Design of pH-responsive antimicrobial peptide melittin analog-camptothecin conjugates for tumor therapy

Melittin,a classical antimicrobial peptide,is a highly potent antitumor agent.However,its significant toxicity seriously hampers its application in tumor therapy.In this study,we developed novel melittin analogs with pH-responsive,cell-penetrating and membranelytic activities by replacing arginine and lysine with histidine.After conjugation with camptothecin(CPT),CPT-AAM-1 and CPT-AAM-2 were capable of killing tumor cells by releasing CPT at low concentrations and disrupting cell membranes at high concentrations under acidic conditions.Notably,we found that the C-terminus of the melittin analogs was more suitable for drug conjugation than the N-terminus.CPT-AAM-1 significantly suppressed melanoma growth in vivo with relatively low toxicity.Collectively,the present study demonstrates that the development of antitumor drugs based on pH-responsive antimicrobial peptide-drug conjugates is a promising strategy.

Protective eff ect and mechanism of nanoantimicrobial peptide ND-C14 against Streptococcus pneumoniae infection

BACKGROUND:Streptococcus pneumoniae(S.pneumoniae)is a common pathogen that causes bacterial pneumonia.However,with increasing bacterial resistance,there is an urgent need to develop new drugs to treat S.pneumoniae infections.Nanodefensin with a 14-carbon saturated fatty acid(ND-C14)is a novel nanoantimicrobial peptide designed by modifying myristic acid at the C-terminus of humanα-defensin 5(HD5)via an amide bond.However,it is unclear whether ND-C14 is effective against lung infections caused by S.pneumoniae.METHODS:In vitro,three groups were established,including the control group,and the HD5 and ND-C14 treatment groups.A virtual colony-count assay was used to evaluate the antibacterial activity of HD5 and ND-C14 against S.pneumoniae.The morphological changes of S.pneumoniae treated with HD5 or ND-C14 were observed by scanning electron microscopy.In vivo,mice were divided into sham,vehicle,and ND-C14 treatment groups.Mice in the sham group were treated with 25μL of phosphate-buffered saline(PBS).Mice in the vehicle and ND-C14 treatment groups were treated with intratracheal instillation of 25μL of bacterial suspension with 2×108 CFU/mL(total bacterial count:5×10^(6) CFU),and then the mice were given 25μL PBS or intratracheally injected with 25μL of ND-C14(including 20μg or 50μg),respectively.Survival rates were evaluated in the vehicle and ND-C14 treatment groups.Bacterial burden in the blood and bronchoalveolar lavage fluid were counted.The lung histology of the mice was assessed.A propidium iodide uptake assay was used to clarify the destructive eff ect of ND-C14 against S.pneumoniae.RESULTS:Compared with HD5,ND-C14 had a better bactericidal eff ect against S.pneumoniae because of its stronger ability to destroy the membrane structure of S.pneumoniae in vitro.In vivo,ND-C14 significantly delayed the death time and improved the survival rate of mice infected with S.pneumoniae.ND-C14 reduced bacterial burden and lung tissue injury.Moreover,ND-C14 had a membrane permeation eff ect on S.pneumoniae,and its destructive ability increased with increasing ND-C14 concentration.CONCLUSION:The ND-C14 may improve bactericidal eff ects on S.pneumoniae both in vitro and in vivo.

Analysis of the Antimicrobial Activity of Antimicrobial Peptides Extracted from Immunized Tenebrio molitor Induced by Different Microbial Treatments Against 26 Pathogens

[Objective] The study was to explore whether antimicrobial activity of the antimicrobial peptides extracted from immunized Tenebrio molitor varied with different pathogens as inducers.[Method]By inducing T.molitor hungry larvaes to generate immune response via feeding with bacteria and with fungi or actinomycete post to pricking,the antimicrobial peptides extracts were obtained by grinding and centrifuging the cultures.Its antimicrobial activity against 26 pathogens was measured by bacteriostatic ring,and evaluated by trisection to four types and inhibitory spectrum.[Result]Both the antimicrobial activity and antimicrobial spectrum of the antimicrobial peptides extracts varied remarkably among different pathogens as inducers.[Conclusion]Bio-control used strains have obvious advantage in inducing the insect to express body fluid immunity material-antimicrobial peptides.

Expression of Exogenous Antimicrobial Peptide and Resistance to Stem Rot Disease of Houttuynia cordata Thunb.

[Objective] This study was to identify the expression of exogenous antimicrobial peptide in transgenic Houttuynia cordata Thunb. plants,and analyze their resistance to stem rot disease. [Methods] SDS-PAGE and Western blot analysis were employed to detect expression of exogenous antimicrobial peptide in transgenic H. cordata plants. Both wild type and transgenic H. cordata plants were inoculated with different concentrations of Rhizoctonia solani spores for detecting their resistance. [Results] The exogenous antimicrobial peptide was detected at translation level. The optimal parameters for detecting the resistance of transgenic H. cordata plants to R. solani was inoculation of spores at a concentration of 3×105 ind./ml and cultured for three days. The results showed that resistance of transgenic H. cordata plants to R. solani was enhanced in comparison with CKs. [Conclusion] Expression of exogenous antimicrobial peptide can enhance the resistance of transgenic H. cordata plants to stem rot disease.

Effects of the Antimicrobial Peptide OH-CATH on Escherichia coli

OH-CATH is a novel cathelicidin identified from king cobra. It showed strong antibacterial activity against various bacteria in the presence of 1% NaCl and no haemolytic activity toward human red blood cells even at a high concentration. OH-CATH might serve as model molecules for the development of antimicrobial drugs. Understanding the action mechanism of OH-CATH and the reason for its selectivity against microbes is very important for this purpose. The bactericidal effect of the king cobra antimicrobial peptide OH-CATH on Gram-negative Escherichia coli （ATCC 25922） is observed by scanning electron microscopy （SEM） and transmitted electron microscopy （TEM）. The SEM and TEM results suggested that the bactericidal mechanism of OH-CATH against Escherichia coli happened in three steps. Firstly, OH-CATH attached to the negatively charged bacterial wall by positively charged amino acid residues. In the second step, the accumulated OH-CATH aggregated and damaged the bacteria membrane in a pore-forming manner. In the last step, with the damage of cell permeability, the contents of the cells were released and eventually cells died.

Purification of Antimicrobial Peptide from Antarctic Krill (Euphausia superba) and its Function Mechanism

The preliminary purification and antimicrobial mechanism of antimicrobial peptide from Antarctic Krill were studied in this paper. The results showed that the molecular weight range of antimicrobial polypeptide （CMCC-1） obtained by cation exchange chromatography was between 245-709D as detected by molecular sieve chromatography, and the minimum inhibition concentration （MIC） of CMCC-1 against Staphylococcus aureus was 5.0mgmL^-1. The antimicrobial mechanism of CMCC-1 was studied with S. aureus as indicator bacterium. Compared with control group, the results of the experimental group in which S. aureus was treated with CMCC-1 were as follows： l） CMCC-1 could inhibit cell division at logarithmic phase. 2） The protein and reducing sugar con- tent, and the conductivity of culture medium increased, and the activity of alkaline phosphatase and [3-galactosidase could be detected in the culture medium. 3） Observation under scanning electron microscope revealed that somatic morphology became irregular, and then somatic surface became coarse. The cell became much smaller, and most somatic ceils gathered. The boundary between cells became dim and finally fused as a whole. 4） Observation under transmission electron microscope showed that the surface of S. aureus became rough and the reproducing ability was restrained. The cell wall became thin and the cytoplasm shrunk. Substances inside cell leaked out, which caused cells death. 5） SDS-PAGE analysis showed that some bands disappeared, and the residual bands became vague. 6） The genomic DNA electrophoresis results showed that the genomic DNA bands ofS. aureus were not degraded but the brightness significantly reduced. Thus, it is supposed that CMCC-1 could destroy the cell wall and membrane of S. aureu, increase the cell membrane permeability and the leaking-out of intracellular substances, and thus cause the death ofS. aureu.

The application of antimicrobial peptides as growth and health promoters for swine

With the widespread ban on the use of antibiotics in swine feed, alternative measures need to be sought to maintain swine health and performance, Antimicrobial peptides （AMPs） are part of the nonspecific defense system and are natural antibiotics produced by plants, insects, mammalians, and micro-organisms as well as by chemical synthesis. Due to their broad microbicidal activity against various fungi, bacteria and enveloped viruses AMPs are a potential alternative to conventional antibiotics for use in swine production. This review focuses on the structure and mechanism of action of AMPs, as well as their effects on performance, immune function and intestinal health in pigs. The aim is to provide support for the application of AMPs as feed additives replacing antibiotics in swine nutrition.

Antimicrobial peptides and the gut microbiome in inflammatory bowel disease

Antimicrobial peptides(AMP)are highly diverse and dynamic molecules that are expressed by specific intestinal epithelial cells,Paneth cells,as well as immune cells in the gastrointestinal(GI)tract.They play critical roles in maintaining tolerance to gut microbiota and protecting against enteric infections.Given that disruptions in tolerance to commensal microbiota and loss of barrier function play major roles in the pathogenesis of inflammatory bowel disease(IBD)and converge on the function of AMP,the significance of AMP as potential biomarkers and novel therapeutic targets in IBD have been increasingly recognized in recent years.In this frontier article,we discuss the function and mechanisms of AMP in the GI tract,examine the interaction of AMP with the gut microbiome,explore the role of AMP in the pathogenesis of IBD,and review translational applications of AMP in patients with IBD.

Resistance of Antimicrobial Peptide Gene Transgenic Rice to Bacterial Blight

Antimicrobial peptide is a polypeptide with antimicrobial activity. Antimicrobial peptide genes Np3 and Np5 from Chinese shrimp （Fenneropenaeus Chinensis） were integrated into Oryza sativa L. subsp, japonica cv. Aichi ashahi by Agrobacterium mediated transformation system. PCR analysis showed that the positive ratios of Np3 and Np5 were 36% and 45% in To generation, respectively. RT-PCR analysis showed that the antimicrobial peptide genes were expressed in T1 generation, and there was no obvious difference in agronomic traits between transgenic plants and non-transgenic plants. Four Np3 and Np5 transgenic lines in T1 generation were inoculated with Xanthomonas oryzae pv. oryzae strain CR4, and all the four transgenic lines had significantly enhanced resistance to bacterial blight caused by the strain CR4. The Np5 transgenic lines also showed higher resistance to bacterial blight caused by strains JS97-2, Zhe 173 and OS-225. It is suggested that transgenic lines with Np5 gene might possess broad spectrum resistance to rice bacterial blight.

Quantitative analysis of a novel antimicrobial peptide in rat plasma by ultra performance liquid chromatography-tandem mass spectrometry

We described the first results of a quantitative ultra performance liquid chromatographytandem mass spectrometry method for a novel antimicrobial peptide （phylloseptin, PSN-1）. Chromatographic separation was accomplished on a Waters bridged ethyl hybrid （BEH） C18 （50mm× 2.1 mm, 1.7 μm） column with acetonitrile-water （25：75, v/v） as isocratic mobile phase. Mass spectrometry detection was performed in the positive electrospray ionization mode and by monitoring of the transitions at m/z 679.6/120, 509.6/120 （PSN-1） and m/z 340.7/165 （Thymopentin, IS）. Protein precipitation was investigated and the recovery was satisfactory （above 82%）. The method was shown to be reproducible and reliable with intra-day precision below 5.3%, inter-day precision below 14.2%, and linear range from 0.02 to 2 lag/mL with r〉0.994. The method was successfully applied to a pharmacokinetic study of PSN-1 in rats after intravenous administration.

Construction of Salmonella Pullorum ghost by co-expression of lysis gene E and the antimicrobial peptide SMAP29 and evaluation of its immune efficacy in specific-pathogen-free chicks

In this study, a safety enhanced Salmonella Pullorum （S. Pullorum） ghost was constructed using an antimicrobial peptide gene, and evaluated for its potential as a Pullorum disease （PD） vaccine candidate. The antimicrobial peptide SMAP29 was co-expressed with lysis gene E to generate S. Pullorum ghosts. No viable bacteria were detectable either in the fermentation culture after induction of gene E- and SMAP29-mediated lysis for 24 h or in the lyophilized ghost products. Specific-pathogen- free （SPF） chicks were intraperitoneally immunized with ghosts at day 7 of age and no mortality, clinical symptoms or signs of PD such as anorexia, depression and diarrhea were observed. On challenge with a virulent S. Pullorum strain at 4 wk post-immunization, a comparatively higher level of protection was observed in the S. Pullorum ghost immunized chickens with a minimum of pathological lesions and bacterial loads compared to the birds in inactivated vaccine groups. In addition, immunization with the S. Pullorum ghosts induced a potent systemic IgG response and was associated with significantly increased levels of cytokine IFN-y and IL-4 and relative percentages of CD4＋ and CD8＋ T lymphocytes. Our results indicate that SMAP29 can be employed as a new secondary lethal protein to enhance the safety of bacterial ghosts, and to prepare a non-living bacterial vaccine candidate that can prevent PD in chickens.

Antimicrobial peptides: mechanism of action, activity and clinical potential

The management of bacterial infections is becoming a major clinical challenge due to the rapid evolution of antibiotic resistant bacteria.As an excellent candidate to overcome antibiotic resistance,antimicrobial peptides(AMPs)that are produced from the synthetic and natural sources demonstrate a broad-spectrum antimicrobial activity with the high specificity and low toxicity.These peptides possess distinctive structures and functions by employing sophisticated mechanisms of action.This comprehensive review provides a broad overview of AMPs from the origin,structural characteristics,mechanisms of action,biological activities to clinical applications.We finally discuss the strategies to optimize and develop AMP-based treatment as the potential antimicrobial and anticancer therapeutics.

Tissue expression and developmental regulation of chicken cathelicidin antimicrobial peptides

Cathelicidins are a major family of antimicrobial peptides present in vertebrate animals with potent microbicidal and immunomodulatory activities. Four cathelicidins, namely fowlicidins 1 to 3 and cathelicidin B1, have been identified in chickens. As a first step to understand their role in early innate host defense of chickens, we examined the tissue and developmental expression patterns of all four cathelicidins. Real-time PCR revealed an abundant expression of four cathelicidins throughout the gastrointestinal, respiratory, and urogenital tracts as well as in all primary and secondary immune organs of chickens. Fowlicidins 1 to 3 exhibited a similar tissue expression pattern with the highest expression in the bone marrow and lung, while cathelicidin B1 was synthesized most abundantly in the bursa of Fabricius. Additionally, a tissue-specific regulatory pattern was evident for all four cathelicidins during the first 28 days after hatching. The expression of fowlicidins 1 to 3 showed an age-dependent increase both in the cecal tonsil and lung, whereas all four cathelicidins were peaked in the bursa on day 4 after hatching, with a gradual decline by day 28. An abrupt augmentation in the expression of fowlicidins 1 to 3 was also observed in the cecum on day 28, while the highest expression of cathelicidin B1 was seen in both the lung and cecal tonsil on day 14. Collectively, the presence of cathelicidins in a broad range of tissues and their largely enhanced expression during development are suggestive of their potential important role in early host defense and disease resistance of chickens.

Expression profiles of antimicrobial peptides (AMPs) and their regulation by Relish

Antimicrobial peptides （AMPs）, as key immune effectors, play important roles in the innate immune system of invertebrates. Different types of AMPs, including Penaeidin, Crustin, ALF （anti- lipopolysaccharide factor） have been identified in different penaeid shrimp; however, systematic analyses on the function of different AMPs in shrimp responsive to different types of bacteria are very limited. In this study, we analyzed the expression profiles of AMPs in the Chinese shrimps, Fenneropenaeus chinensis, simultaneously by real-time RT-PCR （reverse transcription-polymerase chain reaction） when shrimp were challenged with Micrococcus lysodeikticus （Gram-positive, G＋） or Hbrio anguillarium （Gram-negative, G-）. Different AMPs showed different expression profiles when shrimp were injected with one type of bacterium, and one AMP also showed different expression profiles when shrimp were challenged with different bacteria. Furthermore, the expression of these AMPs showed temporal expression profiles, suggesting that different AMPs function coordinately in bacteria-infected shrimp. An RNA interference approach was used to study the function of the Relish transcription factor in regulating the transcription of different AMPs. The current study showed that Relish could regulate the transcription of different AMPs in shrimp. Differential expression profiles of AMPs in shrimp injected with different types of bacteria indicated that a complicated antimicrobial response network existed in shrimp. These data contribute to our understanding of immunity in shrimp and may provide a strategy for the control of disease in shrimp.

Purification and in vitro Activity of an Antimicrobial Peptide from Skin of Rana Temporaria Chensinensis,David

In this study, an antimicrobial component（RTCI） was purified from the skin of Rana temporaria chensinensis, David. Antimicrobial activities of RTCI against clinical multi-drug resistant bacterial strains, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureaus, Klebsiella oxytoca, Klebsiella pneumoniae, Enterococcus fae-calis, and Proteus mirabilis, were measured in vitro by means of minimal inhibitory concentration and time-kill studies. The results indicate that RTCI could inhibit the growth of these bacteria at a proper concentration and suggest that RT-CI shows a better antimicrobial activity to Gram-negative bacterial strains than to Gram-positive bacterial strains.

Advances in Antimicrobial Peptides and Expression Strategy

Antimicrobial peptides are widely distributed in nature,existing in organisms of plants,insects,and vertebrates.It has been approved that antimicrobial peptides have broad spectrum antimicrobial activities,and play a key modulatory role in the innate immune response and tumor inhibiting activity.Due to the special action mechanism,the antimicrobial peptides become a hot field of genetic engineering.In the present paper,the general properties,mechanism of action,application value,existing problems,the latest progress and the expression strategy were discussed.

Hybrid Antimicrobial Peptide Buforin Ⅱ-Cecropin B Exhibits Antimicrobial Activity

[ Objective ] This study aimed to investigate the antimicrobial activity of hybrid antimicrobial peptide buforin II-cecropin B. [ Method ] Gene fragment BC encoding the hybrid antimicrobial peptide Buforin II-Cecropin B was synthesized by SOE-PCR with six primers designed according to the published amino acid sequences. Then, the BC gene fragment was ligated into pET32a vector and expressed in BI21. Antimierobial ability of the crude hybrid antimicrobial peptide was detected with the Oxford cup method. [ Result] The BC gene fragment was correctly amplified by SOE-PCR and ligated into pET32a vector, to construct the expression vector pET32-BC. The hybrid antimicrobial peptide buforin II-cecropin B was expressed in BI21 after induction by IPTG. The optimal induction time was 2 h. Antimicrobial activity assay suggested that the hybrid antimicrobial peptide possessed antimicrobial activity to Escherichia coli and Bacillus subtilis. [ Conclusion] The hybrid antimicrobial peptide buforin II-cecropin B exhibited antimicrobial activity to Gram-negative E. coli and Gram-positive B. subtilis strains.

Antimicrobial peptides act on the rumen the performance of castrated bulls

Background Many countries have already banned the use of antibiotics in animal husbandry,making it extremely difficult to maintain animal health in livestock breeding.In the livestock industry,there is an urgent need to develop alternatives to antibiotics which will not lead to drug resistance on prolonged use.In this study,eighteen castrated bulls were randomly divided into two groups.The control group(CK)was fed the basal diet,while the antimicrobial peptide group(AP)was fed the basal diet supplemented with 8 g of antimicrobial peptides in the basal diet for the experimental period of 270 d.They were then slaughtered to measure production performance,and the ruminal contents were isolated for metagenomic and metabolome sequencing analysis.Result The results showed that antimicrobial peptides could improve the daily weight,carcass weight,and net meat weight of the experimental animals.Additionally,the rumen papillae diameter and the micropapillary density in the AP were significantly greater than those in the CK.Furthermore,the determination of digestive enzymes and fermentation parameters showed that the contents of protease,xylanase,andβ-glucoside in the AP were greater than those in the CK.However,lipase content in the CK was greater than that in the AP.Moreover,the content of acetate,propionate,butyrate,and valerate was found to be greater in AP than those in CK.The metagenomic analysis annotated 1993 differential microorganisms at the species level.The KEGG enrichment of these microorganisms revealed that the enrichment of drug resistance-related pathways was dramatically decreased in the AP,whereas the enrichment of immune-related pathways was significantly increased.There was also a significant reduction in the types of viruses in the AP.187 probiotics with significant differences were found,135 of which were higher in AP than in CK.It was also found that the antimicrobial mechanism of the antimicrobial peptides was quite specific.Seven low-abundance microorganisms(Acinetobactersp.Ac1271,Aequorivita soesokkakensis,Bacillus lacisalsi,Haloferax larsenii,Lysinibacillussp.3DF0063,Parabacteroidessp.217,Streptomycessp.So13.3)were found to regulate growth performance of the bull negatively.Metabolome analysis identified 45 differentially differential metabolites that significantly different between the CK and the AP groups.Seven upregulated metabolites(4-pyridoxic acid,Ala-Phe,3-ureidopropionate,hippuric acid,terephthalic acid,L-alanine,uridine 5-monophosphate)improve the growth performance of the experimental animals.To detect the interactions between the rumen microbiome and metabolism,we associated the rumen microbiome with the metabolome and found that negative regulation between the above 7 microorganisms and 7 metabolites.Conclusions This study shows that antimicrobial peptides can improve the growth performance of animals while resisting viruses and harmful bacteria and are expected to become healthy alternatives to antibiotics.We demonstrated a new antimicrobial peptides pharmacological model.We demonstrated low-abundance microorganisms may play a role by regulating the content of metabolites.

Mechanisms of antimicrobial peptides as characterized by solid-state NMR

Antimicrobial peptides(AMP)are small proteins that play critical roles in host defense against microbe invasion.Many AMPs disrupt the cellular membrane of microbe,while the mechanism of action of AMPs can be very sophisticated.Solid-state NMR(SSNMR)technique is powerful in characterizing the mechanism of AMPs in vivo and in vitro.This review summarizes the recent advance of SSNMR technique in AMP mechanisms characterization.We highlight the sample preparation approaches,the SSNMR spectroscopic methods,and a number of outstanding examples of AMP mechanisms elucidated via SSNMR spectroscopy.

Research progress of plant antimicrobial peptides

Plant antimicrobial peptides are a very large family of antimicrobial peptides,which have strong resistance to various pathogenic microorganisms,especially fungi.With the increasing use of antibiotics,the problems caused by antibiotics,including antibiotic residues and pathogen resistance,are becoming more and more prominent.The research on antimicrobial peptides as new antibiotic substitutes is also a hot spot.This article introduces the action sites and antibacterial mechanisms of several plant antimicrobial peptides,as well as the application of plant antimicrobial peptides in the fields of medicine,agriculture,and food preservation.