Enterohaemorrhagic Escherichia coli produces outer membrane vesicles as an active defence system against antimicrobial peptide LL‐37

Antimicrobial peptides (AMPs) are important components of the innate immune system. Enterohaemorrhagic Escherichia coli (EHEC), a food‐borne pathogen causing serious diarrheal diseases, must overcome attack by AMPs. Here, we show that resistance of EHEC against human cathelicidin LL‐37, a primary AMP, was enhanced by butyrate, which has been shown to act as a stimulant for the expression of virulence genes. The increase of resistance depended on the activation of the ompT gene, which encodes the outer membrane protease OmpT for LL‐37. The expression of the ompT gene was enhanced through the activation system for virulence genes. The increase in ompT expression did not result in an increase in OmpT protease in bacteria but in enhancement of the production of OmpT‐loaded outer membrane vesicles (OMVs), which primarily contributed to the increase in LL‐37‐resistance. Furthermore, a sublethal dosage of LL‐37 stimulated the production of OMVs. Finally, we showed that OMVs produced by OmpT‐positive strains protect the OmpT‐negative strain, which is susceptible to LL‐37 by itself more efficiently than OMVs from the ompT mutant. These results indicate that EHEC enhances the secretion of OmpT‐loaded OMVs in coordination with the activation of virulence genes during infection and blocks bacterial cell attack by LL‐37.     

Short KR‐12 analogs designed from human cathelicidin LL‐37 possessing both antimicrobial and antiendotoxic activities without mammalian cell toxicity

KR‐12 (residues 18–29 of LL‐37) was known to be the smallest peptide of human cathelicidin LL‐37 possessing antimicrobial activity. In order to optimize α‐helical short antimicrobial peptides having both antimicrobial and antiendotoxic activities without mammalian cell toxicity, we designed and synthesized a series of KR‐12 analogs. Highest hydrophobic analogs KR‐12‐a5 and KR‐12‐a6 displayed greater inhibition of lipopolysaccharide (LPS)‐stimulated tumor necrosis factor‐α production and higher LPS‐binding activity. We have observed that antimicrobial activity is independent of charge, but LPS neutralization requires a balance of hydrophobicity and net positive charge. Among KR‐12 analogs, KR‐12‐a2, KR‐12‐a3 and KR‐12‐a4 showed much higher cell specificity for bacteria over erythrocytes and retained antiendotoxic activity, relative to parental LL‐37. KR‐12‐a5 displayed the strongest antiendotoxic activity but almost similar cell specificity as compared with LL‐37. Also, these KR‐12 analogs (KR‐12‐a2, KR‐12‐a3, KR‐12‐a4 and KR‐12‐a5) exhibited potent antimicrobial activity (minimal inhibitory concentration: 4 μM) against methicillin‐resistant Staphylococcus aureus. Taken together, these KR‐12 analogs have the potential for future development as a novel class of antimicrobial and anti‐inflammatory therapeutic agents. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Antimicrobial activity,bactericidal mechanism and LPS‐neutralizing activity of the cell‐penetrating peptide pVEC and its analogs

pVEC is a cell‐penetrating peptide derived from the murine vascular endothelial‐cadherin protein. To evaluate the potential of pVEC as antimicrobial peptide (AMP), we synthesized pVEC and its analogs with Trp and Arg/Lys substitution, and their antimicrobial and lipopolysaccharide (LPS)‐neutralizing activities were investigated. pVEC and its analogs displayed a potent antimicrobial activity (minimal inhibitory concentration: 4–16 μM) against Gram‐positive and Gram‐negative bacteria but no or less hemolytic activity (less than 10% hemolysis) even at a concentration of 200 μM. These peptides induced a near‐complete membrane depolarization (more than 80%) at 4 μM against Staphylococcus aureus and a significant dye leakage (35–70%) from bacterial membrane‐mimicking liposome at a concentration as low as 1 μM. The fluorescence profiles of pVEC and its analogs in dye leakage from liposome and membrane depolarization were similar to those of a frog‐derived AMP, magainin 2. These results suggest that pVEC and its analogs kill bacteria by forming a pore or ion channel in the cytoplasmic membrane. pVEC and its analogs significantly inhibited nitric oxide production or tumor necrosis factor‐α release in LPS‐stimulated mouse macrophage RAW264.7 cells at 10 to 50 μM, in which RAW264.7 were not damaged. Taken together, our results suggest that pVEC and its analogs with potent antimicrobial and LPS‐neutralizing activities can serve as AMPs for the treatment of microbial infection and sepsis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Absence of in vitro innate immunomodulation by insect‐derived short proline‐rich antimicrobial peptides points to direct antibacterial action in vivo

Some antimicrobial peptides (AMPs) have been described to exert immunomodulatory effects, which may contribute to their in vivo antibacterial activity. Very recently, we could show that novel oncocin and apidaecin derivatives are potently antibacterially active in vivo. Therefore, we studied oncocin and apidaecin derivatives for their effects on murine dendritic cells (DC) and macrophages and compared them with well‐known immunomodulatory activities of murine cathelicidin‐related antimicrobial peptide (CRAMP). To characterize the immunomodulatory activity of the peptides on key cells of the innate immune system, we stimulated murine DC and macrophages with the oncocin and apidaecin derivatives alone, or in combination with lipopolysaccharide (LPS). We analyzed the secretion of pro‐inflammatory cytokines, the expression of surface activation markers, and the chemotactic activity of the AMPs. In contrast to LPS, none of the oncocin and apidaecin derivatives alone has an influence on cytokine or surface marker expression by DC and macrophages. Furthermore, the tested oncocin and apidaecin derivatives do not modulate the immune response after LPS stimulation, whereas CRAMP shows a reduction of the LPS‐mediated immune response as expected. All peptides tested are not chemotactic for DC. Together, lack of in vitro immunomodulatory effects by oncocin and apidaecin derivatives on key cells of the innate murine immune system suggests that their potent in vivo antibacterial activity relies on a direct antibacterial effect. This will simplify further pharmaceutical investigation and development of insect peptides as therapeutic compounds against bacterial infections. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Prokaryotic selectivity and LPS-neutralizing activity of short antimicrobial peptides designed from the human antimicrobial peptide LL-37

To develop novel antimicrobial peptides (AMPs) with shorter lengths, improved prokaryotic selectivity and retained lipolysaccharide (LPS)-neutralizing activity compared to human cathelicidin AMP, LL-37, a series of amino acid-substituted analogs based on IG-19 (residues 13-31 of LL-37) were synthesized. Among the IG-19 analogs, the analog a4 showed the highest prokaryotic selectivity, but much lower LPS-neutralizing activity compared to parental LL-37. The analogs, a5, a6, a7 and a8 with higher hydrophobicity displayed LPS-neutralizing activity comparable to that of LL-37, but much lesser prokaryotic selectivity. These results indicate that the proper hydrophobicity of the peptides is crucial to exert the amalgamated property of LPS-neutralizing activity and prokaryotic selectivity. Furthermore, to increase LPS-neutralizing activity of the analog a4 without a remarkable decrease in prokaryotic selectivity, we synthesized Trp-substituted analogs (a4-W1 and a4-W2), in which Phe(5) or Phe(15) of a4 is replaced by Trp. Despite their same prokaryotic selectivity, a4-W2 displayed much higher LPS-neutralizing activity compared to a4-W1. When compared with parental LL-37, a4-W2 showed retained LPS-neutralizing activity and 2.8-fold enhanced prokaryotic selectivity. These results suggest that the effective site for Trp-substitution when designing novel AMPs with higher LPS-neutralizing activity, without a remarkable reduction in prokaryotic selectivity, is the amphipathic interface between the end of the hydrophilic side and the start of the hydrophobic side rather than the central position of the hydrophobic side in their α-helical wheel projection. Taken together, the analog a4-W2 can serve as a promising template for the development of therapeutic agents for the treatment of endotoxic shock and bacterial infection.     

Enhancement of the anti‐inflammatory activity of temporin‐1Tl‐derived antimicrobial peptides by tryptophan,arginine and lysine substitutions

Temporin‐1Tl (TL) is a 13‐residue frog antimicrobial peptide (AMP) exhibiting potent antimicrobial and anti‐inflammatory activity. To develop novel AMP with improved anti‐inflammatory activity and antimicrobial selectivity, we designed and synthesized a series of TL analogs by substituting Trp, Arg and Lys at selected positions. Except for Escherichia coli and Staphylococcus epidermidis, all TL analogs exhibited retained or increased antimicrobial activity against seven bacterial strains including three methicillin‐resistant Staphylococcus aureus strains compared with TL. TL‐1 and TL‐4 showed a little increase in antimicrobial selectivity, while TL‐2 and TL‐3 displayed slightly decreased antimicrobial selectivity because of their about twofold increased hemolytic activity. All TL analogs demonstrated greatly increased anti‐inflammatory activity, evident by their higher inhibition of the production tumor necrosis factor‐α (TNF‐α) and nitric oxide and the mRNA expression of inducible nitric oxide synthase and TNF‐α in lipopolysaccharide (LPS)‐stimulated RAW264.7 macrophage cells, compared with TL. Taken together, the peptide anti‐inflammatory activity is as follows: TL‐2 ≈ TL‐3 ≈ TL‐4 > TL‐1 > TL. In addition, LPS binding ability of the peptides corresponded with their anti‐inflammatory activity. These results apparently suggest that the anti‐inflammatory activity of TL analogs is associated with the direct binding ability between these peptides and LPS. Collectively, our designed TL analogs possess improved anti‐inflammatory activity and retain antimicrobial activity without a significant increase in hemolysis. Therefore, it is evident that our TL analogs constitute promising candidates for the development of peptide therapeutics for gram‐negative bacterial infection. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

New Antibacterial Thiodiketopiperazines from the Deep Sea Sediment‐Derived Fungus Epicoccum nigrum SD‐388

Two new antibacterial thiodiketopiperazine derivatives (TDKPs), 7‐dehydroxyepicoccin H and 7‐hydroxyeutypellazine F, along with seven known TDKP analogs, were isolated and identified from Epicoccum nigrum SD‐388, a deep‐sea‐sediment‐derived fungus. The structures of these compounds were elucidated on the basis of detailed spectroscopic analysis. The absolute configuration of 7‐dehydroxyepicoccin H was established by X‐ray crystallographic analysis, while 7‐hydroxyeutypellazine F was determined by ECD experiment and TDDFT‐ECD calculation. The antibacterial activities against human and aquatic pathogens were evaluated. 7‐Dehydroxyepicoccin H and 7‐hydroxyeutypellazine F displayed inhibitory activities against aquatic pathogens Vibrio vulnificus, V. alginolyticus, and Edwardsiella tarda, with MIC values ranging from 4.0 to 8.0 μg/mL.     

WIPF2 promotes Shigella flexneri actin‐based motility and cell‐to‐cell spread

Shigella flexneri is an intracellular pathogen that disseminates in colonic epithelial cells through actin‐based motility and formation of membrane protrusions at cell–cell contacts, that project into adjacent cells and resolve into vacuoles, from which the pathogen escapes, thereby achieving cell‐to‐cell spread. Actin nucleation at the bacterial pole relies on the recruitment of the nucleation‐promoting factor N‐WASP, which activates the actin nucleator ARP2/3. In cells, the vast majority of N‐WASP exists as a complex with WIP. The involvement of WIP in N‐WASP‐dependent actin‐based motility of various pathogens, including vaccinia virus and S. flexneri, has been highly controversial. Here, we show that WIPF2 was the only WIP family member expressed in the human colonic epithelial cell line HT‐29, and its depletion impaired S. flexneri dissemination. WIPF2 depletion increased the number of cytosolic bacteria lacking actin tails (non‐motile) and decreased the velocity of motile bacteria. This correlated with a decrease in the recruitment of N‐WASP to the bacterial pole, and among N‐WASP‐positive bacteria, a decrease in actin tail‐positive bacteria, suggesting that WIPF2 is required for N‐WASP recruitment and activation at the bacterial pole. In addition, when motile bacteria formed protrusions, WIPF2 depletion decreased the number of membrane protrusions that successfully resolved into vacuoles.     

Antimicrobial activity of human α‐defensin 6 analogs: insights into the physico‐chemical reasons behind weak bactericidal activity of HD6 in vitro

Human α‐defensin 6 (HD6), unlike other mammalian defensins, does not exhibit bactericidal activity, particularly against aerobic bacteria. Monomeric HD6 has a tertiary structure similar to other α‐defensins in the crystalline state. However, the physico‐chemical reasons behind the lack of antibacterial activity of HD6 are yet to be established unequivocally. In this study, we have investigated the antimicrobial activity of HD6 analogs. A linear analog of HD6, in which the distribution of arginine residues was similar to active α‐defensins, shows broad‐spectrum antimicrobial activity, indicating that atypical distribution of arginine residues contributes to the inactivity of HD6. Peptides spanning the N‐terminal cationic segment were active against a wide range of organisms. Antimicrobial potency of these shorter analogs was further enhanced when myristic acid was conjugated at the N‐terminus. Cytoplasmic localization of the analogs without fatty acylation was observed to be necessary for bacterial killing, while they exhibited fungicidal activity by permeabilizing Candida albicans membranes. Myristoylated analogs and the linear full‐length arginine analog exhibited activity by permeabilizing bacterial and fungal membranes. Our study provides insights into the lack of bactericidal activity of HD6 against aerobic bacteria. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Evaluation of free or anchored antimicrobial peptides as candidates for the prevention of orthopaedic device‐related infections

The prevention of implant‐associated infection, one the most feared complications in orthopaedic surgery, remains a major clinical challenge and urges development of effective methods to prevent bacterial colonization of implanted devices. Alpha‐helical antimicrobial peptides (AMPs) may be promising candidates in this respect due to their potent and broad‐spectrum antimicrobial activity, their low tendency to elicit resistance and possible retention of efficacy in the immobilized state. The aim of this study was to evaluate the potential of five different helical AMPs, the cathelicidins BMAP‐27 and BMAP‐28, their (1–18) fragments and the rationally designed, artificial P19(9/G7) peptide, for the prevention of orthopaedic implant infections. Peptides were effective at micromolar concentrations against 22 Staphylococcus and Streptococcus isolates from orthopaedic infections, while only BMAP‐28 and to a lesser extent BMAP‐27 were active against Enterococcus faecalis. Peptides in solution showed activities comparable to those of cefazolin and linezolid, on a molar basis, and also a variable capacity to neutralize bacterial lipopolysaccharide, while devoid of adverse effects on MG‐63 osteoblast cells at concentrations corresponding to the MIC. The (1–18) BMAP fragments and P19(9/G7) were selected for further examination, based on better selectivity indices, and showed effectiveness in the presence of hyaluronic acid and in synovial fluid, while human serum affected their activity to variable extents, with BMAP‐27(1–18) best retaining activity. This peptide was immobilized on streptavidin–resin beads and retained activity against reference Staphylococcus epidermidis and Staphylococcus aureus strains, with negligible toxicity towards osteoblasts, underlining its potential for the development of infection‐resistant biomaterials for orthopaedic application. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

In vitro activity of novel in silico‐developed antimicrobial peptides against a panel of bacterial pathogens

Antimicrobial‐peptide‐based therapies could represent a reliable alternative to overcome antibiotic resistance, as they offer potential advantages such as rapid microbicidal activity and multiple activities against a broad spectrum of bacterial pathogens. Three synthetic antimicrobial peptides (AMPs), AMP72, AMP126, and also AMP2041, designed by using ad hoc screening software developed in house, were synthesized and tested against nine reference strains. The peptides showed a partial β‐sheet structure in 10‐mM phosphate buffer. Low cytolytic activity towards both human cell lines (epithelial, endothelial, and fibroblast) and sheep erythrocytes was observed for all peptides. The antimicrobial activity was dose dependent with a minimum bactericidal concentration (MBC) ranging from 0.17 to 10.12 μM (0.4–18.5 µg/ml) for Gram‐negative and 0.94 to 20.65 μM (1.72‐46.5 µg/ml) for Gram‐positive bacteria. Interestingly, in high‐salt environment, the antibacterial activity was generally maintained for Gram‐negative bacteria. All peptides achieved complete bacterial killing in 20 min or less against Gram‐negative bacteria. A linear time‐dependent membrane permeabilization was observed for the tested peptides at 12.5 µg/ml. In a medium containing Mg²⁺ and Ca²⁺, the peptide combination with EDTA restores the antimicrobial activity particularly for AMP2041. Moreover, in combination with anti‐infective agents (quinolones or aminoglycosides) known to bind divalent cation, AMP126 and AMP2041 showed additive activity in comparison with colistin. Our results suggest the following: (i) there is excellent activity against Gram‐negative bacteria, (ii) there is low cytolytic activity, (iii) the presence of a chelating agent restores the antimicrobial activity in a medium containing Mg²⁺ and Ca²⁺, and (iv) the MBC value of the combination AMPs–conventional antibiotics was lower than the MBC of single agents alone. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Antibacterial potential of Forsythia suspensa polysaccharide against resistant Enterobacter cloacae with SHV‐12 extended‐spectrum β‐lactamase (ESBL)

In this study, a homogenous polysaccharide (FSP), with an average molecular weight of 9.08 × 10⁴ Da, was isolated from Forsythia suspense and its antibacterial potential against Enterobacter cloacae producing SHV‐12 ESBL was investigated. Growth kinetics, in vitro competition and biofilm formation experiments demonstrated that SHV‐12 ESBL contributed to a fitness benefit to E cloacae strain. The antibacterial activity of FSP (2.5, 5.0 and 10.0 μg/mL) was tested against E cloacae bearing SHV‐12 ESBL gene using bacterial sensitivity, agar bioassay and agar well diffusion assays. It was found that the addition of FSP demonstrated potent antibacterial activities against this bacterial as showed by the decrease of bacterial growth and the increase of the inhibition zone diameter. Furthermore, SHV‐12 ESBL gene expression was decreased in E cloacae strain following different FSP treatment in a concentration‐dependent manner. In conclusion, these data showed that FSP exhibited potent good antibacterial activity against E cloacae producing SHV‐12 ESBL via inhibition of SHV‐12 ESBL gene expression, which may promote the development of novel natural antibacterial agents to treat infections caused by this drug‐resistant bacterial pathogen.     

Graphene oxide/silver nanohybrid: Optimization,antibacterial activity and its impregnation on bacterial cellulose as a potential wound dressing based on GO‐Ag nanocomposite‐coated BC

Recently, bacterial cellulose (BC) based wound dressing have raised significant interests in medical fields. However, to our best knowledge, it is apparent that the BC itself has no antibacterial activity. In this study, we optimized graphene oxide‐silver (GO‐Ag) nanohybrid synthesis using Response Surface Methodology and impregnate it to BC and carefully investigate their antibacterial activities against both the Gram‐negative bacteria Escherichia coli and the Gram‐positive bacteria Staphylococcus aureus. We discover that, compared to silver nanoparticles, GO‐Ag nanohybrid with an optimal GO suspension's pH and ratio is much more effective and shows synergistically enhanced, strong antibacterial activities at rather low dose. The GO‐Ag nanohybrid is more toxic to E. coli than that to S. aureus. The antibacterial and mechanical properties of BC/GO‐Ag composite are further investigated.     

Computational study of solution behavior of magainin 2 monomers

Antimicrobial peptides (AMPs) play crucial role as mediators of the primary host defense against microbial invasion. They are considered a promising alternative to antibiotics for multidrug resistant bacterial strains. For complete understanding of the antimicrobial defense mechanism, a detailed knowledge of the dynamics of peptide-membrane interactions, including atomistic studies on AMPs geometry and both peptide and membrane structural changes during the whole process is a prerequisite. We aim at clarifying the conformation dynamics of small linear AMPs in solution as a first step of in silico protocol for establishing a correspondence between certain amino-acid sequence motifs, secondary-structure elements, conformational dynamics in solution and the intensity and mode of interaction with the bacterial membrane. To this end, we use molecular dynamics simulations augmented by well-tempered metadynamics to study the free-energy landscape of two AMPs with close primary structure and different antibacterial activity – the native magainin 2 (MG2) and an analog (MG2m, with substitutions F5Y and F16W) in aqueous solution. We observe that upon solvation, the initial α-helical structures change differently. The native form remains structured, with three shorter α-helical motifs, connected by random coils, while the synthetic analog tends predominantly to a disordered conformation. Our results indicate the importance of the side-chains at positions 5 and 16 for maintaining the solvated peptide conformation. They also provide a modeling background for recent experimental observations, relating the higher α-helical content in solution (peptide pre-folding) in the case of small linear AMPs to a lower antibacterial activity.     

Effects of arginine and leucine substitutions on anti‐endotoxic activities and mechanisms of action of cationic and amphipathic antimicrobial octadecapeptide from rice α‐amylase

Previously, we showed that the antimicrobial cationic and amphipathic octadecapeptide AmyI‐1‐18 from rice α‐amylase (AmyI‐1) inhibited the endotoxic activity of lipopolysaccharide (LPS) from Escherichia coli. In addition, we demonstrated that several AmyI‐1‐18 analogs containing arginine or leucine substitutions, which were designed on the basis of the helical wheel projection of AmyI‐1‐18, exhibited higher antimicrobial activity against human pathogenic microorganisms than AmyI‐1‐18. In the present study, anti‐inflammatory (anti‐endotoxic) activities of five AmyI‐1‐18 analogs containing arginine or leucine substitutions were investigated. Two single arginine‐substituted and two single leucine‐substituted AmyI‐1‐18 analogs inhibited the production of LPS‐induced nitric oxide in mouse macrophages (RAW264) more effectively than AmyI‐1‐18. These data indicate that enhanced cationic and hydrophobic properties of AmyI‐1‐18 are associated with improved anti‐endotoxic activity. In subsequent chromogenic Limulus amebocyte lysate assays, 50% inhibitory concentrations (IC₅₀) of the three AmyI‐1‐18 analogs (G12R, D15R, and E9L) were 0.11–0.13 μm , indicating higher anti‐endotoxic activity than that of AmyI‐1‐18 (IC_50, 0.22 μm ), and specific LPS binding activity. In agreement, surface plasmon resonance analyses confirmed direct LPS binding of three AmyI‐1‐18 analogs. In addition, AmyI‐1‐18 analogs exhibited little or no cytotoxic activity against RAW264 cells, indicating that enhancements of anti‐inflammatory and LPS‐neutralizing activities following replacement of arginine or leucine did not result in significant increases in cytotoxicity. This study shows that the arginine‐substituted and leucine‐substituted AmyI‐1‐18 analogs with improved anti‐endotoxic and antimicrobial activities have clinical potential as dual‐function host defense agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Antibacterial Δ1‐3‐Ketosteroids from the South China Sea Gorgonian Coral Subergorgia rubra

Three new Δ¹‐3‐ketosteroids characterized with a 9‐OH, subergosterones A–C ( 1 – 3 ), together with five known analogs 4 – 8 , were obtained from the gorgonian coral Subergorgia rubra collected from the South China Sea. The structures of 1 – 3 , including their absolute configurations, were determined by comprehensive spectroscopic methods and electronic circular dichroism (ECD) experiments. Compounds 2 and 3 exhibited inhibitory antibacterial activities against Bacillus cereus with MIC values of 1.56 μM .     

Antimicrobial activity of broccoli (Brassica oleracea var. italica) cultivar Avenger against pathogenic bacteria,phytopathogenic filamentous fungi and yeast

Aims

The objective of this study was to show whether the edible part of broccoli has antibacterial and antifungal activity against micro‐organism of importance in human health and vegetable spoilage, and to test if this effect was partially due to antimicrobial peptides (AMPs).

Methods and Results

Crude extracts were obtained from florets and stems of broccoli cultivar Avenger and the inhibitory effect was demonstrated against pathogenic bacteria (Bacillus cereus, Staphylococcus xylosus, Staphylococcus aureus, Shigella flexneri, Shigella sonnei, Proteus vulgaris), phytopathogenic fungi (Colletotrichum gloeosporioides, Asperigillus niger) and yeasts (Candida albicans and Rhodotorula sp.). It was shown that samples treated with proteolytic enzymes had a reduction of approximately 60% in antibacterial activity against Staph. xylosus, suggesting that proteinaceous compounds might play a role in the inhibitory effect. Antimicrobial components in crude extracts were thermoresistant and the highest activity was observed under acidic conditions. It was shown that antifungal activity of broccoli's crude extracts might not be attributed to chitinases.

Conclusions

Organic broccoli cultivar Avenger has antimicrobial activity against pathogenic bacteria, yeast and phytophatogenic fungi. Data suggest that this effect is partially due to AMPs.

Significance and Impact of the Study

Broccoli's crude extracts have activity not only against pathogenic bacteria but also against phytophatogenic fungi of importance in agriculture. We suggest for first time that the inhibitory effect is probably due to AMPs.     

King cobra peptide OH-CATH30 as a potential candidate drug through clinic drug-resistant isolates

Cationic antimicrobial peptides(AMPs) are considered as important candidate therapeutic agents, which exert potent microbicidal properties against bacteria, fungi and some viruses. Based on our previous findings king cobra cathelicidin(OH-CATH) is a 34-amino acid peptide that exerts strong antibacterial and weak hemolytic activity. The aim of this research is to evaluate the efficacy of both OH-CATH30 and its analog D-OH-CATH30 against clinical isolates comparing with routinely utilized antibiotics in vitro.In this study, 584 clinical isolates were tested(spanning 2013-2016) and the efficacy of the candidate peptides and antibiotics were determined by a broth microdilution method according to the CLSI guidelines. Among the 584 clinical isolates, 85% were susceptible to OH-CATH30 and its analogs. Both L-and D-OH-CATH30 showed higher efficacy against(toward) Gram-positive bacteria and stronger antibacterial activity against nearly all Gram-negative bacteria tested compare with antibiotics. The highest bactericidal activity was detected against Acinetobacter spp., including multi-drug-resistant Acinetobacter baumannii(MRAB)and methicillin-resistant Staphylococcus aureus(MRSA). The overall efficacy of OH-CATH30 and its analogs was higher than that of the 9 routinely used antibiotics. OH-CATH30 is a promising candidate drug for the treatment of a wide variety of bacterial infections which are resistant to many routinely used antimicrobial agents.     

Proteomic identification of CBM37-containing cellulases produced by the rumen cellulolytic bacterium <Emphasis Type="Italic">Ruminococcus albus</Emphasis> 20 and their putative involvement in bacterial adhesion to cellulose

The objective of this study was to identify and characterize other proteins than fimbrial proteins potentially involved in R. albus 20 adhesion to cellulose using an adhesion-related antiserum preparation (i.e. anti-Adh serum). From protein fractions of R. albus 20 grown on cellulose, the serum recognized at least 10 cellulose-binding proteins (CBPs), among which homologs of glycoside hydrolases (family 5, 9 and 48) of R. albus 8 (i.e. Cel5G, Cel9B and Cel48A) were identified by a proteomic approach. In strain 20, Cel9B and Cel48A were identified as two major CBPs and as bacterial cell-associated proteins. The anti-Adh serum was also shown to target the C-terminal family 37 carbohydrate-binding module (CBM37) of Cel9B and Cel48A, indicating that this module, unique to R. albus, may play a significant role in bacterial adhesion to cellulose as suggested previously for R. albus 8. Overall, our results support the hypothesis of an adhesion mechanism involving the CBM37 of Cel9B and Cel48A. This adhesion mechanism may not be restricted to these two enzymes but may also involve other CBM37-containing proteins such as Cel5G and the other uncharacterised proteins recognized by the anti-Adh serum. The EMBL accession numbers for the sequences reported in this paper are FM872295 for Cel9B and FM872296 for Cel48A.     

CASPR2 forms a complex with GPR37 via MUPP1 but not with GPR37(R558Q), an autism spectrum disorder‐related mutation

Autism spectrum disorder (ASD) is a developmental brain disorder. Mutations in synaptic components including synaptic adhesion molecules have been found in ASD patients. Contactin‐associated protein‐like 2 (CASPR2) is one of the synaptic adhesion molecules associated with ASD. CASPR2 forms a complex with receptors via interaction with multiple PDZ domain protein 1 (MUPP1). Little is known about the relationship between impaired CASPR2‐MUPP1‐receptor complex and the pathogenesis of ASD. GPR37 is a receptor for survival factors. We recently identified mutations including R558Q in the G‐protein‐coupled receptor 37 (GPR37) gene in ASD patients. The mutated GPR37s accumulate in the endoplasmic reticulum. In this study, we show that GPR37 is a component of the CASPR2‐MUPP1 receptor complex in the mouse brain. CASPR2 and GPR37 mainly interacted with the PDZ3 and PDZ11 domains of MUPP1, respectively. Compared to GPR37, GPR37(R558Q) slightly interacted with MUPP1 and caused dendritic alteration. GPR37, but not GPR37(R558Q) nor GPR37‐deltaC which lacks its PDZ binding domain, was transported to the cell surface by MUPP1. In primary hippocampal neurons, GPR37 co‐localized with MUPP1 and CASPR2 at the synapse, but not GPR37(R558Q). Thus, ASD‐related mutation of GPR37 may cause the impaired CASPR2‐MUPP1‐GPR37 complex on the dendrites associated with one of the pathogenesis of ASD.