C. P. Hollenberg     

Characterization of ATP receptor which mediates norepinephrine release in PC12 cells.

PC12 cells, a rat pheochromocytoma cell line, has been reported to release norepinephrine in response to extracellular ATP in the presence of extracellular Ca2+. The potency order of ATP analogues was adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than adenosine 5'-O-(1-thiotriphosphate) = 2-methylthioadenosine 5'-triphosphate (MeSATP) greater than 2'- and 3'-O-(4-benzoyl-benzoyl)ATP (BzATP) greater than ADP greater than 5-adenylylimidodiphosphate. Adenosine 5'-O-(2-thiodiphosphate), beta, gamma-methyleneadenosine 5'-triphosphate, AMP and adenosine were inactive. The ATP action in the absence of extracellular Ca2+, suggests a small but appreciable contribution of intracellular Ca2+ mobilization, for norepinephrine release. However, for some ATP derivatives, like BzATP, almost no contribution of the phospholipase C-Ca2+ pathway is suggested, based on their low activity in inositol phosphates production. To identify the ATP-receptor protein, PC12 cell membranes were photoaffinity-labeled with [32P]BzATP. SDS-PAGE analysis showed that a 53-kDa protein labeling was inhibited by ATP and its derivatives, as well as by P2-antagonists, suramin and reactive blue 2, which inhibit the nucleotide-induced norepinephrine release. The inhibitory activity of the nucleotides was, in parallel with their potency, to induce norepinephrine release. Despite their inability to release norepinephrine, GTP and GTP gamma S inhibited the BzATP labeling, suggesting the participation of a putative G protein in the ATP-receptor-mediated actions. We suggest that the 53-kDa protein on the PC12 cell surface is an ATP receptor, which mediates the norepinephrine release, depending, mainly, on extracellular Ca2+ gating.     

Role of membranes in the activities of antimicrobial cationic peptides

Cationic amphiphilic peptides that are found throughout nature have very broad-spectrum activities against microbes. The initial sites of interaction are with microbial membranes. Although dogma suggests that their lethal action involves disruption of the cytoplasmic membranes, a number of cationic peptides can traverse intact membranes to interact with internal targets.     

Effects of Opioid Peptides Containing the Sequence of Met5-Enkephalin or Leu5-Enkephalin on Nicotine-Induced Secretion from Bovine Adrenal Chromaffin Cells

Eighteen endogenous opioid peptides, all containing the sequence of either Met5- or Leu5-enkephalin, were tested for their ability to modify nicotine-induced secretion from bovine adrenal chromaffin cells. ATP released from suspensions of freshly isolated cells was measured with the luciferin-luciferase bioluminescence method as an index of secretion. None of the peptides affected 5 microM nicotine-induced ATP release at 10 nM. Three peptides inhibited secretion at 5 microM: dynorphin1-13, dynorphin1-9, and rimorphin inhibited by 65%, 37%, and 29% respectively. Use of peptidase inhibitors (bestatin, thiorphan, bacitracin, or 1,10-phenanthroline) did not result in any of the other peptides showing potent actions on the nicotinic response, although bestatin and thiorphan did enhance the inhibitory actions of dynorphin1-13 and dynorphin1-9 by 20-30%. Nicotine-induced secretion of endogenous catecholamines from bovine chromaffin cells cultured for 3 days was also studied to assess any selective actions of the peptides on adrenaline or noradrenaline cell types. Dynorphin1-13 was 1,000-fold more potent than Leu5-enkephalin at inhibiting endogenous catecholamine secretion. Dynorphin1-13 was slightly more potent at inhibiting noradrenaline release than adrenaline release whereas Leu5-enkephalin showed the opposite selectivity. The structure-activity relationships of opioid peptide actions on the chromaffin cell nicotinic response are discussed in relation to the properties of the adrenal opioid binding sites.     

Ionic, neuronal and endocrine influences on the proopiomelanocortin system of the hypothalamus.

Increasing evidence supports a neurotransmitter or a neuromodulator action for peptides derived from proopiomelanocortin in the hypothalamus. Peptide release involves sodium, potassium and calcium ion channels and is dependent on the presence of extracellular calcium ions at the time of depolarisation of neuronal membranes. Dopaminergic and gamma-aminobutyric acid-containing neuronal systems inhibit POMC-derived peptide release from the hypothalamus through D2-dopamine and GABAA receptors, respectively. Serotoninergic mechanisms exert a biphasic effect on peptide release being directly stimulatory at low concentrations of serotonin and indirectly inhibitory at higher concentrations via interactions with the endogenous dopaminergic system. Cholinergic and glutamergic drugs stimulate peptide release through nicotinic and N-methyl-D-aspartate receptors, respectively. Finally, circulating steroids regulate the hypothalamic POMC system with testosterone stimulating POMC gene expression whilst oestradiol and glucocorticoids induce an inhibitory control.     

Effects of histatin 5 modifications on antifungal activity and kinetics of proteolysis

Histatin 5 (Hst‐5) is an antimicrobial peptide with strong antifungal activity against Candida albicans, an opportunistic pathogen that is a common cause of oral thrush. The peptide is natively secreted by human salivary glands and shows promise as an alternative therapeutic against infections caused by C. albicans. However, Hst‐5 can be cleaved and inactivated by a family of secreted aspartic proteases (Saps) produced by C. albicans. Single‐residue substitutions can significantly affect the proteolytic resistance of Hst‐5 to Saps and its antifungal activity; the K17R substitution increases resistance to proteolysis, while the K11R substitution enhances antifungal activity. In this work, we showed that the positive effects of these two single‐residue modifications can be combined in a single peptide, K11R–K17R, with improved proteolytic resistance and antifungal activity. We also investigated the effect of additional single‐residue substitutions, with a focus on the effect of addition or removal of negatively charged residues, and found Sap‐dependent effects on degradation. Both single‐ and double‐substitutions affected the kinetics of proteolytic degradation of the intact peptide and of the fragments formed during degradation. Our results demonstrate the importance of considering proteolytic stability and not just antimicrobial activity when designing peptides for potential therapeutic applications.     

Membrane association, electrostatic sequestration, and cytotoxicity of Gly-Leu-rich peptide orthologs with differing functions

The skins of closely related frog species produce Gly-Leu-rich peptide orthologs that have very similar sequences, hydrophobicities, and amphipathicities but differ markedly in their net charge and membrane-damaging properties. Cationic Gly-Leu-rich peptides are hemolytic and very potent against microorganisms. Peptides with no net charge have only hemolytic activity. We have used ancestral protein reconstruction and peptide analogue design to examine the roles of electrostatic and hydrophobic interactions in the biological activity and mode of action of functionally divergent Gly-Leu-rich peptides. The structure and interaction of the peptides with anionic and zwitterionic model membranes were investigated by circular dichroism with 2-dimyristoyl-sn-glycero-3-phosphatidylcholine or 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol vesicles and surface plasmon resonance with immobilized bilayers. The results, combined with antimicrobial assays, the kinetics of bacterial killing, and membrane permeabilization assays, reveal that Gly, Val, Thr, and Ile can all be accommodated in an amphipathic alpha helix when the helix is in a membrane environment. Binding to anionic and zwitterionic membranes fitted to a 2-stage interaction model (adsorption to the membrane followed by membrane insertion). The first step is governed by hydrophobic interactions between the nonpolar surface of the peptide helix and the membranes. The strong binding of Gly-Leu-rich cationic peptides to anionic membranes is due to the second binding step and involves short-range Coulombic interactions that prolong the residence time of the membrane-inserted peptide. The data demonstrate that evolution has positively selected charge-altering nucleotide substitutions to generate an orthologous cationic variant of neutral hemolytic peptides that bind to and permeate bacterial cell membranes.