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The recent progressive increase in the incidence of invasive fungal infections, especially in immunocompromised patients, makes the search for new therapies crucial in the face of the growing drug resistance of prevalent nosocomial yeast strains. The latest research focuses on the active compounds of natural origin, inhibiting fungal growth, and preventing the formation of fungal biofilms. Antimicrobial peptides are currently the subject of numerous studies concerning effective antifungal therapy. In the present study, the antifungal properties of two synthetic peptides (ΔM3, ΔM4) derived from an insect antimicrobial peptide – cecropin D – were investigated. The fungicidal activity of both compounds was demonstrated against the yeast forms of Candida albicans, Candida tropicalis, and Candida parapsilosis, reaching a MFC99.9 in the micromolar range, while Candida glabrata showed greater resistance to these peptides. The scanning electron microscopy revealed a destabilization of the yeast cell walls upon treatment with both peptides; however, their effectiveness was strongly modified by the presence of salt or plasma in the yeast environment. The transition of C. albicans cells from yeast to filamentous form, as well as the formation of biofilms, was effectively reduced by ΔM4. Mature biofilm viability was inhibited by a higher concentration of this peptide and was accompanied by increased ROS production, activation of the GPX3 and SOD5 genes, and finally, increased membrane permeability. Furthermore, both peptides showed a synergistic effect with caspofungin in inhibiting the metabolic activity of C. albicans cells, and an additive effect was also observed for the mixtures of peptides with amphotericin B. The results indicate the possible potential of the tested peptides in the prevention and treatment of candidiasis.

Microbial keratitis is a common cause of ocular pain and visual impairment worldwide. The ocular surface has a relatively paucicellular microbial community, mostly found in the conjunctiva, while the cornea would be considered relatively sterile. However, in patients with microbial keratitis, the cornea can be infected with multiple pathogens includingStaphylococcus aureus,Pseudomonas aeruginosa, andFusariumsp. Treatment with topical antimicrobials serves as the standard of care for microbial keratitis, however, due to high rates of pathogen resistance to current antimicrobial medications, alternative therapeutic strategies must be developed. Multiple studies have characterized the expression and activity of antimicrobial peptides (AMPs), endogenous peptides with key antimicrobial and wound healing properties, on the ocular surface. Recent studies and clinical trials provide promise for the use of AMPs as therapeutic agents. This article reviews the repertoire of AMPs expressed at the ocular surface, how expression of these AMPs can be modulated, and the potential for harnessing the AMPs as potential therapeutics for patients with microbial keratitis.

Halotolerant bacteria produce a wide range of bioactive compounds with important applications in agriculture for abiotic stress amelioration and plant growth promotion. In the present study, 17 biosynthetic gene clusters (BGCs) were identified in Exiguobacterium profundum PHM11 belonging to saccharides, desmotamide, pseudaminic acid, dipeptide aldehydes, and terpene biosynthetic pathways representing approximately one-sixth of genomes. The terpene biosynthetic pathway was conserved in Exiguobacterium spp. while the E. profundum PHM11 genome confirms the presence of the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway for the isopentenyl diphosphate (IPP) synthesis. Further, 2,877 signal peptides (SPs) were identified using the PrediSi server, out of which 592 proteins were prophesied for the secretion having a transmembrane helix (TMH). In addition, antimicrobial peptides (AMPs) were also identified using BAGEL4. The transcriptome analysis of PHM11 under salt stress reveals the differential expression of putative secretion and transporter genes having SPs and TMH. Priming of the rice, wheat and maize seeds with PHM11 under salt stress led to improvement in the root length, root diameters, surface area, number of links and forks, and shoot length. The study shows that the presence of BGCs, SPs, and secretion proteins constituting TMH and AMPs provides superior competitiveness in the environment and make E. profundum PHM11 a suitable candidate for plant growth promotion under salt stress.

Antimicrobial resistance is a major concern to public health demanding effective alternative strategies to disease control and prevention. Modulation of endogenous host defense peptide (HDP) synthesis has emerged as a promising antibiotic alternative approach. This study investigated a potential synergy between sugars and butyrate in inducing HDP gene expression in chickens. Our results revealed that sugars differentially regulated HDP expression in both gene- and sugar-specific manners in chicken HD11 macrophage cells. Among eight mono- and disaccharides tested, all were potent inducers of avian β-defensin 9 (AvBD9) gene (pCATHB1) gene expression. The expression of AvBD14 gene, on the other hand, was minimally influenced by sugars. Moreover, all sugars exhibited a strong synergy with butyrate in enhancing AvBD9 expression, while only galactose, trehalose, and lactose were synergistic with butyrate in CATHB1 induction. No synergy in AvBD14 induction was observed between sugars and butyrate. Although lactose augmented the expression of nearly all HDP genes, its synergy with butyrate was only seen with several, but not all, HDP genes. Mucin-2 gene was also synergistically induced by a combination of lactose and butyrate. Furthermore, lactose synergized with butyrate to induce AvBD9 expression in chicken jejunal explants (pAvBD9 induction. Collectively, a combination of butyrate and a sugar with both HDP-inducing and barrier protective activities holds the promise to be developed as an alternative to antibiotics for disease control and prevention.

Antimicrobial peptides (AMPs) constitute a broad range of bioactive compounds in diverse organisms, including fish. They are effector molecules for the innate immune response, against pathogens, tissue damage and infections. Still, AMPs from African Catfish, Clarias gariepinus, skin mucus are largely unexplored despite their possible therapeutic role in combating antimicrobial resistance. In this study, African Catfish Antimicrobial peptides (ACAPs) were identified from the skin mucus of African Catfish, C. gariepinus. Native peptides were extracted from fish mucus scrapings in 10% acetic acid (v/v) and ultra-filtered using 5 kDa molecular weight cut-off membrane. The extract was purified using C18 Solid-Phase Extraction. The antibacterial activity was determined using the Agar Well Diffusion method and broth-dilution method utilizing Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922). Thereafter, Sephadex G-25 gel filtration was further utilized in bio-guided isolation of the most active fractions prior to peptide identification using Orbitrap Fusion Lumos Tribrid Mass Spectrometry. The skin mucus extracted from African Catfish from all the three major lakes of Uganda exhibited antimicrobial activity on E. coli and S. aureus. Lake Albert’s C. gariepinus demonstrated the best activity with the lowest MIC of 2.84 and 0.71 μg/ml on S. aureus and E. coli, respectively. Sephadex G-25 peak I mass spectrometry analysis (Data are available via ProteomeXchange with identifier PXD029193) alongside in silico analysis revealed seven short peptides (11–16 amino acid residues) of high antimicrobial scores (0.561–0.905 units). In addition, these peptides had a low molecular weight (1005.57–1622.05 Da) and had percentage hydrophobicity above 54%. Up to four of these AMPs demonstrated α-helix structure conformation, rendering them amphipathic. The findings of this study indicate that novel AMPs can be sourced from the skin mucus of C. gariepinus. Such AMPs are potential alternatives to the traditional antibiotics and can be of great application to food and pharmaceutical industries; however, further studies are still needed to establish their drug-likeness and safety profiles.

Among chronic skin autoinflammatory diseases, Hidradenitis Suppurativa (HS) stands out for its chronicity, highly variable condition, and profound impact on the patients’ quality of life. HS is characterized by suppurative skin lesions in diverse body areas, including deep-seated painful nodules, abscesses, draining sinus, and bridged scars, among others, with typical topography. To date, HS is considered a refractory disease and medical treatments aim to reduce the incidence, the infection, and the pain of the lesions. For this purpose, different classes of drugs, including anti-inflammatory molecules, antibiotics and biological drugs are being used. Antimicrobial peptides (AMPs), also called defense peptides, emerge as a new class of therapeutic compounds, with broad-spectrum antimicrobial action, in addition to reports on their anti-inflammatory, healing, and immunomodulating activity. Such peptides are present in prokaryotes and eukaryotes, as part of the innate eukaryotic immune system. It has been proposed that a deregulation in the expression of AMPs in human epithelial tissues of HS patients may be associated with the etiology of this skin disease. In this scenario, plant AMPs stand out for their richness, diversity of types, and broad antimicrobial effects, with potential application for topical systemic use in patients affected by HS. This work was supported by a Biomolecular Analyses for Tailored Medicine in AcneiNversa (BATMAN) project, funded by ERA PerMed (JTC_2018), by a grant from the Institute for Maternal and Child Health IRCCS ‘Burlo Garofolo’/Italian Ministry of Health (RC16/2018), and by a Starting Grant (SG-2019-12369421) funded by Italian Ministry of Health.

Antimicrobial peptides are an important component of many organisms’ innate immune system, with a good inhibitory or killing effect against the invading pathogens. As a type of biological polypeptide with natural immune activities, antimicrobial peptides have a broad spectrum of antibacterial, antiviral, and antitumor activities. Nevertheless, these peptides cause no harm to the organisms themselves. Compared with traditional antibiotics, antimicrobial peptides have the advantage of not producing drug resistance and have a unique antibacterial mechanism, which has attracted widespread attention. In this study, marine invertebrates were classified into arthropods, annelids, mollusks, cnidarians, and tunicata. We then analyzed the types, sources and antimicrobial activities of the antimicrobial peptides in each group. We also reviewed the immune mechanism from three aspects: membrane-targeted direct killing effects, non-membrane targeting effects and immunomodulatory effects. Finally, we discussed their applications and the existing problems facing antimicrobial peptides in actual production. The results are expected to provide theoretical support for future research and applications of antimicrobial peptides in marine invertebrates.

Antimicrobial peptides (AMPs) are ubiquitous amongst living organisms and are part of the innate immune system with the ability to kill pathogens directly or indirectly by modulating the immune system. AMPs have potential as a novel therapeutic against bacteria due to their quick-acting mechanism of action that prevents bacteria from developing resistance. Additionally, there is a dire need for therapeutics with activity specifically against Gram-negative bacterial infections that are intrinsically difficult to treat, with or without acquired drug resistance. Development of new antibiotics has slowed in recent years and novel therapeutics (like AMPs) with a focus against Gram-negative bacteria are needed. We designed eight novel AMPs, termed PHNX peptides, using ab initio computational design (database filtering technology combined with the novel positional analysis on APD3 dataset of AMPs with activity against Gram-negative bacteria) and assessed their theoretical function using published machine learning algorithms, and finally, validated their activity in our laboratory. These AMPs were tested to establish their minimum inhibitory concentration (MIC) and half-maximal effective concentration (EC50) under CLSI methodology against antibiotic resistant and antibiotic susceptible Escherichia coli and Staphylococcus aureus. Laboratory-based experimental results were compared to computationally predicted activities for each of the peptides to ascertain the accuracy of the computational tools used. PHNX-1 demonstrated antibacterial activity (under high and low-salt conditions) against antibiotic resistant and susceptible strains of Gram-positive and Gram-negative bacteria and PHNX-4 to -8 demonstrated low-salt antibacterial activity only. The AMPs were then evaluated for cytotoxicity using hemolysis against human red blood cells and demonstrated some hemolysis which needs to be further evaluated. In this study, we successfully developed a design methodology to create synthetic AMPs with a narrow spectrum of activity where the PHNX AMPs demonstrated higher antibacterial activity against Gram-negative bacteria compared to Gram-positive bacteria. Thus, these peptides present novel synthetic peptides with a potential for therapeutic use. Based on our findings, we propose upfront selection of the peptide dataset for analysis, an additional step of positional analysis to add to the ab initio database filtering technology (DFT) method, and we present laboratory data on the novel, synthetically designed AMPs to validate the results of the computational approach. We aim to conduct future in vivo studies which could establish these AMPs for clinical use.

Bacillus velezensis ML122-2 is an antimicrobial-producing strain isolated from the leaf of Assam tea or Miang [Camellia sinensis var. assamica (J.W.Mast.) Kitam.]. The cell-free supernatant (CFS) of strain ML122-2 exhibits a broad-spectrum antimicrobial activity against various Gram-positive and Gram-negative bacteria as well as the mold Penicillium expansum. The genome of B. velezensis ML122-2 was sequenced and in silico analysis identified three potential bacteriocin-associated gene clusters, that is, those involved in the production of mersacidin, amylocyclicin, and LCI. Furthermore, six gene clusters exhibiting homology (75–100% DNA sequence identity) to those associated with the secondary metabolites bacilysin, bacillibactin, surfactin, macrolactin H, bacillaene, and plipastatin were identified. Individual antimicrobial activities produced by B. velezensis ML122-2 were purified and characterized by Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis, revealing three antimicrobial peptides with molecular masses corresponding to surfactin, plipastatin, and amylocyclicin. Transcriptional analysis of specific genes associated with mersacidin (mrsA), amylocyclicin (acnA), plipastatin (ppsA), and surfactin (srfAA) production by B. velezensis ML122-2 showed that the first was not transcribed under the conditions tested, while the latter three were consistent with the presence of the associated peptides as determined by mass spectrometry analysis. These findings demonstrate that B. velezensis ML122-2 has the genetic capacity to produce a wide range of antimicrobial activities that may support a specific community structure and highlight the biotechnological properties of Assam tea.

The predominance of bacterial taxa in the gut, was examined in view of the putative antimicrobial peptide sequences (AMPs) within their proteomes. The working assumption was that compatible bacteria would share homology and thus immunity to their putative AMPs, while competing taxa would have dissimilarities in their proteome-hidden AMPs. A network–based method (“Bacterial Wars”) was developed to handle sequence similarities of predicted AMPs among UniProt-derived protein sequences from different bacterial taxa, while a resulting parameter (“Die” score) suggested which taxa would prevail in a defined microbiome. T he working hypothesis was examined by correlating the calculated Die scores, to the abundance of bacterial taxa from gut microbiomes from different states of health and disease. Eleven publicly available 16S rRNA datasets and a dataset from a full shotgun metagenomics served for the analysis. The overall conclusion was that AMPs encrypted within bacterial proteomes affected the predominance of bacterial taxa in chemospheres.

The global spread of antibiotic-resistant infections has meant that there is an urgent need to develop new antimicrobial alternatives. In this study, we developed a strategy to boost and/or synergize the activity of conventional antibiotics by combination with antimicrobial peptides tagged with the bulky non-natural amino acid β-naphthylalanine (Nal) to their N- or C-terminus. A checkerboard method was used to evaluate synergistic effects of the parent peptide and the Nal-tagged peptides. Moreover, boron-dipyrro-methene labeled vancomycin was used to characterize the synergistic mechanism of action between the peptides and vancomycin on the bacterial strains. These Nal-tagged antimicrobial peptides also reduced the antibiotic-induced release of lipopolysaccharide from Gram-negative bacteria by more than 99.95%. Our results demonstrate that Nal-tagged peptides could help in developing antimicrobial peptides that not only have enhanced antibacterial activities but also increase the synergistic effects with conventional antibiotics against antibiotic-resistant bacteria.

Eco-friendly analogs of Trichogin GA IV, a short peptaibol produced by Trichoderma longibrachiatum, were assayed against Pyricularia oryzae, the causal agent of rice blast disease. In vitro and in vivo screenings allowed us to identify six peptides able to reduce by about 70% rice blast symptoms. One of the most active peptides was selected for further studies. Microscopy analyses highlighted that the treated fungal spores could not germinate and the fluorescein-labeled peptide localized on the spore cell wall and in the agglutinated cytoplasm. Transcriptomic analysis was carried out on P. oryzae mycelium 3 h after the peptide treatment. We identified 1,410 differentially expressed genes, two-thirds of which upregulated. Among these, we found genes involved in oxidative stress response, detoxification, autophagic cell death, cell wall biogenesis, degradation and remodeling, melanin and fatty acid biosynthesis, and ion efflux transporters. Molecular data suggest that the trichogin analogs cause cell wall and membrane damages and induce autophagic cell death. Ultrastructure observations on treated conidia and hyphae confirmed the molecular data. In conclusion, these selected peptides seem to be promising alternative molecules for developing effective bio-pesticides able to control rice blast disease.

Antibiotic resistance is emerging as a hot issue with the abuse and overuse of antibiotics, and the shortage of effective antimicrobial agents against multidrug resistant bacteria creates a huge problem to treat the threatening nosocomial skin and soft tissue infection. Antimicrobial peptides (AMPs) exhibite enormous potential as one of the most promising candidates of antibiotic to fight against pathogenic infections because of its unique membrane penetration mechanism to kill pathogens, whereas the clinical application of AMPs still faces the challenges of production cost, stability, safety, and design strategy. Herein, a series of Trp-rich peptides was designed following the principle of paired Trp plated at the ith and ith+4 position on the backbone of peptides, based on the template (VKKX)4, where X represents W, A, or L, to study the effect of intramolecular aromatic interactions on the bioactivity of AMPs. Through comparing the antimicrobial performance, hemolysis, cytotoxicity, and stability, VW5 which is equipped with the characters of direct antimicrobial efficacy (GM=1.68μM) and physical destruction of bacterial membrane (SEM and electron microscopy) stood out from the engineering peptides. VW5 also performed well in mice models, which could significantly decrease the bacterial colony (VW5 vs infection group, 12.72±2.26 vs 5.52±2.01×109CFU/abscess), the area of dermo-necrosis (VW5 vs infection group, 0.74±0.29 vs 1.86±0.98mm2) and the inflammation cytokine levels at the abscess site without causing toxicity to the skin. Overall, this study provides a strategy and template to diminish the randomness in the exploration and design of novel peptides.

With the increasing number of drug-resistant bacteria, there is an urgent need for new antimicrobial agents, and antimicrobial peptides (AMPs), which exist in the human non-specific immune system, are one of the most promising candidates. It is an effective optimization strategy to modify antimicrobial peptides (AMPs) according to the distribution of amino acids and hydrophobic characteristics. The addition of bacterial pheromones to the N short peptide can increase the ability to recognize bacteria. In this study, we designed and synthesized AMP1–6 by amino acid substitution of mBjAMP1. Additionally, P-6, S-6, and L-6 were designed and synthesized by adding bacterial pheromones based on 1–6. Functional tests showed that the four AMPs had the ability to kill Gram-negative Vibrio anguillarum, Pseudomonas mendocina, and Vibrio parahaemolyticus, and Gram-positive Micrococcus luteus and Listeria monocytogenes. Additionally, all four AMPs induced permeabilization and depolarization of bacterial cell membranes and increased intracellular reactive oxygen species (ROS) levels. Importantly, they had little or no mammalian cytotoxicity. At the same time, 1–6 and L-6 protected the stability of intestinal flora in Sebastes schlegelii and increased the relative abundance of Lactobacillaceae. In summary, our results indicate that the designed AMPs have broad application prospects as a new type of polypeptide antimicrobial agent.

Bacteriocins are a highly diverse group of antimicrobial peptides that have been identified in a wide range of commensal and probiotic organisms, especially those resident in host microbiomes. Rising antibiotic resistance have fueled renewed research into new drug scaffolds such as antimicrobial peptides for use in therapeutics. In this investigation, we examined mung bean seeds for endophytes possessing activity against human and plant pathogens. We isolated a novel strain of Bacillus safensis, from the contents of surface-sterilized mung bean seed, which we termed B. safensis C3. Genome sequencing of C3 identified three distinct biosynthetic systems that produce bacteriocin-based peptides. C3 exhibited antibacterial activity against Escherichia coli, Xanthomonas axonopodis, and Pseudomonas syringae. Robust antimicrobial activity of B. safensis C3 was observed when C3 was co-cultured with Bacillus subtilis. Using the cell-free supernatant of C3 and cation exchange chromatography, we enriched a product that retained antimicrobial activity against B. subtilis. The peptide was found to be approximately 3.3 kDa in size by mass spectrometry, and resistant to proteolysis by Carboxypeptidase Y and Endoproteinase GluC, suggesting that it is a modified variant of an AS-48 like bacteriocin. Our findings open new avenues into further development of novel bacteriocin-based scaffolds for therapeutic development, as well as further investigations into how our discoveries of bacteriocin-producing plant commensal microorganisms may have the potential for an immediate impact on the safety of food supplies.

New methods for antimicrobial design are critical for combating pathogenic bacteria in the post-antibiotic era. Fortunately, competition within complex communities has led to the natural evolution of antimicrobial peptide (AMP) sequences that have promising bactericidal properties. Unfortunately, the identification, characterization, and production of AMPs can prove complex and time consuming. Here, we report a peptide generation framework, PepVAE, based around variational autoencoder (VAE) and antimicrobial activity prediction models for designing novel AMPs using only sequences and experimental minimum inhibitory concentration (MIC) data as input. Sampling from distinct regions of the learned latent space allows for controllable generation of new AMP sequences with minimal input parameters. Extensive analysis of the PepVAE-generated sequences paired with antimicrobial activity prediction models supports this modular design framework as a promising system for development of novel AMPs, demonstrating controlled production of AMPs with experimental validation of predicted antimicrobial activity.

The incidence of zoonotic Staphylococcus pseudintermedius and Microsporum canis infections is rapidly growing worldwide in the context of an increasing frequency of close contact between animals and humans, presenting challenges in both human and veterinary medicine. Moreover, the development of microbial resistance and emergence of recalcitrant biofilms, accompanied by the insufficiency of new antimicrobial agents, have become major obstacles in treating superficial skin infections caused by various microbes including S. pseudintermedius and M. canis. Over recent years, the prospects of antimicrobial peptides as emerging antimicrobials to combat microbial infections have been demonstrated. In our study, two novel short-chain peptides, namely, allomyrinasin and andricin B, produced by Allomyrina dichotoma and Andrias davidianus, were revealed to exhibit potent antimicrobial efficacy against clinical isolates of S. pseudintermedius and M. canis with remarkable and rapid fungicidal and bactericidal effects, while allomyrinasin exhibited inhibition of biofilm formation and eradication of mature biofilm. These peptides displayed synergistic activity when combined with amoxicillin and terbinafine against S. pseudintermedius and M. canis. Cytoplasmic leakage via cytomembrane permeabilization serves as a mechanism of action. Extremely low hemolytic activity and serum stability in vitro, as well as superior anti-infective efficacy in reducing bacterial counts and relieving the inflammatory response in vivo, were detected. The potent antibacterial, antifungal, and anti-inflammatory activities of allomyrinasin and andricin B might indicate promising anti-infective alternatives for the treatment of S. pseudintermedius and M. canis infections in the context of human and veterinary medicine.

An increase in antibiotic resistance has led to escalating the need for the development of alternate therapy. Antimicrobial peptides (AMPs) are at the forefront of replacing conventional antibiotics, showing slower development of drug resistance, antibiofilm activity, and the ability to modulate the host immune response. The ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens that jeopardize most conventional antibiotics are known to be involved in severe respiratory tract, bloodstream, urinary tract, soft tissue, and skin infections. Among them, S. aureus is an insidious microbe and developed resistance against conventional antibiotics. In the present study, an AMP (named as peptide-Ba49) isolated from Bacillus subtilis subsp. spizizenii strain from Allium cepa (the common onion) exhibited strong antibacterial efficacy against S. aureus ATCC 25923. The mode of action of this peptide-Ba49 on S. aureus was deciphered through various sensitive probes, i.e., DiSC3 (5) and H2DCFDA, suggesting the peptide-Ba49 to be acting upon through change in membrane potential and by triggering the production of reactive oxygen species (ROS). This induced disruption of the cell membrane was further supported by morphological studies using scanning electron microscopy (SEM). Investigations on a possible post-antibiotic effect (PAE) of peptide-Ba49 showed prolonged PAE against S. aureus. Furthermore, the peptide-Ba49 prevented the formation of S. aureus biofilm at low concentration and showed its potential to degrade the mature biofilm of S. aureus. The peptide-Ba49 also exhibited intracellular killing potential against S. aureus ATCC 25923 in the macrophage cells, and moreover, peptide-Ba49 was found to bolster the fibroblast cell migration in the scratch assay at low concentration, exhibiting a wound healing efficacy of this peptide. These studies demonstrated that peptide-Ba49 isolated from the strain B. subtilis subsp. spizizenii could be a therapeutic candidate to combat the pathogenic S. aureus infections.

The security issue of human health is faced with dispiriting threats from multidrug-resistant bacteria infections induced by the abuse and misuse of antibiotics. Over decades, the antimicrobial peptides (AMPs) hold great promise as a viable alternative to treatment with antibiotics due to their peculiar antimicrobial mechanisms of action, broad-spectrum antimicrobial activity, lower drug residue, and ease of synthesis and modification. However, they universally express a series of disadvantages that hinder their potential application in the biomedical field (e.g., low bioavailability, poor protease resistance, and high cytotoxicity) and extremely waste the abundant resources of AMP database discovered over the decades. For all these reasons, the nanostructured antimicrobial peptides (Ns-AMPs), based on a variety of nanosystem modification, have made up for the deficiencies and pushed the development of novel AMP-based antimicrobial therapies. In this review, we provide an overview of the advantages of Ns-AMPs in improving therapeutic efficacy and biological stability, reducing side effects, and gaining the effect of organic targeting and drug controlled release. Then the different material categories of Ns-AMPs are described, including inorganic material nanosystems containing AMPs, organic material nanosystems containing AMPs, and self-assembled AMPs. Additionally, this review focuses on the Ns-AMPs for the effect of biological activities, with emphasis on antimicrobial activity, biosecurity, and biological stability. The “state-of-the-art” antimicrobial modes of Ns-AMPs, including controlled release of AMPs under a specific environment or intrinsic antimicrobial properties of Ns-AMPs, are also explicated. Finally, the perspectives and conclusions of the current research in this field are also summarized.

Butterflies represent one of the largest animal groups on Earth, yet antimicrobial peptides (AMPs) of this group are less studied in comparison with their moth counterparts. This study employed an integrated bioinformatics approach to survey natural AMPs from publicly available genomic datasets. Numerous AMPs, including cecropins, defensins, and moricins, were identified and subsequently used as templates for the design of a series of synthetic AMPs that mimicked the naturally occurring sequences. Despite differing biological effects among the various sequences, the synthetic AMPs exhibited potent antibacterial and antifungal activities in vitro and in vivo, without inducing hemolysis, which implied their therapeutic potential in infectious diseases. Electron and confocal fluorescence microscopies revealed that the AMPs induced distinct morphological and biophysical changes on microbial cell membranes and nuclei, suggesting that the antimicrobial effects were related to a mechanism of membrane penetration and nucleic acid binding by the peptides. In conclusion, this study not only offers insights into butterfly AMPs but also provides a practical strategy for high-throughput natural AMP discoveries that will have implications for future research in this area.

The ClpX ATPase is critical for resistance to cell envelope targeting antibiotics in Bacillus anthracis, however, it is unclear whether this is due to its function as an independent chaperone or as part of the ClpXP protease. In this study, we demonstrate that antibiotic resistance is due to formation of the ClpXP protease through construction of a ClpX complementation plasmid that is unable to interact with ClpP. Additionally, we genetically disrupted both clpP genes, clpP1 and clpP2, found in B. anthracis Sterne and find that the loss of either increases susceptibility to cell envelope targeting antimicrobials, although neither has as strong of a phenotype as loss of clpX and neither clpP gene is essential for virulence in a G. mellonella model of infection. Lastly, we looked at changes to cell envelope morphology that could contribute to increased antibiotic sensitivity. We find no difference in cell charge or cell lysis, although we do see increased hydrophobicity in the ΔclpX strain, decreased cellular density and slightly thinner cells walls. We also see significant cell division defects in ΔclpX, although only when cells are grown in the mammalian cell culture medium, RPMI. We conclude that the intrinsic resistance of B. anthracis to cell wall active antimicrobials is dependent on formation of the ClpXP protease and that this could be due, at least in part, to the role of ClpX in regulating cell envelope morphology.

Brucellais a facultatively intracellular bacterial pathogen and the cause of worldwide zoonotic infections, infamous for its ability to evade the immune system and persist chronically within host cells. Despite the frequent association with attenuation in other Gram-negative bacteria, a rough lipopolysaccharide phenotype is retained byBrucella canisandBrucella ovis, which remain fully virulent in their natural canine and ovine hosts, respectively. While these natural rough strains lack the O-polysaccharide they, like their smooth counterparts, are able to evade and manipulate the host immune system by exhibiting low endotoxic activity, resisting destruction by complement and antimicrobial peptides, entering and trafficking within host cells along a similar pathway, and interfering with MHC-II antigen presentation.B. canisandB. ovisappear to have compensated for their roughness by alterations to their outer membrane, especially in regards to outer membrane proteins.B. canis, in particular, also shows evidence of being less proinflammatoryin vivo, suggesting that the rough phenotype may be associated with an enhanced level of stealth that could allow these pathogens to persist for longer periods of time undetected. Nevertheless, much additional work is required to understand the correlates of immune protection against the natural roughBrucellaspp., a critical step toward development of much-needed vaccines. This review will highlight the significance of rough lipopolysaccharide in the context of both natural disease and host–pathogen interactions with an emphasis on natural roughBrucellaspp. and the implications for vaccine development.

Antibiotic-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) has become a serious threat to public health worldwide. Cationic α-helical antimicrobial peptides (CαAMPs) have attracted much attention as promising solutions in post-antibiotic era. However, strong hemolytic activity and in vivo inefficacy have hindered their pharmaceutical development. Here, we attempt to address these obstacles by investigating BmKn2 and BmKn2-7, two scorpion-derived CαAMPs with the same hydrophobic face and a distinct hydrophilic face. Through structural comparison, mutant design and functional analyses, we found that while keeping the hydrophobic face unchanged, increasing the number of alkaline residues (i.e., Lys + Arg residues) on the hydrophilic face of BmKn2 reduces the hemolytic activity and broadens the antimicrobial spectrum. Strikingly, when keeping the total number of alkaline residues constant, increasing the number of Lys residues on the hydrophilic face of BmKn2-7 significantly reduces the hemolytic activity but does not influence the antimicrobial activity. BmKn2-7K, a mutant of BmKn2-7 in which all of the Arg residues on the hydrophilic face were replaced with Lys, showed the lowest hemolytic activity and potent antimicrobial activity against antibiotic-resistant ESKAPE pathogens. Moreover, in vivo experiments indicate that BmKn2-7K displays potent antimicrobial efficacy against both the penicillin-resistant S. aureus and the carbapenem- and multidrug-resistant A. baumannii, and is non-toxic at the antimicrobial dosages. Taken together, our work highlights the significant functional disparity of Lys vs Arg in the scorpion-derived antimicrobial peptide BmKn2-7, and provides a promising lead molecule for drug development against ESKAPE pathogens.

Searching for new antimicrobials is a pressing issue to conquer the emergence of multidrug-resistant (MDR) bacteria and fungi. Antimicrobial peptides (AMPs) usually have antimicrobial mechanisms different from those of traditional antibiotics and bring new hope in the discovery of new antimicrobials. In addition to antimicrobial activity, stability and target selectivity are important concerns to decide whether an antimicrobial peptide can be applied in vivo. Here, we used a simple de novo designed peptide, pepD2, which contains only three kinds of amino acid residues (W, K, L), as an example to evaluate how the residues and modifications affect the antimicrobial activity against Acinetobacter baumannii, stability in plasma, and toxicity to human HEK293 cells. We found that pepI2 with a Leu→Ile substitution can decrease the minimum bactericidal concentrations (MBC) against A. baumannii by one half (4 μg/mL). A D-form peptide, pepdD2, in which the D-enantiomers replaced the L-enantiomers of the Lys(K) and Leu(L) residues, extended the peptide half-life in plasma by more than 12-fold. PepD3 is 3-residue shorter than pepD2. Decreasing peptide length did not affect antimicrobial activity but increased the IC50 to HEK293 cells, thus increased the selectivity index (SI) between A. baumannii and HEK293 cells from 4.7 to 8.5. The chain length increase of the N-terminal acyl group and the Lys→Arg substitution greatly enhanced the hemolytic activity, hence those modifications are not good for clinical application. Unlike colistin, the action mechanism of our peptides relies on negatively charged lipids rather than lipopolysaccharides. Therefore, not only gram-negative bacteria but also gram-positive bacteria can be killed by our peptides.

Lactobacillus casei (L. casei), a normal resident of the gastrointestinal tract of mammals, has been extensively studied over the past few decades for its probiotic properties in clinical and animal models. Some studies have shown that some bacterium of Lactobacillus stimulate the production of antimicrobial peptides in intestinal cells to clear enteric pathogens, however, which antimicrobial peptides are produced by L. casei stimulation and its function are still not completely understood. In this study, we investigated the changes of antimicrobial peptides’ expression after intragastric administration of L. casei to mice. The bioinformatics analysis revealed there were nine genes strongly associated with up-regulated DEGs. But, of these, only the antimicrobial peptide mReg3a gene was continuously up-regulated, which was also confirmed by qRT-PCR. We found out the mReg3a expressed in engineering E.coli promoted cell proliferation and wound healing proved by CCK-8 assay and wound healing assay. Moreover, the tight junction proteins ZO-1 and E-cadherin in mReg3a treatment group were significantly higher than that in the control group under the final concentration of 0.2 mg/ml both in Porcine intestinal epithelial cells (IPEC-J2) and Mouse intestinal epithelial cells (IEC-6) (p < 0.05). Surprisingly, the recombinant mReg3a not only inhibited Enterotoxigenic Escherichia coli (ETEC), but also reduced the copy number of the piglet diarrheal viruses, porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine rotavirus (PoRV), indicating the antimicrobial peptides mReg3a may be feed additives to resist the potential of the intestinal bacterial and viral diarrhea disease.

The ClpX ATPase is critical for resistance to cell envelope targeting antibiotics in Bacillus anthracis, however, it is unclear whether this is due to its function as an independent chaperone or as part of the ClpXP protease. In this study, we demonstrate that antibiotic resistance is due to formation of the ClpXP protease through construction of a ClpX complementation plasmid that is unable to interact with ClpP. Additionally, we genetically disrupted both clpP genes, clpP1 and clpP2, found in B. anthracis Sterne and find that the loss of either increases susceptibility to cell envelope targeting antimicrobials, although neither has as strong of a phenotype as loss of clpX and neither clpP gene is essential for virulence in a G. mellonella model of infection. Lastly, we looked at changes to cell envelope morphology that could contribute to increased antibiotic sensitivity. We find no difference in cell charge or cell lysis, although we do see increased hydrophobicity in the ΔclpX strain, decreased cellular density and slightly thinner cells walls. We also see significant cell division defects in ΔclpX, although only when cells are grown in the mammalian cell culture medium, RPMI. We conclude that the intrinsic resistance of B. anthracis to cell wall active antimicrobials is dependent on formation of the ClpXP protease and that this could be due, at least in part, to the role of ClpX in regulating cell envelope morphology.

The honey bee is one of the most important pollinators in the agricultural system and is responsible for pollinating a third of all food we eat. Sacbrood virus (SBV) is a member of the virus family Iflaviridae and affects honey bee larvae and causes particularly devastating disease in the Asian honey bees, Apis cerana. Chinese Sacbrood virus (CSBV) is a geographic strain of SBV identified in China and has resulted in mass death of honey bees in China in recent years. However, the molecular mechanism underlying SBV infection in the Asian honey bee has remained unelucidated. In this present study, we employed high throughput next-generation sequencing technology to study the host transcriptional responses to CSBV infection in A. cerana larvae, and were able to identify genome-wide differentially expressed genes associated with the viral infection. Our study identified 2,534 differentially expressed genes (DEGs) involved in host innate immunity including Toll and immune deficiency (IMD) pathways, RNA interference (RNAi) pathway, endocytosis, etc. Notably, the expression of genes encoding antimicrobial peptides (abaecin, apidaecin, hymenoptaecin, and defensin) and core components of RNAi such as Dicer-like and Ago2 were found to be significantly upregulated in CSBV infected larvae. Most importantly, the expression of Sirtuin target genes, a family of signaling proteins involved in metabolic regulation, apoptosis, and intracellular signaling was found to be changed, providing the first evidence of the involvement of Sirtuin signaling pathway in insects’ immune response to a virus infection. The results obtained from this study provide novel insights into the molecular mechanism and immune responses involved in CSBV infection, which in turn will contribute to the development of diagnostics and treatment for the diseases in honey bees.

Increasing prevalence of antimicrobial resistance (AMR) has posed a major health concern worldwide, and the addition of new antimicrobial agents is diminishing due to overexploitation of plants and microbial resources. Inevitably, alternative sources and new strategies are needed to find novel biomolecules to counter AMR and pandemic circumstances. The association of plants with microorganisms is one basic natural interaction that involves the exchange of biomolecules. Such a symbiotic relationship might affect the respective bio-chemical properties and production of secondary metabolites in the host and microbes. Furthermore, the discovery of taxol and taxane from an endophytic fungus, Taxomyces andreanae from Taxus wallachiana, has stimulated much research on endophytes from medicinal plants. A gram-positive endophytic bacterium, Paenibacillus peoriae IBSD35, was isolated from the stem of Millettia pachycarpa Benth. It is a rod-shaped, motile, gram-positive, and endospore-forming bacteria. It is neutralophilic as per Joint Genome Institute’s (JGI) IMG system analysis. The plant was selected based on its ethnobotany history of traditional uses and highly insecticidal properties. Bioactive molecules were purified from P. peoriae IBSD35 culture broth using 70% ammonium sulfate and column chromatography techniques. The biomolecule was enriched to 151.72-fold and the yield percentage was 0.05. Peoriaerin II, a highly potent and broad-spectrum antimicrobial peptide against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Candida albicans ATCC 10231 was isolated. LC-MS sequencing revealed that its N-terminal is methionine. It has four negatively charged residues (Asp + Glu) and a total number of two positively charged residues (Arg + Lys). Its molecular weight is 4,685.13 Da. It is linked to an LC-MS/MS inferred biosynthetic gene cluster with accession number A0A2S6P0H9, and blastp has shown it is 82.4% similar to fusaricidin synthetase of Paenibacillus polymyxa SC2. The 3D structure conformation of the BGC and AMP were predicted using SWISS MODEL homology modeling. Therefore, combining both genomic and proteomic results obtained from P. peoriae IBSD35, associated with M. pachycarpa Benth., will substantially increase the understanding of antimicrobial peptides and assist to uncover novel biological agents.

Neisseria meningitidisis a gram-negative bacterium that often asymptomatically colonizes the human nasopharyngeal tract. These bacteria cross the epithelial barrier can cause life-threatening sepsis and/or meningitis. Antimicrobial peptides are one of the first lines of defense against invading bacterial pathogens. Human beta-defensin 2 (hBD2) is an antimicrobial peptide with broad antibacterial activity, although its mechanism of action is poorly understood. Here, we investigated the effect of hBD2 onN. meningitidis. We showed that hBD2 binds to and kills actively growing meningococcal cells. The lethal effect was evident after 2 h incubation with the peptide, which suggests a slow killing mechanism. Further, the membrane integrity was not changed during hBD2 treatment. Incubation with lethal doses of hBD2 decreased the presence of diplococci; the number and size of bacterial microcolonies/aggregates remained constant, indicating that planktonic bacteria may be more susceptible to the peptide. Meningococcal DNA bound hBD2 in mobility shift assays and inhibited the lethal effect of hBD2 in a dose-dependent manner both in suspension and biofilms, supporting the interaction between hBD2 and DNA. Taken together, the ability of meningococcal DNA to bind hBD2 opens the possibility that extracellular DNA due to bacterial lysis may be a means ofN. meningitidisto evade immune defenses.

Biofilms form a complex layer with defined structures, that attach on biotic or abiotic surfaces, are tough to eradicate and tend to cause some resistance against most antibiotics. Several studies confirmed that biofilm-producing bacteria exhibit higher resistance compared to the planktonic form of the same species. Antibiotic resistance factors are well understood in planktonic bacteria which is not so in case of biofilm producing forms. This may be due to the lack of available drugs with known resistance mechanisms for biofilms. Existing antibiotics cannot eradicate most biofilms, especially of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). Insects produce complex and diverse set of chemicals for survival and defense. Antimicrobial peptides (AMPs), produced by most insects, generally have a broad spectrum of activity and the potential to bypass the resistance mechanisms of classical antibiotics. Besides, AMPs may well act synergistically with classical antibiotics for a double-pronged attack on infections. Thus, AMPs could be promising alternatives to overcome medically important biofilms, decrease the possibility of acquired resistance and treatment of multidrug-resistant pathogens including ESKAPE. The present review focuses on insect-derived AMPs with special reference to anti-biofilm-based strategies. It covers the AMP composition, pathways and mechanisms of action, the formation of biofilms, impact of biofilms on human diseases, current strategies as well as therapeutic options to combat biofilm with antimicrobial peptides from insects. In addition, the review also illustrates the importance of bioinformatics tools and molecular docking studies to boost the importance of select bioactive peptides those can be developed as drugs, as well as suggestions for further basic and clinical research.

Pine needles are used in several East Asian countries as food or traditional medicine. It contains functional components that exhibit a wide spectrum of pharmacological effects such as antioxidant, antimicrobial, anti-diabetic, and anti-inflammatory activities. We determined and characterized the novel antimicrobial peptides (AMPs) isolated from Pinus densiflora Sieb. et Zucc. The four active pine-needle (PN) peptides showed antimicrobial activity against foodborne bacteria with minimum inhibitory concentration (MIC) values within the range of 8–128 μg/ml. PN peptides showed no detectable hemolytic activity or cytotoxicity at the antimicrobial concentrations. The N-terminal amino acid sequence of the PN5 was identified using Edman degradation and Antimicrobial Peptide Database (APD) homology analysis showed that it was not identical to any other plant peptide. This suggests that PN5 can serve as an alternative therapeutic agent to be used in the food industry.

The world is facing a significant increase in infections caused by drug-resistant infectious agents. In response, various strategies have been recently explored to treat them, including the development of bacteriocins. Bacteriocins are a group of antimicrobial peptides produced by bacteria, capable of controlling clinically relevant susceptible and drug-resistant bacteria. Bacteriocins have been studied to be able to modify and improve their physicochemical properties, pharmacological effects, and biosafety. This manuscript focuses on the research being developed on the biosafety of bacteriocins, which is a topic that has not been addressed extensively in previous reviews. This work discusses the studies that have tested the effect of bacteriocins against pathogens and assess their toxicity using in vivo models, including murine and other alternative animal models. Thus, this work concludes the urgency to increase and advance the in vivo models that both assess the efficacy of bacteriocins as antimicrobial agents and evaluate possible toxicity and side effects, which are key factors to determine their success as potential therapeutic agents in the fight against infections caused by multidrug-resistant microorganisms.

Antimicrobial resistance is an increasing threat to global health and challenges the way we treat infections. Peptides containing the PCNA interacting motif APIM (APIM-peptides) were recently shown to bind to the bacterial PCNA homolog, the beta (β)-clamp, and to have both antibacterial and anti-mutagenic activities. In this study we explore the antibacterial effects of these peptides on Staphylococcus epidermidis, a bacterial species commonly found in prosthetic joint infections (PJI). Drug-resistant bacterial isolates from PJIs often lead to difficult-to-treat chronic infections. We show that APIM-peptides have a rapid bactericidal effect which when used at sublethal levels also increase the efficacy of gentamicin. In addition, APIM-peptides reduce development and eliminate already existing S. epidermidis biofilm. To study the potential use of APIM-peptides to prevent PJI, we used an in vivo bone graft model in rats where APIM-peptide, gentamicin, or a combination of the two was added to cement. The bone grafts containing cement with the combination was more effective than cement containing only gentamicin, which is the current standard of care. In summary, these results suggest that APIM-peptides can be a promising new drug candidate for anti-infective implant materials to use in the fight against resistant bacteria and chronic PJI.

Antimicrobial peptides (AMPs) or host defense peptides protect the host against various pathogens such as yeast, fungi, viruses and bacteria. AMPs also display immunomodulatory properties ranging from the modulation of inflammatory responses to the promotion of wound healing. More interestingly, AMPs cause cell disruption through non-specific interactions with the membrane surface of pathogens. This is most likely responsible for the low or limited emergence of bacterial resistance against many AMPs. Despite the increasing number of antibiotic-resistant bacteria and the potency of novel AMPs to combat such pathogens, only a few AMPs are in clinical use. Therefore, the current review describes (i) the potential of AMPs as alternatives to antibiotics, (ii) the challenges toward clinical implementation of AMPs and (iii) strategies to improve the success rate of AMPs in clinical trials, emphasizing the lessons we could learn from these trials.

Fusarium graminearum is the etiological agent of Fusarium head blight (FHB), a disease that produces a significant decrease in wheat crop yield and it is further aggravated by the presence of mycotoxins in the affected grains that may cause health problems to humans and animals. Plant defensins and defensin-like proteins are antimicrobial peptides (AMPs); they are small basic, cysteine-rich peptides (CRPs) ubiquitously expressed in the plant kingdom and mostly involved in host defence. They present a highly variable sequence but a conserved structure. The γ-core located in the C-terminal region of plant defensins has a conserved β-hairpin structure and is a well-known determinant of the antimicrobial activity among disulphide-containing AMPs. Another conserved motif of plant defensins is the α-core located in the N-terminal region, not conserved among the disulphide-containing AMPs, it has not been yet extensively studied. In this report, we have cloned the putative antimicrobial protein DefSm2, expressed in flowers of the wild plant Silybum marianum. The cDNA encodes a protein with two fused basic domains of an N-terminal defensin domain (DefSm2-D) and a C-terminal Arg-rich and Lys-rich domain. To further characterize the DefSm2-D domain, we built a 3D template-based model that will serve to support the design of novel antifungal peptides. We have designed four potential antifungal peptides: two from the DefSm2-D α-core region (SmAPα1-21 and SmAPα10-21) and two from the γ-core region (SmAPγ27-44 and SmAPγ29-35). We have chemically synthesized and purified the peptides and further characterized them by electrospray ionization mass spectrometry (ESI-MS) and Circular dichroism (CD) spectroscopy. SmAPα1-21, SmAPα10-21, and SmAPγ27-44 inhibited the growth of the phytopathogen F. graminearum at low micromolar concentrations. Conidia exposure to the fungicidal concentration of the peptides caused membrane permeabilization to the fluorescent probe propidium iodide (PI), suggesting that this is one of the main contributing factors in fungal cell killing. Furthermore, conidia treated for 0.5h showed cytoplasmic disorganization as observed by transmission electron microscopy (TEM). Remarkably, the peptides derived from the α-core induced morphological changes on the conidia cell wall, which is a promising target since its distinctive biochemical and structural organization is absent in plant and mammalian cells., Centro de Investigación de Proteínas Vegetales, Centro de Investigaciones en Fitopatología

The skin functions as a protective barrier to inhibit the entry of foreign pathogens, all the while hosting a diverse milieu of microorganisms. Over time, skin cells, immune cells, cytokines, and microbes interact to integrate the processes of maintaining the skin’s physical and immune barrier. In the present study, the basal expression of two immunologically divergent mouse strains C57BL/6 and BALB/c, as well as a strain on the C57 background lacking IL-6, was characterized. Additionally, cutaneous antimicrobial gene expression profiles and skin bacterial microbiome were assessed between strains. Total RNA sequencing was performed on untreated C57BL/6 (control), BALB/c, and IL-6-deficient skin samples and found over 3,400 genes differentially modulated between strains. It was found that each strain modulated its own transcriptional “profile” associated with skin homeostasis and also influenced the overall bacterial colonization as indicated by the differential phyla present on each strain. Together, these data not only provide a comprehensive view of the transcriptional changes in homeostatic skin of different mouse strains but also highlight the possible influence of the strain differences (e.g., Th1/Th2 balance) as well as a role for IL-6 in overall skin immunity and resident microbial populations.

Foodborne illness caused by consumption of food contaminated with Salmonella is one of the most common causes of diarrheal disease and affects millions of people worldwide. The rising emergence and spread of antimicrobial resistance, especially in some serotypes of Salmonella, has raised a great awareness of public health issues worldwide. To ensure safety of the food processing chain, the development of new food preservatives must be expedited. Recently, thermal- and pH-stable antimicrobial peptides have received much attention for use in food production, and represent safe alternatives to chemical preservatives. A 12-mer cathelicidin-derived, α-helical cationic peptide, P7, displayed rapid killing activity, against strains of drug-resistant foodborne Salmonella enterica serovar Typhimurium and its monophasic variant (S. enterica serovar 4,5,12:i:-) and had minimal toxicity against mouse fibroblast cells. P7 tended to form helical structure in the membrane-mimic environments as evaluated by circular dichroism (CD) spectroscopy. The action mode of P7 at the membrane-level was affirmed by the results of flow cytometry, and confocal, scanning and transmission electron microscopy. P7 killed bacteria through binding to bacterial membranes, penetration and the subsequent accumulation in S. enterica serovar Typhimurium cytoplasm. This induced membrane depolarization, permeabilization, and sequential leakage of intracellular substances and cell death. Except for sensitivity to proteolytic digestive enzymes, P7 maintained its inhibitory activity against S. enterica serovar Typhimurium in the presence of different conditions [various salts, extreme pHs and heat (even at 100°C)]. Moreover, the peptide is unlikely to induce bacterial resistance in vitro. Taken together, this study demonstrated that the membrane-permeabilizing P7 peptide has much potential as a new antimicrobial agent for use in food processing and preservation.

The plant microbiome is a vastly underutilized resource for identifying new genes and bioactive compounds. Here, we used Pseudomonas sp. EDOX, isolated from the leaf endosphere of a tomato plant grown on a small farm in the Netherlands. To get more insight into its biosynthetic potential, the genome of Pseudomonas sp. EDOX was sequenced and subjected to bioinformatic analyses. The genome sequencing analysis identified strain EDOX as a member of the Pseudomonas mediterranea. In silico analysis for secondary metabolites identified a total of five non-ribosomally synthesized peptides synthetase (NRPS) gene clusters, related to the biosynthesis of syringomycin, syringopeptin, anikasin, crochelin A, and fragin. Subsequently, we purified and characterized several cyclic lipopeptides (CLPs) produced by NRPS, including some of the already known ones, which have biological activity against several plant and human pathogens. Most notably, mass spectrometric analysis led to the discovery of two yet unknown CLPs, designated medipeptins, consisting of a 22 amino acid peptide moiety with varying degrees of activity against Gram-positive and Gram-negative pathogens. Furthermore, we investigated the mode of action of medipeptin A. The results show that medipeptin A acts as a bactericidal antibiotic against Gram-positive pathogens, but as a bacteriostatic antibiotic against Gram-negative pathogens. Medipeptin A exerts its potent antimicrobial activity against Gram-positive bacteria via binding to both lipoteichoic acid (LTA) and lipid II as well as by forming pores in membranes. Collectively, our study provides important insights into the biosynthesis and mode of action of these novel medipeptins from P. mediterranea EDOX.

Antimicrobial peptides (AMPs) are essential effectors of the host innate immune system and they represent promising molecules for the treatment of multidrug resistant microbes. A better understanding of microbial resistance to these defense peptides is thus prerequisite for the control of infectious diseases. Here, using a random mutagenesis approach, we identify the fliK gene, encoding an internal molecular ruler that controls flagella hook length, as an essential element for Bacillus thuringiensis resistance to AMPs in Drosophila. Unlike its parental strain, that is highly virulent to both wild-type and AMPs deficient mutant flies, the fliK deletion mutant is only lethal to the latter’s. In agreement with its conserved function, the fliK mutant is non-flagellated and exhibits highly compromised motility. However, comparative analysis of the fliK mutant phenotype to that of a fla mutant, in which the genes encoding flagella proteins are interrupted, indicate that B. thuringiensis FliK-dependent resistance to AMPs is independent of flagella assembly. As a whole, our results identify FliK as an essential determinant for B. thuringiensis virulence in Drosophila and provide new insights on the mechanisms underlying bacteria resistance to AMPs.

Amphipathicity has traditionally been considered to be essential for the de novo design or systematic optimization of antimicrobial peptides (AMPs). However, the current research methods to study the relationship between amphiphilicity and antimicrobial activity are inappropriate, because the key parameters (hydrophobicity, positive charge, etc.) and secondary structure of AMPs are changed. To systematically and accurately study the effects of amphiphilicity on antimicrobial properties of AMPs, we designed parallel series of AMPs with a different order of amino acids in a sequence composed only of Arg and either Trp (WR series) or Leu (LR series), under conditions in which other vital parameters were fixed. Furthermore, based on the WR and LR peptides that can form stable amphiphilic β-sheet structures in the anionic membrane-mimetic environment, we found that high β-sheet amphipathic was accompanied by strong antimicrobial activity. Of such peptides, W5 ([RW]4W) and L5 ([RL]4L) with a nicely amphipathic β-sheet structure possessed the optimal therapeutic index. W5 and L5 also exhibited high stability in vitro and a potent membrane-disruptive mechanism. These results suggest that the alternate arrangement of hydrophobic and hydrophilic residues to form a stable amphipathic β-sheet structure is an essential factor that significantly affects the antimicrobial properties.

A new haloalkaliphilic species of Wenzhouxiangella, strain AB-CW3, was isolated from a system of hypersaline alkaline soda lakes in the Kulunda Steppe using cells of Staphylococcus aureus as growth substrate. AB-CW3’s complete, circular genome was assembled from combined nanopore and Illumina sequencing and its proteome was determined for three different experimental conditions. AB-CW3 is an aerobic gammaproteobacterium feeding mainly on proteins and peptides. Unique among Wenzhouxiangella, it uses a flagellum for motility, fimbria for cell attachment and is capable of complete denitrification. AB-CW3 can use proteins derived from living or dead cells of Staphylococcus and other Gram-positive bacteria as the carbon and energy source. It encodes and expresses production of a novel Lantibiotic, a class of antimicrobial peptides which have so far only been found to be produced by Gram-positive bacteria. AB-CW3 likely excretes this peptide via a type I secretion system encoded upstream of the genes for production of the Lanthipeptide. Comparison of AB-CW3’s genome to 18 other Wenzhouxiangella genomes from marine, hypersaline, and soda lake habitats indicated one or two transitions from marine to soda lake environments followed by a transition of W. marina back to the oceans. Only 19 genes appear to set haloalkaliphilic Wenzhouxiangella apart from their neutrophilic relatives. As strain AB-CW3 is only distantly related to other members of the genus, we propose to provisionally name it “Wenzhouxiangella alkaliphila”.

The transition of antimicrobial peptides (AMPs) from the laboratory to market has been severely hindered by their instability toward proteases in biological systems. In the present study, we synthesized derivatives of the cationic AMP Pep05 (KRLFKKLLKYLRKF) by substituting L-amino acid residues with D- and unnatural amino acids, such as D-lysine, D-arginine, L-2,4-diaminobutanoic acid (Dab), L-2,3-diaminopropionic acid (Dap), L-homoarginine, 4-aminobutanoic acid (Aib), and L-thienylalanine, and evaluated their antimicrobial activities, toxicities, and stabilities toward trypsin, plasma proteases, and secreted bacterial proteases. In addition to measuring changes in the concentration of the intact peptides, LC-MS was used to identify the degradation products of the modified AMPs in the presence of trypsin and plasma proteases to determine degradation pathways and examine whether the amino acid substitutions afforded improved proteolytic resistance. The results revealed that both D- and unnatural amino acids enhanced the stabilities of the peptides toward proteases. The derivative DP06, in which all of the L-lysine and L-arginine residues were replaced by D-amino acids, displayed remarkable stability and mild toxicity in vitro but only slight activity and severe toxicity in vivo, indicating a significant difference between the in vivo and in vitro results. Unexpectedly, we found that the incorporation of a single Aib residue at the N-terminus of compound UP09 afforded remarkably enhanced plasma stability and improved activity in vivo. Hence, this derivative may represent a candidate AMP for further optimization, providing a new strategy for the design of novel AMPs with improved bioavailability.

Lantibiotics are a growing class of antimicrobial peptides, which possess antimicrobial activity against mainly Gram-positive bacteria including the highly resistant strains such as methicillin-resistant Staphylococcus aureus or vancomycin-resistant enterococci. In the last decades numerous lantibiotics were discovered in natural habitats or designed with bioengineering tools. In this study, we present an insight in the antimicrobial potential of the natural occurring lantibiotic nisin H from Streptococcus hyointestinalis as well as the variant nisin H F1I. We determined the yield of the heterologously expressed peptide and quantified the cleavage efficiency employing the nisin protease NisP. Furthermore, we analyzed the effect on the modification via mass spectrometry analysis. With standardized growth inhibition assays we benchmarked the activity of pure nisin H and the variant nisin H F1I, and their influence on the activity of the nisin immunity proteins NisI and NisFEG from Lactococcus lactis and the nisin resistance proteins SaNSR and SaNsrFP from Streptococcus agalactiae COH1. We further checked the antibacterial activity against clinical isolates of Staphylococcus aureus, Enterococcus faecium and Enterococcus faecalis via microdilution method. In summary, nisin H and the nisin H F1I variant possessed better antimicrobial potency than the natural nisin A.

Antimicrobial peptides (AMPs) are a class of small peptides that widely exist in nature and they are an important part of the innate immune system of different organisms. AMPs have a wide range of inhibitory effects against bacteria, fungi, parasites and viruses. The emergence of antibiotic-resistant microorganisms and the increasing of concerns about the use of antibiotics resulted in the development of AMPs, which have a good application prospect in medicine, food, animal husbandry, agriculture and aquaculture. This review introduces the progress of research on AMPs comprehensively and systematically, including their classification, mechanism of action, design methods, environmental factors affecting their activity, application status, prospects in various fields and problems to be solved. The research progress on antivirus peptides, especially anti-coronavirus (COVID-19) peptides, has been introduced given the COVID-19 pandemic worldwide in 2020.

Pathogenic microorganisms and their chronic pathogenicity are significant concerns in biomedical research. Biofilm-linked persistent infections are not easy to treat due to resident multidrug-resistant microbes. Low efficiency of various treatments and in vivo toxicity of available antibiotics drive the researchers toward the discovery of many effective natural anti-biofilm agents. Natural extracts and natural product-based anti-biofilm agents are more efficient than the chemically synthesized counterparts with lesser side effects. The present review primarily focuses on various natural anti-biofilm agents, i.e., phytochemicals, biosurfactants, antimicrobial peptides, and microbial enzymes along with their sources, mechanism of action via interfering in the quorum-sensing pathways, disruption of extracellular polymeric substance, adhesion mechanism, and their inhibitory concentrations existing in literature so far. This study provides a better understanding that a particular natural anti-biofilm molecule exhibits a different mode of actions and biofilm inhibitory activity against more than one pathogenic species. This information can be exploited further to improve the therapeutic strategy by a combination of more than one natural anti-biofilm compounds from diverse sources.

With the alarming increase of infections caused by pathogenic multidrug-resistant bacteria over the last decades, antimicrobial peptides (AMPs) have been investigated as a potential treatment for those infections, directly through their lytic effect or indirectly, due to their ability to modulate the immune system. There are still concerns regarding the use of such molecules in the treatment of infections, such as cell toxicity and host factors that lead to peptide inhibition. To overcome these limitations, different approaches like peptide modification to reduce toxicity and peptide combinations to improve therapeutic efficacy are being tested. Human defense peptides consist of an important part of the innate immune system, against a myriad of potential aggressors, which have in turn developed different ways to overcome the AMPs microbicidal activities. Since the antimicrobial activity of AMPs vary between Gram-positive and Gram-negative species, so do the bacterial resistance arsenal. This review discusses the mechanisms exploited by Gram-positive bacteria to circumvent killing by antimicrobial peptides. Specifically, the most clinically relevant genera, Streptococcus spp., Staphylococcus spp., Enterococcus spp. and Gram-positive bacilli, have been explored.

Antimicrobial peptides (AMPs) are essential effectors of the host innate immune system and they represent promising molecules for the treatment of multidrug resistant microbes. A better understanding of microbial resistance to these defense peptides is thus prerequisite for the control of infectious diseases. Here, using a random mutagenesis approach, we identify the fliK gene, encoding an internal molecular ruler that controls flagella hook length, as an essential element for Bacillus thuringiensis resistance to AMPs in Drosophila. Unlike its parental strain, that is highly virulent to both wild-type and AMPs deficient mutant flies, the fliK deletion mutant is only lethal to the latter’s. In agreement with its conserved function, the fliK mutant is non-flagellated and exhibits highly compromised motility. However, comparative analysis of the fliK mutant phenotype to that of a fla mutant, in which the genes encoding flagella proteins are interrupted, indicate that B. thuringiensis FliK-dependent resistance to AMPs is independent of flagella assembly. As a whole, our results identify FliK as an essential determinant for B. thuringiensis virulence in Drosophila and provide new insights on the mechanisms underlying bacteria resistance to AMPs.

Broad-spectrum antimicrobial peptides (AMPs) kill bacteria indiscriminately, increasing the possibility of an ecological imbalance in the microbiota. To solve this problem, new types of AMPs, which kill pathogenic bacteria without breaking the micro-ecological balance of the body, were proposed. Here, we successfully designed a targeting AMP, S2, which is a fusion peptide composed of a species-specific targeting domain and broad-spectrum AMP domain. In the current study, S2 showed specific killing activity against Staphylococcus aureus, and almost no resistance induced compared to penicillin. Mechanism studies indicated that S2 killed S. aureus by destroying the bacterial membrane. Meanwhile, S2 possessed excellent salt-tolerance properties and biocompatibility. Importantly, S2 exhibited perfect treatment efficacy against an S. aureus subcutaneous infection model and remained nontoxic. In conclusion, this study provides a promising strategy for designing specific AMPs against growing bacterial infections.

Antimicrobial peptides (AMPs) play pivotal roles in protecting against microbial infection in fish. However, AMPs from topmouth culter (Erythroculter ilishaeformis) are rarely known. In our study, we isolated an AMP from the head kidney of topmouth culter, which belonged to liver-expressed antimicrobial peptide 2 (LEAP-2) family. Topmouth culter LEAP-2 showed inhibitory effects on aquatic bacterial growth, including antibiotic-resistant bacteria, with minimal inhibitory concentration values ranging from 18.75 to 150 μg/ml. It was lethal forAeromonas hydrophila(resistant to ampicillin), and took less than 60 min to killA. hydrophilaat a concentration of 5 × MIC. Scanning electron microscope (SEM) and SYTOX Green uptake assay indicated that it impaired the integrity of bacterial membrane by eliciting pore formation, thereby increasing the permeabilization of bacterial membrane. In addition, it showed none inducible drug resistance to aquatic bacteria. Interestingly, it efficiently delayed ampicillin-induced drug resistance inVibrio parahaemolyticus(sensitive to ampicillin) and sensitized ampicillin-resistant bacteria to ampicillin. The chequerboard assay indicated that topmouth culter LEAP-2 generated synergistic effects with ampicillin, indicating the combinational usage potential of topmouth culter LEAP-2 with antibiotics. As expected, topmouth culter LEAP-2 significantly alleviated ampicillin-resistantA. hydrophilainfectionin vivo, and enhanced the therapeutic efficacy of ampicillin againstA. hydrophila in vivo. Our findings provide a fish innate immune system-derived peptide candidate for the substitute of antibiotics and highlight its potential for application in antibiotic-resistant bacterial infection in aquaculture industry.

Studies of rumen microbial ecology suggest that the capacity to produce antimicrobial peptides could be a useful trait in species competing for ecological niches in the ruminal ecosystem. However, little is known about the synthesis of lasso peptides by ruminal microorganisms. Here we analyzed the distribution and diversity of lasso peptide gene clusters in 425 bacterial genomes from the rumen ecosystem. Genome mining was performed using antiSMASH 5, BAGEL4, and a database of well-known precursor sequences. The genomic context of the biosynthetic clusters was investigated to identify putative lasA genes and protein sequences from enzymes of the biosynthetic machinery were evaluated to identify conserved motifs. Metatranscriptome analysis evaluated the expression of the biosynthetic genes in the rumen microbiome. Several incomplete (n = 23) and complete (n = 11) putative lasso peptide clusters were detected in the genomes of ruminal bacteria. The complete gene clusters were exclusively found within the phylum Firmicutes, mainly (48%) in strains of the genus Butyrivibrio. The analysis of the genetic organization of complete putative lasso peptide clusters revealed the presence of co-occurring genes, including kinases (85%), transcriptional regulators (49%), and glycosyltransferases (36%). Moreover, a conserved pattern of cluster organization was detected between strains of the same genus/species. The maturation enzymes LasB, LasC, and LasD showed regions highly conserved, including the presence of a transglutaminase core in LasB, an asparagine synthetase domain in LasC, and an ABC-type transporter system in LasD. Phylogenetic trees of the essential biosynthetic proteins revealed that sequences split into monophyletic groups according to their shared single common ancestor. Metatranscriptome analyses indicated the expression of the lasso peptides biosynthetic genes within the active rumen microbiota. Overall, our in silico screening allowed the discovery of novel biosynthetic gene clusters in the genomes of ruminal bacteria and revealed several strains with the genetic potential to synthesize lasso peptides, suggesting that the ruminal microbiota represents a potential source of these promising peptides.