In recent decades, pathogenic fungi have become a serious threat to human health, leading to major efforts aimed at characterizing
new agents for improved treatments. Promising in this context are antimicrobial peptides produced by animals and plants as
part of innate immune systems. Here, we describe an antifungal defensin, NaD1, with activity against the major human pathogen
Candida albicans, characterize the mechanism of killing, and identify protection strategies used by the fungus to survive defensin treatment.
The mechanism involves interaction between NaD1 and the fungal cell surface followed by membrane permeabilization, entry into
the cytoplasm, hyperproduction of reactive oxygen species, and killing induced by oxidative damage. By screening C. albicans mutant libraries, we identified that the high-osmolarity glycerol (HOG) pathway has a unique role in protection against NaD1,
while several other stress-responsive pathways are dispensable. The involvement of the HOG pathway is consistent with induction
of oxidative stress by NaD1. The HOG pathway has been reported to have a major role in protection of fungi against osmotic
stress, but our data indicate that osmotic stress does not contribute significantly to the adverse effects of NaD1 on C. albicans. Our data, together with previous studies with human beta-defensins and salivary histatin 5, indicate that inhibition of
the HOG pathway holds promise as a broad strategy for increasing the activity of antimicrobial peptides against C. albicans.
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... NaD1 induces ROS and NO as the final step in a three-step mechanism of action against fungal cells. Initiation of the process involves interactions with cell wall components such as glycosylated proteins or 1,3-β-glucan, which drives energy-dependant import (step 2), allowing lipid binding and ROS/NO production (step 3) ( Figure 3B) [15,23,48,49]. The induction of ROS/NO by NaD1 occurs via interaction with yeast mitochondria, as Saccharomyces cerevisiae with an inactive mitochondria respiratory chain are more resistant to NaD1 than wild-type fungi . ...
... Initiation of the process involves interactions with cell wall components such as glycosylated proteins or 1,3-β-glucan, which drives energy-dependant import (step 2), allowing lipid binding and ROS/NO production (step 3) ( Figure 3B) [15,23,48,49]. The induction of ROS/NO by NaD1 occurs via interaction with yeast mitochondria, as Saccharomyces cerevisiae with an inactive mitochondria respiratory chain are more resistant to NaD1 than wild-type fungi . NaD1 binds cardiolipin (an abundant mitochondrial inner membrane lipid) which, along with the mitochondrial respiratory chain components in yeast, may provide explanation of the mechanism of ROS/NO generation by NaD1 [15,48,50]. ...
... The induction of ROS/NO by NaD1 occurs via interaction with yeast mitochondria, as Saccharomyces cerevisiae with an inactive mitochondria respiratory chain are more resistant to NaD1 than wild-type fungi . NaD1 binds cardiolipin (an abundant mitochondrial inner membrane lipid) which, along with the mitochondrial respiratory chain components in yeast, may provide explanation of the mechanism of ROS/NO generation by NaD1 [15,48,50]. ...
Defensins are a class of host defence peptides (HDPs) that often harbour antimicrobial and anticancer activities, making them attractive candidates as novel therapeutics. In comparison with current antimicrobial and cancer treatments, defensins uniquely target specific membrane lipids via mechanisms distinct from other HDPs. Therefore, defensins could be potentially developed as therapeutics with increased selectivity and reduced susceptibility to the resistance mechanisms of tumour cells and infectious pathogens. In this review, we highlight recent advances in defensin research with a particular focus on membrane lipid-targeting in cancer and infection settings. In doing so, we discuss strategies to harness lipid-binding defensins for anticancer and anti-infective therapies.
... There is compelling evidence that defensins can induce ROS accumulation within the targeted fungal cells. This has been notably demonstrated for RsAFP2 in C. albicans [160,161], for NaD1 in C. albicans [162,163] or in F. oxysporum  and for HsAFP1 in C. albicans . It should be noted that internalization is not required for inducing ROS production as RsAFP2, which is not internalized, induces the production of ROS . ...
Crops are threatened by numerous fungal diseases that can adversely affect the availability and quality of agricultural commodities. In addition, some of these fungal phytopathogens have the capacity to produce mycotoxins that pose a serious health threat to humans and livestock. To facilitate the transition towards sustainable environmentally friendly agriculture, there is an urgent need to develop innovative methods allowing a reduced use of synthetic fungicides while guaranteeing optimal yields and the safety of the harvests. Several defensins have been reported to display antifungal and even—despite being under-studied—antimycotoxin activities and could be promising natural molecules for the development of control strategies. This review analyses pioneering and recent work addressing the bioactivity of defensins towards fungal phytopathogens; the details of approximately 100 active defensins and defensin-like peptides occurring in plants, mammals, fungi and invertebrates are listed. Moreover, the multi-faceted mechanism of action employed by defensins, the opportunity to optimize large-scale production procedures such as their solubility, stability and toxicity to plants and mammals are discussed. Overall, the knowledge gathered within the present review strongly supports the bright future held by defensin-based plant protection solutions while pointing out the obstacles that still need to be overcome to translate defensin-based in vitro research findings into commercial products.
... Another CRP group of interest comprises antifungal plant defensins. Their heterologous production has been achieved in both E. coli (Bleackley et al., 2016) and P. pastoris (Hayes et al., 2013). The evaluation of both the afpA-and paf-cassette for the production of these CRPs in filamentous fungi will be considered in the near future. ...
Fungal antifungal proteins (AFPs) have attracted attention as novel biofungicides. Their exploitation requires safe and cost-effective producing biofactories. Previously, Penicillium chrysogenum and Penicillium digitatum produced recombinant AFPs with the use of a P. chrysogenum-based expression system that consisted of the paf gene promoter, signal peptide (SP)-pro sequence and terminator. Here, the regulatory elements of the afpA gene encoding the highly produced PeAfpA from Penicillium expansum were developed as an expression system for AFP production through the FungalBraid platform. The afpA cassette was tested to produce PeAfpA and P. digitatum PdAfpB in P. chrysogenum and P. digitatum, and its efficiency was compared to that of the paf cassette. Recombinant PeAfpA production was only achieved using the afpA cassette, being P. chrysogenum a more efficient biofactory than P. digitatum. Conversely, P. chrysogenum only produced PdAfpB under the control of the paf cassette. In P. digitatum, both expression systems allowed PdAfpB production, with the paf cassette resulting in higher protein yields. Interestingly, these results did not correlate with the performance of both promoters in a luciferase reporter system. In conclusion, AFP production is a complex outcome that depends on the regulatory sequences driving afp expression, the fungal biofactory and the AFP sequence.
... Evidence has demonstrated the existence of AMPs acting on the fungal membrane and on its components without having always clear information about their mechanism of action. Often these peptides affect the permeability of the membrane leading to ROS accumulation, oxidative stress damages, ATP release and the activation of stress-response pathways as HOG and MAPK cascade . On the other hand, just a few AMPs have been revealed to interact with membrane components like glucosylceramides and β-1,3-glucans or with enzymes involved in the production of membrane components as the inositol phosphoryl ceramide synthase which is essential for the sphingolipid biosynthesis . ...
Despite the great strides in healthcare during the last century, some challenges still remained unanswered. The development of multi-drug resistant bacteria, the alarming growth of fungal infections, the emerging/re-emerging of viral diseases are yet a worldwide threat. Since the discovery of natural antimicrobial peptides able to broadly hit several pathogens, peptide-based therapeutics have been under the lenses of the researchers. This review aims to focus on synthetic peptides and elucidate their multifaceted mechanisms of action as antiviral, antibacterial and antifungal agents. Antimicrobial peptides generally affect highly preserved structures, e.g., the phospholipid membrane via pore formation or other constitutive targets like peptidoglycans in Gram-negative and Gram-positive bacteria, and glucan in the fungal cell wall. Additionally, some peptides are particularly active on biofilm destabilizing the microbial communities. They can also act intracellularly, e.g., on protein biosynthesis or DNA replication. Their intracellular properties are extended upon viral infection since peptides can influence several steps along the virus life cycle starting from viral receptor-cell interaction to the budding. Besides their mode of action, improvements in manufacturing to increase their half-life and performances are also taken into consideration together with advantages and impairments in the clinical usage. Thus far, the progress of new synthetic peptide-based approaches is making them a promising tool to counteract emerging infections.
... This response is independent of Sko1. Hog1 is required for protection against the effect of NaD1 though the (Hayes et al. 2013) ...
While fungi are widely occupying nature, many species are responsible for devastating mycosis in humans. Such niche diversity explains how quick fungal adaptation is necessary to endow the capacity of withstanding fluctuating environments and to cope with host-imposed conditions. Among all the molecular mechanisms evolved by fungi, the most studied one is the activation of the phosphorelay signalling pathways, of which the high osmolarity glycerol (HOG) pathway constitutes one of the key molecular apparatus underpinning fungal adaptation and virulence. In this review, we summarize the seminal knowledge of the HOG pathway with its more recent developments. We specifically described the HOG-mediated stress adaptation, with a particular focus on osmotic and oxidative stress, and point out some lags in our understanding of its involvement in the virulence of pathogenic species including, the medically important fungi Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus, compared to the model yeast Saccharomyces cerevisiae. Finally, we also highlighted some possible applications of the HOG pathway modifications to improve the fungal-based production of natural products in the industry.
... Synthetic NmDef02 (monomer) also showed no activity (unpublished results). Other plant defensins, such as NaD1, require a dimeric conformation to show antifungal antiviral activity (Hayes et al. 2018(Hayes et al. , 2013Lay et al. 2019;Poon et al. 2014). The structure of NmDef02 is unknown but considered to be a defensin, based on molecular modeling predictions (Fig. 1c) and its primary structure, which contains eight cysteines that probably form four disulfide bridges. ...
Antibiotic resistance.is one of the biggest challenges sciences faces today. It is extremely urgent to develop new antimicrobial compounds to control infections in the "post-antibiotic era”. Plant defensins belong to a large family of small cationic antimicrobial peptides and are an integral part of the innate immune system of plants. The gene coding for the mature peptide NmDef02 (isolated from Nicotiana megalosiphon) was cloned into a pSMT3 vector, generating the plasmid pSMT3-DEF02, used to express the protein SUMO-NmDef02 in Escherichia coli, strain Shuffle T7 Express lysY. The soluble chimeric protein was purified by Ni–NTA affinity chromatography and cleaved by the UPL1 protease. The sample was re-applied to a Ni–NTA and approximately 20 mg of NmDef02 was obtained from the fermentation of 1 L of E. coli culture. The purified proteins were analyzed by SDS-PAGE under reduction condition and its identity was confirmed by Western blotting, using anti-histidine and anti-NmDef02 antibodies. NmDef02 defensin showed antimicrobial activity against plant and human pathogens. The recombinant fusion strategy could be an approach to produce bioactive recombinant NmDef02.
... In our study, the time-kill of r-javanicin against C. neoformans was initially examined. Unlike the fungicidal activity of other defensins even completely killed yeast appears in minutes to a few hours [18,19], the activity of r-javanicin was dramatically low starting from 4 h to over 24 h. Based on the morphological change of cells, SEM is a primary tool for analyzing distinctive membrane morphology after peptide exposure [20,21]. ...
The occurrence of Cryptococcus neoformans, the human fungal pathogen that primarily infects immunocompromised individuals, has been progressing at an alarming rate. The increased incidence of infection of C. neoformans with antifungal drugs resistance has become a global concern. Potential antifungal agents with extremely low toxicity are urgently needed. Herein, the biological activities of recombinant javanicin (r-javanicin) against C. neoformans were evaluated. A time-killing assay was performed and both concentration- and time-dependent antifungal activity of r-javanicin were indicated. The inhibitory effect of the peptide was initially observed at 4 h post-treatment and ultimately eradicated within 36 to 48 h. Fungal outer surface alteration was characterized by the scanning electron microscope (SEM) whereas a negligible change with slight shrinkage of external morphology was observed in r-javanicin treated cells. Confocal laser scanning microscopic analysis implied that the target(s) of r-javanicin is conceivably resided in the cell thereby allowing the peptide to penetrate across the membrane and accumulate throughout the fungal body. Finally, cryptococcal cells coped with r-javanicin were preliminarily investigated using label-free mass spectrometry-based proteomics. Combined with microscopic and proteomics analysis, it was clearly elucidated the peptide localized in the intracellular compartment where carbohydrate metabolism and energy production associated with glycolysis pathway and mitochondrial respiration, respectively, were principally interfered. Overall, r-javanicin would be an alternative candidate for further development of antifungal agents.
Plants encounter variable stresses in the environment which lead to huge crop losses worldwide. Environmental stresses that a plant can undergo are categorized into two categories as (a) biotic and (b) abiotic stress. Biotic stresses include attacks by different insects, nematodes, and microbial pathogens like fungi, bacteria, and viruses. While on the other hand, abiotic stresses include high salinity, heat, cold, drought, osmotic stress, and heavy metal. Plants are quite susceptible to both kinds of stressful situations and have adopted different mechanisms to encounter these situations. Plants sense these stresses and stimulated specific stress responses thereby activating different stress response signaling pathways and generating appropriate cellular responses helping in combating these stresses. This chapter gives an overview of the major stresses, plants encounter during growth and transgenic implications that have been made to modify these stress-tolerant properties to produce crops with improved crop yield and minimize crop losses.
Candida albicans is the principal opportunistic fungal pathogen in nosocomial settings and resistance to antifungal drugs is on the rise. Antimicrobial peptides from natural sources are promising novel therapeutics against C. albicans. OsDef2 defensin was previously found to be active against only Gram‐positive bacteria, whereas derived fragments Os and its cysteine‐free analogue, Os‐C, are active against Gram‐positive and Gram‐negative bacteria at low micromolar concentrations. In this study, OsDef2‐derived analogues and fragments were screened for anticandidal activity with the aim to identify peptides with antifungal activity and in so doing obtain a better understanding of the structural requirements for activity and modes of action. Os, Os‐C and Os(11–22)NH2, a Os‐truncated carboxy‐terminal‐amidated fragment, had the most significant antifungal activities, with minimum fungicidal concentrations (MFCs) in the micromolar range (6–28 μM). C. albicans killing was rapid and occurred within 30–60 min. Further investigations showed all three peptides interacted with cell wall derived polysaccharides while both Os and Os(11–22)NH2 permeabilized fungal liposomes. Confocal laser scanning microscopy confirmed that Os‐C and Os(11–22)NH2 could enter the cytosol of live cells and subsequent findings suggest that the uptake of Os and Os‐C, in contrast to Os(11–22)NH2, is energy dependent. Although Os, Os‐C and Os(11–22)NH2 induced the production of reactive oxygen species (ROS), co‐incubation with ascorbic acid revealed that only ROS generated by Os‐C and to a lesser extent Os(11–22)NH2 resulted in cell death. Overall, Os, Os‐C and Os(11–22)NH2 are promising candidacidal agents. C‐terminal derivative of OsDef2, Os and shorter derivatives Os‐C and Os(11–22)NH2 are active against Candida albicans.
Antimicrobial peptides (AMPs) are distributed across all kingdoms of life and are an indispensable component of host defenses. They consist of predominantly short cationic peptides with a wide variety of structures and targets. Given the ever-emerging resistance of various pathogens to existing antimicrobial therapies, AMPs have recently attracted extensive interest as potential therapeutic agents. As the discovery of new AMPs has increased, many databases specializing in AMPs have been developed to collect both fundamental and pharmacological information. In this review, we summarize the sources, structures, modes of action, and classifications of AMPs. Additionally, we examine current AMP databases, compare valuable computational tools used to predict antimicrobial activity and mechanisms of action, and highlight new machine learning approaches that can be employed to improve AMP activity to combat global antimicrobial resistance.
Plant defensins represent a large class of structurally similar peptides found throughout the plant kingdom. Despite a conserved cysteine spacing pattern and three-dimensional structure, their sequences are highly divergent and they display a range of activities including antifungal and antibacterial activities, enzyme inhibitory activities as well as roles in heavy metal tolerance and development. The vast number of sequences along with their diverse range of activities makes it impossible to test the activity and assign function to all plant defensins. However, as the number of characterized defensins increases, in depth sequence analysis may allow us to predict the function of newly identified peptides. In this review, we analyze the sequences of defensins whose activities have been described and group these based on similarity using a maximum-likelihood phylogenetic tree. We also compare the amino acids that have been described as essential for the activity of various plant defensins between these groups. While many more plant defensins will need to be characterized before we can develop rules to predict the activity of novel sequences, this approach may prove useful in identifying structure–function relationships.
Antimicrobial peptides are a vital component of the innate immune system of all eukaryotic organisms and many of these peptides have potent antifungal activity. They have potential application in the control of fungal pathogens that are a serious threat to both human health and food security. Development of antifungal peptides as therapeutics requires an understanding of their mechanism of action on fungal cells. To date, most research on antimicrobial peptides has focused on their activity against bacteria. Several antimicrobial peptides specifically target fungal cells and are not active against bacteria. Others with broader specificity often have different mechanisms of action against bacteria and fungi. This review focuses on the mechanism of action of naturally occurring antifungal peptides from a diverse range of sources including plants, mammals, amphibians, insects, crabs, spiders, and fungi. While antimicrobial peptides were originally proposed to act via membrane permeabilization, the mechanism of antifungal activity for these peptides is generally more complex and often involves entry of the peptide into the cell.
Life-threatening infectious diseases are on their way to cause a worldwide crisis, as treating them effectively is becoming increasingly difficult due to the emergence of antibiotic resistant strains. Antimicrobial peptides (AMPs) form an ancient type of innate immunity found universally in all living organisms, providing a principal first-line of defense against the invading pathogens. The unique diverse function and architecture of AMPs has attracted considerable attention by scientists, both in terms of understanding the basic biology of the innate immune system, and as a tool in the design of molecular templates for new anti-infective drugs. AMPs are gene-encoded short (<100 amino acids), amphipathic molecules with hydrophobic and cationic amino acids arranged spatially, which exhibit broad spectrum antimicrobial activity. AMPs have been the subject of natural evolution, as have the microbes, for hundreds of millions of years. Despite this long history of co-evolution, AMPs have not lost their ability to kill or inhibit the microbes totally, nor have the microbes learnt to avoid the lethal punch of AMPs. AMPs therefore have potential to provide an important breakthrough and form the basis for a new class of antibiotics. In this review, we would like to give an overview of cationic antimicrobial peptides, origin, structure, functions, and mode of action of AMPs, which are highly expressed and found in humans, as well as a brief discussion about widely abundant, well characterized AMPs in mammals, in addition to pharmaceutical aspects and the additional functions of AMPs.
Plant defensins are active against plant and human pathogenic fungi (such as Candida albicans) and baker's yeast. However, they are non-toxic to human cells, providing a possible source for treatment of fungal infections. In this study, we characterized the mode of action of the antifungal plant defensin HsAFP1 from coral bells by screening the Saccharomyces cerevisiae deletion mutant library for mutants with altered HsAFP1 sensitivity and verified the obtained genetic data by biochemical assays in S. cerevisiae and C. albicans. We identified 84 genes, which when deleted conferred at least fourfold hypersensitivity or resistance to HsAFP1. A considerable part of these genes were found to be implicated in mitochondrial functionality. In line, sodium azide, which blocks the respiratory electron transport chain, antagonized HsAFP1 antifungal activity, suggesting that a functional respiratory chain is indispensable for HsAFP1 antifungal action. Since mitochondria are the main source of cellular reactive oxygen species (ROS), we investigated the ROS-inducing nature of HsAFP1. We showed that HsAFP1 treatment of C. albicans resulted in ROS accumulation. As ROS accumulation is one of the phenotypic markers of apoptosis in yeast, we could further demonstrate that HsAFP1 induced apoptosis in C. albicans. These data provide novel mechanistic insights in the mode of action of a plant defensin.
Human β-defensins 2 and 3 are small cationic peptides with antimicrobial activity against the fungal pathogen Candida albicans. We found that hog1 and pbs2 mutants were hypersensitive to treatment with these peptides, pointing to a role of the high-osmolarity glycerol (HOG) pathway
in the response to defensin-induced cell injury.
The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2-hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast-to-hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analysed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24-ceramides in membranes of RsAFP2-treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation.
Human cathelicidin LL37 and its fragments LL13-37 and LL17-32 exhibited similar potencies in inhibiting growth of the yeast Candida albicans. After treatment with 0.5 μM and 5 μM LL13-37, the hyphae changed from a uniformly thick to an increasingly slender appearance, with budding becoming less normal in appearance and cell death could be detected. Only the yeast form and no hyphal form could be observed following exposure to 50 μM LL13-37. LL13-37 at a concentration of 5 μM was able to permeabilize the membrane of yeast form as well as hyphal form of C. albicans since the nuclear stain SYTOX Green was localized in both forms. Mycelia treated with LL13-37 stained with SYTOX Green, but did not stain with MitoTracker deep red, indicating that the mitochondria were adversely affected by LL13-37. Bimane-labeled LL13-37 was able to enter some of the hyphae, but not all hyphae were affected, suggesting that LL37 impaired membrane permeability characteristics in some of the hyphae. Reactive oxygen species was detectable in the yeast form of C. albicans cells after treatment with LL13-37 but not in the untreated cells. The results suggest that the increased membrane permeability caused by LL13-37 might not be the sole cause of cell death. It might lead to the uptake of the peptide, which might have some intracellular targets.
In recent years, studies have demonstrated the function of many antimicrobial peptides against an extensive number of microorganisms that have been isolated from different plant species and that have been used as models for the study of various cellular processes linked to these peptides' activities. Recently, a new defensin from Phaseolus vulgaris (L.) seeds, named PvD(1,) was isolated and characterized. PvD(1) was purified through anion exchange and phase-reverse chromatography. PvD(1)'s antifungal activity was tested. A SYTOX Green uptake assay revealed that the defensin PvD(1) is capable of causing membrane permeabilization in the filamentous fungi Fusarium oxysporum, Fusarium solani, and Fusarium laterithium and in yeast strains Candida parapsilosis, Pichia membranifaciens, Candida tropicalis, Candida albicans, Kluyveromyces marxiannus, and Saccharomyces cerevisiae at a concentration of 100 μg/ml. Ultrastructural analysis of C. albicans and C. guilliermondii cells treated with this defensin revealed disorganization of both cytoplasmic content and the plasma membrane. PvD(1) is also able to inhibit glucose-stimulated acidification of the medium by yeast cells and filamentous fungi, as well as to induce the production of reactive oxygen species and nitric oxide in C. albicans and F. oxysporum cells.