Hyperpolarisation of Mitochondrial Membranes Is a Critical Component of the Antifungal Mechanism of the Plant Defensin, Ppdef1.

Plant defensins are a large family of small cationic proteins with diverse functions and mechanisms of action, most of which assert antifungal activity against a broad spectrum of fungi. The partial mechanism of action has been resolved for a small number of members of plant defensins, and studies have revealed that many act by more than one mechanism. The plant defensin Ppdef1 has a unique sequence and long loop 5 with fungicidal activity against a range of human fungal pathogens, but little is known about its mechanism of action. We screened the S. cerevisiae non-essential gene deletion library and identified the involvement of the mitochondria in the mechanism of action of Ppdef1. Further analysis revealed that the hyperpolarisation of the mitochondrial membrane potential (MMP) activates ROS production, vacuolar fusion and cell death and is an important step in the mechanism of action of Ppdef1, and it is likely that a similar mechanism acts in Trichophyton rubrum.

The Plant Defensin Ppdef1 Is a Novel Topical Treatment for Onychomycosis.

Onychomycosis, or fungal nail infection, causes not only pain and discomfort but can also have psychological and social consequences for the patient. Treatment of onychomycosis is complicated by the location of the infection under the nail plate, meaning that antifungal molecules must either penetrate the nail or be applied systemically. Currently, available treatments are limited by their poor nail penetration for topical products or their potential toxicity for systemic products. Plant defensins with potent antifungal activity have the potential to be safe and effective treatments for fungal infections in humans. The cystine-stabilized structure of plant defensins makes them stable to the extremes of pH and temperature as well as digestion by proteases. Here, we describe a novel plant defensin, Ppdef1, as a peptide for the treatment of fungal nail infections. Ppdef1 has potent, fungicidal activity against a range of human fungal pathogens, including Candida spp., Cryptococcus spp., dermatophytes, and non-dermatophytic moulds. In particular, Ppdef1 has excellent activity against dermatophytes that infect skin and nails, including the major etiological agent of onychomycosis Trichophyton rubrum. Ppdef1 also penetrates human nails rapidly and efficiently, making it an excellent candidate for a novel topical treatment of onychomycosis.

Metabolomic Analysis of Extracellular Vesicles from the Cereal Fungal Pathogen Fusarium graminearum.

Fusarium graminearum (F. graminearum) is a filamentous fungus that infects cereals such as corn, wheat, and barley, with serious impact on yield as well as quality when the grain is contaminated with mycotoxins. Despite the huge impact of F. graminearum on food security and mammalian health, the mechanisms used by F. graminearum to export virulence factors during infection are not fully understood and may involve non-classical secretory pathways. Extracellular vesicles (EVs) are lipid-bound compartments produced by cells of all kingdoms that transport several classes of macromolecules and are implicated in cell-cell communication. EVs produced by human fungal pathogens carry cargo that facilitate infection, leading us to ask whether plant fungal pathogens also deliver molecules that increase virulence via EVs. We examined the metabolome of the EVs produced by F. graminearum to determine whether they carry small molecules that could modulate plant-pathogen interactions. We discovered that EVs from F. graminearum were produced in liquid medium-containing inducers of trichothecene production, but in lower quantities compared to other media. Nanoparticle tracking analysis and cryo-electron microscopy revealed that the EVs were morphologically similar to EVs from other organisms; hence, the EVs were metabolically profiled using LC-ESI-MS/MS. This analysis revealed that EVs carry 2,4-dihydroxybenzophenone (BP-1) and metabolites that have been suggested by others to have a role in host-pathogen interactions. BP-1 reduced the growth of F. graminearum in an in vitro assay, suggesting that F. graminearum might use EVs to limit metabolite self-toxicity.

Membrane binding properties of plant defensins.

The membrane interaction characteristics of five antifungal plant defensin peptides: NaD1, and the related HXP4 and L5, as well as NaD2 and the related ZmD32 were studied. These peptides were chosen to cover a broad range of cationic charges with little structural variations, allowing for assessment of the role of charge in their membrane interactions. Membrane permeabilizing activity against C. albicans was confirmed and quantified for benchmarking purposes. Viscoelastic characteristics of the membrane interactions were studied in typical neutral and charged model membranes using quartz crystal microbalance with dissipation (QCM-D. Frequency-dissipation fingerprinting analysis of the QCM-D results revealed that all of the peptides were able to bind to all studied model membranes albeit with slightly different viscoelastic character for each membrane type. However, characteristic disruption patterns were not observed suggesting that the membrane disrupting activity of these defensins is mostly specific to fungal membranes, and that increasing the peptide charge does not enhance their action. The results also show that the presence of specific sterols has a profound effect on the ability of the peptides to disrupt the membrane.

Extracellular Vesicles from Fusarium graminearum Contain Protein Effectors Expressed during Infection of Corn.

Fusarium graminearum (Fgr) is a devastating filamentous fungal pathogen that causes diseases in cereals, while producing mycotoxins that are toxic for humans and animals, and render grains unusable. Low efficiency in managing Fgr poses a constant need for identifying novel control mechanisms. Evidence that fungal extracellular vesicles (EVs) from pathogenic yeast have a role in human disease led us to question whether this is also true for fungal plant pathogens. We separated EVs from Fgr and performed a proteomic analysis to determine if EVs carry proteins with potential roles in pathogenesis. We revealed that protein effectors, which are crucial for fungal virulence, were detected in EV preparations and some of them did not contain predicted secretion signals. Furthermore, a transcriptomic analysis of corn (Zea mays) plants infected by Fgr revealed that the genes of some of the effectors were highly expressed in vivo, suggesting that the Fgr EVs are a mechanism for the unconventional secretion of effectors and virulence factors. Our results expand the knowledge on fungal EVs in plant pathogenesis and cross-kingdom communication, and may contribute to the discovery of new antifungals.

Fungal Extracellular Vesicles in Pathophysiology.

Fungal pathogens are a concern in medicine and agriculture that has been exacerbated by the emergence of antifungal-resistant varieties that severely threaten human and animal health, as well as food security. This had led to the search for new and sustainable treatments for fungal diseases. Innovative solutions require a deeper understanding of the interactions between fungal pathogens and their hosts, and the key determinants of fungal virulence. Recently, a link has emerged between the release of extracellular vesicles (EVs) and fungal virulence that may contribute to finding new methods for fungal control. Fungal EVs carry pigments, carbohydrates, protein, nucleic acids and other macromolecules with similar functions as those found in EVs from other organisms, however certain fungal features, such as the fungal cell wall, impact EV release and cargo. Fungal EVs modulate immune responses in the host, have a role in cell-cell communication and transport molecules that function in virulence. Understanding the function of fungal EVs will expand our knowledge of host-pathogen interactions and may provide new and specific targets for antifungal drugs and agrichemicals.

Size-exclusion chromatography allows the isolation of EVs from the filamentous fungal plant pathogen Fusarium oxysporum f. sp. vasinfectum (Fov).

Extracellular vesicles (EVs) are nano-sized compartments involved in cell communication and macromolecule transport that are well characterized in mammalian organisms. Fungal EVs transport virulence-related cargo and modulate the host immune response, but most work has been focused on human yeast pathogens. Additionally, the study of EVs from filamentous fungi has been hindered by the lack of protein markers and efficient isolation methods. In this study we performed the isolation and proteomic characterization of EVs from the filamentous cotton pathogen Fusarium oxysporum f. sp. vasinfectum (Fov). EVs were recovered from two different growth media, Czapek Dox and Saboraud's dextrose broth, and purified by size-exclusion chromatography. Our results show that the EV proteome changes depending on the growth medium but EV production remains constant. EVs contained proteins involved in polyketide synthesis, cell wall modifications, proteases and potential effectors. These results support a role in modulation of host-pathogen interactions for Fov EVs.

Improving the Digestibility of Plant Defensins to Meet Regulatory Requirements for Transgene Products in Crop Protection.

Despite the use of chemical fungicides, fungal diseases have a major impact on the yield and quality of plant produce globally and hence there is a need for new approaches for disease control. Several groups have examined the potential use of antifungal plant defensins for plant protection and have produced transgenic plants expressing plant defensins with enhanced resistance to fungal disease. However, before they can be developed commercially, transgenic plants must pass a series of strict regulations to ensure that they are safe for human and animal consumption as well as the environment. One of the requirements is rapid digestion of the transgene protein in the gastrointestinal tract to minimize the risk of any potential allergic response. Here, we examine the digestibility of two plant defensins, NaD1 from Nicotiana alata and SBI6 from soybean, which have potent antifungal activity against major cereal pathogens. The native defensins were not digestible in simulated gastrointestinal fluid assays. Several modifications to the sequences enhanced the digestibility of the two small proteins without severely impacting their antifungal activity. However, these modified proteins did not accumulate as well as the native proteins when transiently expressed in planta, suggesting that the protease-resistant structure of plant defensins facilitates their stability in planta.

Histidine-Rich Defensins from the Solanaceae and Brasicaceae Are Antifungal and Metal Binding Proteins.

Plant defensins are best known for their antifungal activity and contribution to the plant immune system. The defining feature of plant defensins is their three-dimensional structure known as the cysteine stabilized alpha-beta motif. This protein fold is remarkably tolerant to sequence variation with only the eight cysteines that contribute to the stabilizing disulfide bonds absolutely conserved across the family. Mature defensins are typically 46-50 amino acids in length and are enriched in lysine and/or arginine residues. Examination of a database of approximately 1200 defensin sequences revealed a subset of defensin sequences that were extended in length and were enriched in histidine residues leading to their classification as histidine-rich defensins (HRDs). Using these initial HRD sequences as a query, a search of the available sequence databases identified over 750 HRDs in solanaceous plants and 20 in brassicas. Histidine residues are known to contribute to metal binding functions in proteins leading to the hypothesis that HRDs would have metal binding properties. A selection of the HRD sequences were recombinantly expressed and purified and their antifungal and metal binding activity was characterized. Of the four HRDs that were successfully expressed all displayed some level of metal binding and two of four had antifungal activity. Structural characterization of the other HRDs identified a novel pattern of disulfide linkages in one of the HRDs that is predicted to also occur in HRDs with similar cysteine spacing. Metal binding by HRDs represents a specialization of the plant defensin fold outside of antifungal activity.

Protein markers for Candida albicans EVs include claudin-like Sur7 family proteins.

Background: Fungal extracellular vesicles (EVs) have been implicated in host-pathogen and pathogen-pathogen communication in some fungal diseases. In depth research into fungal EVs has been hindered by the lack of specific protein markers such as those found in mammalian EVs that have enabled sophisticated isolation and analysis techniques. Despite their role in fungal EV biogenesis, ESCRT proteins such as Vps23 (Tsg101) and Bro1 (ALIX) are not present as fungal EV cargo. Furthermore, tetraspanin homologs are yet to be identified in many fungi including the model yeast S. cerevisiae. Objective: We performed de novo identification of EV protein markers for the major human fungal pathogen Candida albicans with adherence to MISEV2018 guidelines. Materials and methods: EVs were isolated by differential ultracentrifugation from DAY286, ATCC90028 and ATCC10231 yeast cells, as well as DAY286 biofilms. Whole cell lysates (WCL) were also obtained from the EV-releasing cells. Label-free quantitative proteomics was performed to determine the set of proteins consistently enriched in EVs compared to WCL. Results: 47 proteins were consistently enriched in C. albicans EVs. We refined these to 22 putative C. albicans EV protein markers including the claudin-like Sur7 family (Pfam: PF06687) proteins Sur7 and Evp1 (orf19.6741). A complementary set of 62 EV depleted proteins was selected as potential negative markers. Conclusions: The marker proteins for C. albicans EVs identified in this study will be useful tools for studies on EV biogenesis and cargo loading in C. albicans and potentially other fungal species and will also assist in elucidating the role of EVs in C. albicans pathogenesis. Many of the proteins identified as putative markers are fungal specific proteins indicating that the pathways of EV biogenesis and cargo loading may be specific to fungi, and that assumptions made based on studies in mammalian cells could be misleading. Abbreviations: A1 - ATCC10231; A9 - ATCC90028; DAY B - DAY286 biofilm; DAY Y - DAY286 yeast; EV - extracellular vesicle; Evp1 - extracellular vesicle protein 1 (orf19.6741); GO - gene ontology; Log2(FC) - log2(fold change); MCC - membrane compartment of Can1; MDS - multidimensional scaling; MISEV - minimal information for studies of EVs; sEVs - small EVs; SP - signal peptide; TEMs - tetraspanin enriched microdomains; TM - transmembrane; VDM - vesicle-depleted medium; WCL - whole cell lysate.

Ptychographic imaging of NaD1 induced yeast cell death.

Characterising and understanding the mechanisms involved in cell death are especially important to combating threats to human health, particularly for the study of antimicrobial peptides and their effectiveness against pathogenic fungi. However, imaging these processes often relies on the use of synthetic molecules which bind to specific cellular targets to produce contrast. Here we study yeast cell death, induced by the anti-fungal peptide, NaD1. By treating yeast as a model organism we aim to understand anti-fungal cell death processes without relying on sample modification. Using a quantitative phase imaging technique, ptychography, we were able to produce label free images of yeast cells during death and use them to investigate the mode of action of NaD1. Using this technique we were able to identify a significant phase shift which provided a clear signature of yeast cell death. Additionally, ptychography identifies cell death much earlier than a comparative fluorescence study, providing new insights into the cellular changes that occur during cell death. The results indicate ptychography has great potential as a means of providing additional information about cellular processes which otherwise may be masked by indirect labelling approaches.

Screening the Saccharomyces cerevisiae Nonessential Gene Deletion Library Reveals Diverse Mechanisms of Action for Antifungal Plant Defensins.

Plant defensins are a large family of proteins, most of which have antifungal activity against a broad spectrum of fungi. However, little is known about how they exert their activity. The mechanisms of action of only a few members of the family have been investigated and, in most cases, there are still a number of unknowns. To gain a better understanding of the antifungal mechanisms of a set of four defensins, NaD1, DmAMP1, NbD6, and SBI6, we screened a pooled collection of the nonessential gene deletion set of Saccharomyces cerevisiae Strains with increased or decreased ability to survive defensin treatment were identified based on the relative abundance of the strain-specific barcode as determined by MiSeq next-generation sequencing. Analysis of the functions of genes that are deleted in strains with differential growth in the presence of defensin provides insight into the mechanism of action. The screen identified a novel role for the vacuole in the mechanisms of action for defensins NbD6 and SBI6. The effect of these defensins on vacuoles was further confirmed by using confocal microscopy in both S. cerevisiae and the cereal pathogen Fusarium graminearum These results demonstrate the utility of this screening method to identify novel mechanisms of action for plant defensins.

Antibacterial and antifungal activity of defensins from the Australian paralysis tick, Ixodes holocyclus.

Tick innate immunity involves humoral and cellular responses. Among the humoral effector molecules in ticks are the defensins which are a family of small peptides with a conserved γ-core motif that is crucial for their antimicrobial activity. Defensin families have been identified in several hard and soft tick species. However, little is known about the presence and antimicrobial activity of defensins from the Australian paralysis tick Ixodes holocyclus. In this study the I. holocyclus transcriptome was searched for the presence of defensins. Unique and non-redundant defensin sequences were identified and designated as holosins 1 - 5. The antimicrobial activity of holosins 2 and 3 and of the predicted γ-cores of holosins 1-4 (HoloTickCores 1-4), was assessed using Gram-negative and Gram-positive bacteria as well as the fungus Fusarium graminearum and the yeast Candida albicans. All holosins had molecular features that are conserved in other tick defensins. Furthermore holosins 2 and 3 were very active against the Gram-positive bacteria Staphylococcus aureus and Listeria grayi. Holosins 2 and 3 were also active against F. graminearum and C. albicans and 5 µM of peptide abrogate the growth of these microorganisms. The activity of the synthetic γ-cores was lower than that of the mature defensins apart from HoloTickCore 2 which had activity comparable to mature holosin 2 against the Gram-negative bacterium Escherichia coli. This study reveals the presence of a multigene defensin family in I. holocyclus with wide antimicrobial activity.

Salt-Tolerant Antifungal and Antibacterial Activities of the Corn Defensin ZmD32.

Pathogenic microbes are developing resistance to established antibiotics, making the development of novel antimicrobial molecules paramount. One major resource for discovery of antimicrobials is the arsenal of innate immunity molecules that are part of the first line of pathogen defense in many organisms. Gene encoded cationic antimicrobial peptides are a major constituent of innate immune arsenals. Many of these peptides exhibit potent antimicrobial activity in vitro. However, a major hurdle that has impeded their development for use in the clinic is the loss of activity at physiological salt concentrations, attributed to weakening of the electrostatic interactions between the cationic peptide and anionic surfaces of the microbial cells in the presence of salt. Using plant defensins we have investigated the relationship between the charge of an antimicrobial peptide and its activity in media with elevated salt concentrations. Plant defensins are a large class of antifungal peptides that have remarkable stability at extremes of pH and temperature as well as resistance to protease digestion. A search of a database of over 1200 plant defensins identified ZmD32, a defensin from Zea mays, with a predicted charge of +10.1 at pH 7, the highest of any defensin in the database. Recombinant ZmD32 retained activity against a range of fungal species in media containing elevated concentrations of salt. In addition, ZmD32 was active against Candida albicans biofilms as well as both Gram negative and Gram-positive bacteria. This broad spectrum antimicrobial activity, combined with a low toxicity on human cells make ZmD32 an attractive lead for development of future antimicrobial molecules.

Fungal Extracellular Vesicles with a Focus on Proteomic Analysis.

Extracellular vesicles (EVs) perform crucial functions in cell-cell communication. The packaging of biomolecules into membrane-enveloped vesicles prior to release into the extracellular environment provides a mechanism for coordinated delivery of multiple signals at high concentrations that is not achievable by classical secretion alone. Most of the understanding of the biosynthesis, composition, and function of EVs comes from mammalian systems. Investigation of fungal EVs, particularly those released by pathogenic yeast species, has revealed diverse cargo including proteins, lipids, nucleic acids, carbohydrates, and small molecules. Fungal EVs are proposed to function in a variety of biological processes including virulence and cell wall homeostasis with a focus on host-pathogen interactions. EVs also carry signals between fungal cells allowing for a coordinated attack on a host during infection. Research on fungal EVs in still in its infancy. Here a review of the literature thus far with a focus on proteomic analysis is provided with respect to techniques, results, and prospects.

The evolution, function and mechanisms of action for plant defensins.

Plant defensins are an extensive family of small cysteine rich proteins characterised by a conserved cysteine stabilised alpha beta protein fold which resembles the structure of insect and vertebrate defensins. However, secondary structure and disulphide topology indicates two independent superfamilies of defensins with similar structures that have arisen via an extreme case of convergent evolution. Defensins from plants and insects belong to the cis-defensin superfamily whereas mammalian defensins belong to the trans-defensin superfamily. Plant defensins are produced by all species of plants and although the structure is highly conserved, the amino acid sequences are highly variable with the exception of the cysteine residues that form the stabilising disulphide bonds and a few other conserved residues. The majority of plant defensins are components of the plant innate immune system but others have evolved additional functions ranging from roles in sexual reproduction and development to metal tolerance. This review focuses on the antifungal mechanisms of plant defensins. The activity of plant defensins is not limited to plant pathogens and many of the described mechanisms have been elucidated using yeast models. These mechanisms are more complex than simple membrane permeabilisation induced by many small antimicrobial peptides. Common themes that run through the characterised mechanisms are interactions with specific lipids, production of reactive oxygen species and induction of cell wall stress. Links between sequence motifs and functions are highlighted where appropriate. The complexity of the interactions between plant defensins and fungi helps explain why this protein superfamily is ubiquitous in plant innate immunity.

The interaction with fungal cell wall polysaccharides determines the salt tolerance of antifungal plant defensins.

The fungal cell wall is the first point of contact between fungal pathogens and host organisms. It serves as a protective barrier against biotic and abiotic stresses and as a signal to the host that a fungal pathogen is present. The fungal cell wall is made predominantly of carbohydrates and glycoproteins, many of which serve as binding receptors for host defence molecules or activate host immune responses through interactions with membrane-bound receptors. Plant defensins are a large family of cationic antifungal peptides that protect plants against fungal disease. Binding of the plant defensin NaD1 to the fungal cell wall has been described but the specific component of the cell wall with which this interaction occurred was unknown. The effect of binding was also unclear, that is whether the plant defensin used fungal cell wall components as a recognition motif for the plant to identify potential pathogens or if the cell wall acted to protect the fungus against the defensin. Here we describe the interaction between the fungal cell wall polysaccharides chitin and ß-glucan with NaD1 and other plant defensins. We discovered that the ß-glucan layer protects the fungus against plant defensins and the loss of activity experienced by many cationic antifungal peptides at elevated salt concentrations is due to sequestration by fungal cell wall polysaccharides. This has limited the development of cationic antifungal peptides for the treatment of systemic fungal diseases in humans as the level of salt in serum is enough to inactivate most cationic peptides.

Extracellular Vesicles From the Cotton Pathogen Fusarium oxysporum f. sp. vasinfectum Induce a Phytotoxic Response in Plants.

Extracellular vesicles (EVs) represent a system for the coordinated secretion of a variety of molecular cargo including proteins, lipids, nucleic acids, and metabolites. They have an essential role in intercellular communication in multicellular organisms and have more recently been implicated in host-pathogen interactions. Study of the role for EVs in fungal biology has focused on pathogenic yeasts that are major pathogens in humans. In this study we have expanded the investigation of fungal EVs to plant pathogens, specifically the major cotton pathogen Fusarium oxysporum f. sp. vasinfectum. EVs isolated from F. oxysporum f. sp. vasinfectum culture medium have a morphology and size distribution similar to EVs from yeasts such as Candida albicans and Cryptococcus neoformans. A unique feature of the EVs from F. oxysporum f. sp. vasinfectum is their purple color, which is predicted to arise from a napthoquinone pigment being packaged into the EVs. Proteomic analysis of F. oxysporum f. sp. vasinfectum EVs revealed that they are enriched in proteins that function in synthesis of polyketides as well as proteases and proteins that function in basic cellular processes. Infiltration of F. oxysporum f. sp. vasinfectum EVs into the leaves of cotton or N. benthamiana plants led to a phytotoxic response. These observations lead to the hypothesis that F. oxysporum f. sp. vasinfectum EVs are likely to play a crucial role in the infection process.

Resistance to the Plant Defensin NaD1 Features Modifications to the Cell Wall and Osmo-Regulation Pathways of Yeast.

Over the last few decades, the emergence of resistance to commonly used antifungal molecules has become a major barrier to effective treatment of recurrent life-threatening fungal diseases. Resistance combined with the increased incidence of fungal diseases has created the need for new antifungals, such as the plant defensin NaD1, with different mechanisms of action to broaden treatment options. Antimicrobial peptides produced in plants and animals are promising new molecules in the arsenal of antifungal agents because they have different mechanisms of action to current antifungals and are often targeted specifically to fungal pathogens (van der Weerden et al., 2013). A key step in the development of novel antifungals is an understanding of the potential for the fungus to develop resistance. Here, we have used the prototypic plant defensin NaD1 in serial passages with the model fungus Saccharomyces cerevisiae to examine the evolution of resistance to plant antifungal peptides. The yeast strains did develop tolerance to NaD1, but it occurred more slowly than to the clinically used antifungal caspofungin. Sequencing the genomes of the strains with increased tolerance failed to identify any 'hotspot' mutations associated with increased tolerance to NaD1 and led to the identification of 12 genes that are involved in resistance. Characterization of the strains with increased tolerance to NaD1 also revealed changes in tolerance to abiotic stressors. Resistance developed slowly via an accumulation of single nucleotide mutations and had a fitness penalty associated with it. One of the genes identified FPS1, revealed that there is a common mechanism of resistance to NaD1 that involves the osmotic stress response pathway. These data indicate that it is more difficult to generate resistance to antimicrobial peptides such as NaD1 compared to small molecule antifungals.

X-ray structure of a carpet-like antimicrobial defensin-phospholipid membrane disruption complex.

Defensins are cationic antimicrobial peptides expressed throughout the plant and animal kingdoms as a first line of defense against pathogens. Membrane targeting and disruption is a crucial function of many defensins, however the precise mechanism remains unclear. Certain plant defensins form dimers that specifically bind the membrane phospholipids phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate, thereby triggering the assembly of defensin-lipid oligomers that permeabilize cell membranes. To understand this permeabilization mechanism, here we determine the crystal structure of the plant defensin NaD1 bound to PA. The structure reveals a 20-mer that adopts a concave sheet- or carpet-like topology where NaD1 dimers form one face and PA acyl chains form the other face of the sheet. Furthermore, we show that Arg39 is critical for PA binding, oligomerization and fungal cell killing. These findings identify a putative defensin-phospholipid membrane attack configuration that supports a longstanding proposed carpet mode of membrane disruption.