Rapid Assessment of Susceptibility of Bacteria and Erythrocytes to Antimicrobial Peptides by Single-Cell Impedance Cytometry.

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight antibiotic-resistant infections. In most cases, they kill bacteria by making their membrane permeable and therefore exhibit low propensity to induce bacterial resistance. In addition, they are often selective, killing bacteria at concentrations lower than those at which they are toxic to the host. However, clinical applications of AMPs are hindered by a limited understanding of their interactions with bacteria and human cells. Standard susceptibility testing methods are based on the analysis of the growth of a bacterial population and therefore require several hours. Moreover, different assays are required to assess the toxicity to host cells. In this work, we propose the use of microfluidic impedance cytometry to explore the action of AMPs on both bacteria and host cells in a rapid manner and with single-cell resolution. Impedance measurements are particularly well-suited to detect the effects of AMPs on bacteria, due to the fact that the mechanism of action involves perturbation of the permeability of cell membranes. We show that the electrical signatures of Bacillus megaterium cells and human red blood cells (RBCs) reflect the action of a representative antimicrobial peptide, DNS-PMAP23. In particular, the impedance phase at high frequency (e.g., 11 or 20 MHz) is a reliable label-free metric for monitoring DNS-PMAP23 bactericidal activity and toxicity to RBCs. The impedance-based characterization is validated by comparison with standard antibacterial activity assays and absorbance-based hemolytic activity assays. Furthermore, we demonstrate the applicability of the technique to a mixed sample of B. megaterium cells and RBCs, which paves the way to study AMP selectivity for bacterial versus eukaryotic cells in the presence of both cell types.

Antimicrobial Peptides Therapy: An Emerging Alternative for Treating Drug-Resistant Bacteria.

Microbial resistance to antibiotics is an ancient and dynamic issue that has brought a situation reminiscent of the pre-antibiotic era to the limelight. Currently, antibiotic resistance and the associated infections are widespread and pose significant global health and economic burden. Thus, the misuse of antibiotics, which has increased resistance, has necessitated the search for alternative therapeutic agents for combating resistant pathogens. Antimicrobial peptides (AMPs) hold promise as a viable therapeutic approach against drug-resistant pathogens. AMPs are oligopeptides with low molecular weight. They have broad-spectrum antimicrobial activities against pathogenic microorganisms. AMPs are nonspecific and target components of microbes that facilitate immune response by acting as the first-line defense mechanisms against invading pathogenic microbes. The diversity and potency of AMPs make them good candidates for alternative use. They could be used alone or in combination with several other biomaterials for improved therapeutic activity. They can also be employed in vaccine production targeting drug-resistant pathogens. This review covers the opportunities and advances in AMP discovery and development targeting antimicrobial resistance (AMR) bacteria. Briefly, it presents an overview of the global burden of the antimicrobial resistance crisis, portraying the global magnitude, challenges, and consequences. After that, it critically and comprehensively evaluates the potential roles of AMPs in addressing the AMR crisis, highlighting the major potentials and prospects.

First-in-Class Cyclic Temporin L Analogue: Design, Synthesis, and Antimicrobial Assessment.

The pharmacodynamic and pharmacokinetic properties of bioactive peptides can be modulated by introducing conformational constraints such as intramolecular macrocyclizations, which can involve either the backbone and/or side chains. Herein, we aimed at increasing the α-helicity content of temporin L, an isoform of an intriguing class of linear antimicrobial peptides (AMPs), endowed with a wide antimicrobial spectrum, by the employment of diverse side-chain tethering strategies, including lactam, 1,4-substituted [1,2,3]-triazole, hydrocarbon, and disulfide linkers. Our approach resulted in a library of cyclic temporin L analogues that were biologically assessed for their antimicrobial, cytotoxic, and antibiofilm activities, leading to the development of the first-in-class cyclic peptide related to this AMP family. Our results allowed us to expand the knowledge regarding the relationship between the α-helical character of temporin derivatives and their biological activity, paving the way for the development of improved antibiotic cyclic AMP analogues.

Recombinant production of Trx-Ib-AMP4 and Trx-E50-52 antimicrobial peptides and antimicrobial synergistic assessment on the treatment of methicillin-resistant Staphylococcus aureus under in vitro and in vivo situations.

PURPOSE: The production of alternative novel antimicrobial agents is considered an efficient way to cope with multidrug resistance among pathogenic bacteria. E50-52 and Ib-AMP4 antimicrobial peptides (AMPs) have illustrated great proven antibacterial effects. The aim of this study was recombinant production of these AMPs and investigation of their synergistic effects on methicillin-resistant Staphylococcus aureus (MRSA). METHOD: At first, the codon optimized sequences of the Ib-AMP4 (UniProt: 024006 (PRO_0000020721), and E50-52 (UniProtKB: P85148) were individually ligated into the pET-32α vector and transformed into E. coli. After the optimization of production and purification steps, the MIC (Minimum inhibitory concentration), time kill and growth kinetic tests of recombinant proteins were determined against MRSA. Finally, the in vivo wound healing efficiency was tested. RESULTS AND CONCLUSION: The recorded MIC of recombinant Trx-Ib-AMP4, Trx-E50-52 against MRSA bacterium were 0.375 and 0.0875 mg/mL respectively. The combination application of the produced AMPs by the checkerboard method confirmed their synergic activity. The results of the time-kill showed sharply decrease of the number of viable cells with over five time reductions in log10 CFU/mL by the combination of Trx-E50-52 and Trx-IbAMP4 at 2 × MIC within 240 min. The growth kinetic results confirmed the combination of Trx-E50-52 and Trx-IbAMP4 had much greater success in the reduction of over 50 % of MRSA suspensions' turbidity within the first hour. Wound healing assay and histological analysis of infected mice treated with Trx-Ib-AMP4 or Trx-E50-52 compared with those treated with a combination of Trx-Ib-AMP4 and Trx-E50-52 showed significant synergic effects.

Antibacterial effects assessment on some livestock pathogens, thermal stability and proposing a probable reason for different levels of activity of thanatin.

There is a continuing need to prevent the increasing use of common antibiotic and find the replacement to combat the drug/antibiotic resistant bacteria such as antimicrobial peptides (AMPs) such as thanatin peptide. In this study, recombinant thanatin peptide was expressed in the HEK293 cell line. Then the antimicrobial properties of this peptide on some poultry and farm animal's pathogen strains were assessed. The thermal-stability of thanatin was predicted in various temperatures through in silico analysis. Afterwards, according to Minimum Inhibitory Concentration (MIC) results, Escherichia coli and Pseudomonas aeruginosa were chosen to test the hypothesis of LptA/LptD-thanatin interaction, computationally. Relative amino acid sequences and crystallography structures were retrieved and missed tertiary structures were predicted. The interaction of thanatin with LptA and LptD of Escherichia coli and Pseudomonas aeruginosa were analyzed subsequently. The antibacterial activity of thanatin peptide was evaluated between 6.25 and 100 µg/mL using minimum inhibitory concentration. Also, the amounts of minimum bactericidal concentrations (MBC) were between 12.5 and 200 µg/mL. The bioinformatics analysis followed by the in vitro assessment, demonstrated that thanatin would be thermally stable in the body temperature of poultry and farm animals. Thanatin could penetrate to the outer membrane domain of LptD in Escherichia coli and it could block the transition path of this protein while the entrance of LptD in Pseudomonas aeruginosa was blocked for thanatin by extra residues in comparison with Escherichia coli LptD. In addition, the quality of interaction, with regard to the number and distance of interactions which leads to higher binding energy for thanatin and LptD of Escherichia coli was much better than Pseudomonas aeruginosa. But the site and quality of interaction for thanatin and LptA was almost the same for Escherichia coli and Pseudomonas aeruginosa. Accordingly, thanatin can prevent the assembly of LptA periplasmic bridge in both pathogens. The antibacterial and thermal stability of the thanatin peptide suggested that thanatin peptide might serve as a natural alternative instead of common antibiotics in the veterinary medicine. The outcome of this in silico study supports the MIC results. Therefore, a probable reason for different level of activity of thanatin against Escherichia coli and Pseudomonas aeruginosa might be the quality of LptA/LptD-thanatin interaction.

Using In Vivo Assessment on Host Defense Peptide Mimicking Polymer-Modified Surfaces for Combating Implant Infections.

Infections have accounted for the majority of failures in implants over the past decades. Host defense peptide mimicking polymers have been considered as one of the promising antimicrobial candidates for their cost-effective synthesis, broad-spectrum antimicrobial activity, low propensity to induce drug resistance, and remarkable biocompatibility. In this review, covalent-grafting strategies are mainly discussed to tether host defense peptide mimicking polymers on surfaces, aiming to obtain potent antimicrobial activity. In addition to the antimicrobial function, we review the antimicrobial mechanism of these polymer-modified antimicrobial surfaces in precedent literatures. We also review the in vivo subcutaneous implant infection models that are critical assessments for potential biomedical applications. In the end, we provide our perspective on the future development of this field, especially for biomedical applications.

Shortened derivatives from native antimicrobial peptide LyeTx I: In vitro and in vivo biological activity assessment.

In the continuing search for novel antibiotics, antimicrobial peptides are promising molecules, due to different mechanisms of action compared to classic antibiotics and to their selectivity for interaction with microorganism cells rather than with mammalian cells. Previously, our research group has isolated the antimicrobial peptide LyeTx I from the venom of the spider Lycosa erythrognatha. Here, we proposed to synthesize three novel shortened derivatives from LyeTx I (LyeTx I mn; LyeTx I mnΔK; LyeTx I mnΔKAc) and to evaluate their toxicity and biological activity as potential antimicrobial agents. Peptides were synthetized by Fmoc strategy and circular dichroism analysis was performed, showing that the three novel shortened derivatives may present membranolytic activity, like the original LyeTx I, once they folded as an alpha helix in 2.2.2-trifluorethanol and sodium dodecyl sulfate. In vitro assays revealed that the shortened derivative LyeTx I mnΔK presents the best score between antimicrobial (↓ MIC) and hemolytic (↑ EC50) activities among the synthetized shortened derivatives, and LUHMES cell-based NeuriTox test showed that it is less neurotoxic than the original LyeTx I (EC50 [LyeTx I mnΔK] ⋙ EC50 [LyeTx I]). In vivo data, obtained in a mouse model of septic arthritis induced by Staphylococcus aureus, showed that LyeTx I mnΔK is able to reduce infection, as demonstrated by bacterial recovery assay (∼10-fold reduction) and scintigraphic imaging (less technetium-99m labeled-Ceftizoxime uptake by infectious site). Infection reduction led to inflammatory process and pain decreases, as shown by immune cells recruitment reduction and threshold nociception increment, when compared to positive control group. Therefore, among the three shortened peptide derivatives, LyeTx I mnΔK is the best candidate as antimicrobial agent, due to its smaller amino acid sequence and toxicity, and its greater biological activity.

Multichannel bacterial discrimination based on recognition and disintegration disparity of short antimicrobial peptides.

In this paper, we present a facile and rapid multichannel approach for the simultaneous detection and discrimination of multiple bacterial species. The proposed assay employed four short antimicrobial peptides (SAMPs) for recognition due to their disparity in antibacterial activity against different bacterial strains, and utilized fluorescence measurements to explicate the bacterial recognition and disintegration disparity of the SAMPs. Then, linear discriminant analysis (LDA) was used to effectively discriminate and classify the observed characteristic fluorescence patterns of SAMPs towards target bacteria, exhibiting excellent bacterial discrimination and classification accuracy. This is the first report on the use of SAMPs as recognition units for simultaneous multiple bacterial detection and discrimination. The presented approach was simple, fast, highly repeatable, and required no labelling processes. According to the corresponding LDA discriminant results, six different target bacterial species could be effectively identified and discriminated within 30 min.

Nanoantibiotics containing membrane-active human cathelicidin LL-37 or synthetic ceragenins attached to the surface of magnetic nanoparticles as novel and innovative therapeutic tools: current status and potential future applications.

Nanotechnology-based therapeutic approaches have attracted attention of scientists, in particular due to the special features of nanomaterials, such as adequate biocompatibility, ability to improve therapeutic efficiency of incorporated drugs and to limit their adverse effects. Among a variety of reported nanomaterials for biomedical applications, metal and metal oxide-based nanoparticles offer unique physicochemical properties allowing their use in combination with conventional antimicrobials and as magnetic field-controlled drug delivery nanocarriers. An ever-growing number of studies demonstrate that by combining magnetic nanoparticles with membrane-active, natural human cathelicidin-derived LL-37 peptide, and its synthetic mimics such as ceragenins, innovative nanoagents might be developed. Between others, they demonstrate high clinical potential as antimicrobial, anti-cancer, immunomodulatory and regenerative agents. Due to continuous research, knowledge on pleiotropic character of natural antibacterial peptides and their mimics is growing, and it is justifying to stay that the therapeutic potential of nanosystems containing membrane active compounds has not been exhausted yet.

Making plants into cost-effective bioreactors for highly active antimicrobial peptides.

As antibiotic-resistant bacterial pathogens become an ever-increasing concern, antimicrobial peptides (AMPs) have grown increasingly attractive as alternatives. Potentially, plants could be used as cost-effective AMP bioreactors; however, reported heterologous AMP expression is much lower in plants than in E. coli expression systems and often results in plant cytotoxicity, even for AMPs fused to carrier proteins. This suggests that there may be a physical characteristic of the previously described heterologous AMPs which impedes efficient expression in plants. Using a meta-analysis of protein databases, this study has determined that native plant AMPs were significantly less cationic than AMPs native to other taxa. To apply this finding to plant expression, the transient expression of 10 different heterologous AMPs, ranging in charge from +7 to -5, was tested in the tobacco, Nicotiana benthamiana. Elastin-like polypeptide (ELP) was used as the carrier protein for AMP expression. ELP fusion allowed for a simple, cost-effective temperature shift purification. Using this system, all five anionic AMPs expressed well, with two at unusually high levels (375 and 563 µg/gfw). Furthermore, antimicrobial activity against Staphylococcus epidermidis was an order of magnitude greater (average minimum inhibitory concentration MIC of 0.26µM) than that typically seen for AMPs expressed in E. coli systems and was associated with the uncleaved fusion peptide. In summary, this study describes a means of expressing AMP fusions in plants in high yield, purified by a simple temperature-shift protocol, resulting in a fusion peptide with high antimicrobial activity and without the need for a peptide cleavage step.

Comparative assessment of genotypic and phenotypic correlates of Staphylococcus pseudintermedius strains isolated from dogs with otitis externa and healthy dogs.

Staphylococcus pseudintermedius is considered a primary pathogen of canine skin and soft tissue infections, and the rapid emergence of methicillin-resistant S. pseudintermedius worldwide is a major issue. In the current study, genotypic and phenotypic correlates associated with S. pseudintermedius causing canine otitis externa were evaluated using 41 S. pseudintermedius strains isolated from dogs with otitis externa (n = 26) and healthy dogs (n = 15). The S. pseudintermedius strains were subjected to a comparative analysis of (i) genotypes (multilocus sequence typing, agr, and spa types), (ii) methicillin resistance and SCCmec types, (iii) multidrug resistance (MDR), (iv) biofilm formation, and (v) susceptibility to canine cathelicidin (K9CATH). A high degree of genetic diversity was observed in both groups of S. pseudintermedius strains, regardless of methicillin resistance. Almost all methicillin-resistant strains (>95%) harbored SCCmec V and displayed MDR. Although there was no difference in biofilm formation, S. pseudintermedius strains derived from otitis externa exhibited enhanced resistance to cationic antimicrobial peptide (K9CATH) compared with strains from healthy dogs. The high degree of heterogeneity in MLST, agr, and spa types prevented the identification of correlations between any specific genotype and virulence phenotype in otitis externa caused by S. pseudintermedius, These findings provide an important basis for monitoring and treating canine skin and soft tissue infections in Korea.

Designing and optimizing new antimicrobial peptides: all targets are not the same.

Because the resistance of microorganisms to the available antibiotics is a growing healthcare problem worldwide, the search for new antimicrobial peptides (AMPs) that provide useful therapeutic options has been increasing in importance. Many initial candidates have had to be discarded after having advanced to the preclinical and clinical stages. This has led to substantial losses in terms of time and money. For that reason, the essential characteristics of AMPs (i.e. their activity, selectivity, stability in physiological conditions and low production cost) must be considered in their design. In addition, peptides could be active against several kinds of cells with activity and selectivity resulting from interaction with multiple target cell components, which sometimes are present in mammalian cells as well. Thus, the cellular composition is important in the AMP-target cell interaction and must be considered in the design of AMPs, too. This review describes general aspects of AMP design, limitations concerning their therapeutic application, and optimization strategies for overcoming such limitations.

Mammalian antimicrobial peptide protegrin-4 self assembles and forms amyloid-like aggregates: Assessment of its functional relevance.

Protegrin-4 (PG-4) is a member of the porcine leukocyte protegrins family of cysteine-rich antimicrobial peptides (AMPs) isolated from Sus scrofa. It consists of 18 amino acid residues and works as a part of innate immune system. In this study, we examined the intrinsic aggregation propensity of this AMP using multiple computational algorithms, namely, TANGO, AGGRESCAN, FOLDAMYLOID, AMYLPRED, and ZYGGREGATOR, and found that the peptide is predicted to have a high propensity for the ß sheet formation that disposes this peptide to be amyloidogenic. Under in vitro conditions, PG-4 formed visible aggregates and displayed the hallmark properties of typical amyloids such as enhanced binding of Congo red, increased fluorescence with Thioflavin-T, and fibrillar morphology under transmission electron microscopy. Then we examined its antimicrobial activity against Bacillus subtilis and found that the aggregated peptide retained its antimicrobial activity. Additionally, the aggregates remain non-toxic to the HEK293 and Caco2 cells. Our study suggests that the inherent aggregation properties of AMP can rationally be explored as a potential source of peptide-based antimicrobials with enhanced stability.

Electrical detection of pathogenic bacteria in food samples using information visualization methods with a sensor based on magnetic nanoparticles functionalized with antimicrobial peptides.

Outbreaks of foodborne diseases demand simple, rapid techniques for detecting pathogenic bacteria beyond the standard methods that are not applicable to routine analysis in the food industry and in the points of food consumption. In this work, we developed a sensitive, rapid and low-cost assay for detecting Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Salmonella typhimurium (S. typhi) in potable water and apple juice. The assay is based on electrical impedance spectroscopy measurements with screen-printed interdigitated electrodes coupled with magnetite nanoparticles functionalized with the antimicrobial peptide melittin (MLT). The data were analyzed with the information visualization methods Sammon's Mapping and Interactive Document Map to distinguish samples at two levels of contamination from food suitable for consumption. With this approach it has been possible to detect E. coli concentration down to 1 CFU mL-1 in potable water and 3.5 CFU mL-1 in apple juice without sample preparation, within only 25 min. This approach may serve as a low-cost, quick screening procedure to detect bacteria-related food poisoning, especially if the impedance data of several sensing units are combined.

Design and Assessment of Anti-Biofilm Peptides: Steps Toward Clinical Application.

Highly antibiotic resistant, microbial communities, referred to as biofilms, cause various life-threatening infections in humans. At least two-thirds of all clinical infections are biofilm associated, and antibiotic therapy regularly fails to cure patients. Anti-biofilm peptides represent a promising approach to treat these infections by targeting biofilm-specific characteristics such as highly conserved regulatory mechanisms. They are being considered for clinical application and we discuss here key factors in discovery, design, and application, particularly the implementation of host-mimicking conditions, that are required to enable the successful advancement of potent anti-biofilm peptides from the bench to the clinic.

Structural and Functional Assessment of mBjAMP1, an Antimicrobial Peptide from Branchiostoma japonicum, Revealed a Novel α-Hairpinin-like Scaffold with Membrane Permeable and DNA Binding Activity.

Here we describe the three-dimensional structure and antimicrobial mechanism of mBjAMP1, an antimicrobial peptide (AMP) isolated from Branchiostoma japonicum. The structure of mBjAMP1 was determined by 2D solution NMR spectroscopy and revealed a novel α-hairpinin-like scaffold stabilized by an intramolecular disulfide bond. mBjAMP1 showed effective growth inhibition and bactericidal activities against pathogenic bacteria but was not cytotoxic to mammalian cells. Antimicrobial mechanism studies using fluorescence-based experiments demonstrated that mBjAMP1 did not disrupt membrane integrity. Laser-scanning confocal microscopy indicated that mBjAMP1 is able to penetrate the bacterial cell membrane without causing membrane disruption. Moreover, gel retardation assay suggested that mBjAMP1 directly binds to bacterial DNA as an intracellular target. Collectively, mBjAMP1 may inhibit biological functions by binding to DNA or RNA after penetrating the bacterial cell membrane, thereby causing cell death. These results suggest that mBjAMP1 may present a promising template for the development of peptide-based antibiotics.

Assessment of the In Vivo Activity of SPR741 in Combination with Azithromycin against Multidrug-Resistant Enterobacteriaceae Isolates in the Neutropenic Murine Thigh Infection Model.

SPR741 is a novel agent with structural similarity to polymyxins that is capable of potentiating the activities of various classes of antibiotics. Previously published studies indicated that although Enterobacteriaceae isolates had minimal susceptibilities to azithromycin (AZM), the in vitro antimicrobial activity of AZM against Enterobacteriaceae was enhanced when it was combined with SPR741. The current study evaluated the in vivo activity of human-simulated regimens (HSR) of AZM equivalent to clinical doses of 500 mg given intravenously (i.v.) every 24 h (q24h) and SPR741 equivalent to clinical doses of 400 mg q8h i.v. (1-h infusion), alone and in combination, against multidrug-resistant (MDR) Enterobacteriaceae We studied 30 MDR Enterobacteriaceae isolates expressing a wide spectrum of ß-lactamases (ESBL, NDM, VIM, and KPC), including a subset of isolates positive for genes conferring macrolide resistance (mphA, mphE, ermB, and msr). In vivo activity was assessed as the change in log10 CFU per thigh at 24 h compared with 0 h. Treatment with AZM alone was associated with net growth of 2.60 ± 0.83 log10 CFU/thigh. Among isolates with AZM MICs of ≤16 mg/liter, treatment with AZM-SPR741was associated with an average reduction in bacterial burden of -0.53 ± 0.82 log10 CFU/thigh, and stasis to 1-log kill was observed in 9/11 isolates (81.8%). Combination therapy with an AZM-SPR741 HSR showed promising in vivo activity against MDR Enterobacteriaceae isolates with AZM MICs of ≤16 mg/liter, including those producing a variety of ß-lactamases. These data support a potential role for AZM-SPR741 in the treatment of infections due to MDR Enterobacteriaceae.

Prediction of antimicrobial activity of large pool of peptides using quasi-SMILES.

The purpose of this study was the estimation of ability of the so-called optimal descriptors calculated to be a tool to predict the antimicrobial activity of large pool of peptides. Traditional simplified molecular input-line entry system (SMILES) is an efficient tool to represent the molecular structure of different compounds. Quasi-SMILES represents an extension of traditional SMILES. This approach provides the possibility to involve different eclectic conditions related to analyzed endpoint in the modelling process. In addition, the quasi-SMILES can be used to represent structure of peptides via abbreviations of corresponding amino acids. In this study, quasi-SMILES represents sequences of amino acids in peptides that were tested as the basis to predict antimicrobial activity of 1581 peptides. Predictive potential of binary classification for antimicrobial activity for different splits is quite good when it comes to the training, invisible training, calibration, and validation sets. For the external validation sets, the statistical criteria are ranged: (i) sensitivity 0.82-097; (ii) specificity 0.88-0.99; (iii) accuracy 0.87-0.98; and (iv) Matthews correlation coefficient 0.73-0.97. The suggested optimal descriptors calculated with data on composition of amino acids in peptides can be a tool to predict antimicrobial activity of peptides.

Activity of Antimicrobial Peptide Aggregates Decreases with Increased Cell Membrane Embedding Free Energy Cost.

Antimicrobial peptides (AMPs) are a promising alternative to antibiotics for mitigating bacterial infections, in light of increasing bacterial resistance to antibiotics. However, predicting, understanding, and controlling the antibacterial activity of AMPs remain a significant challenge. While peptide intramolecular interactions are known to modulate AMP antimicrobial activity, peptide intermolecular interactions remain elusive in their impact on peptide bioactivity. Herein, we test the relationship between AMP intermolecular interactions and antibacterial efficacy by controlling AMP intermolecular hydrophobic and hydrogen bonding interactions. Molecular dynamics simulations and Gibbs free energy calculations in concert with experimental assays show that increasing intermolecular interactions via interpeptide aggregation increases the energy cost for the peptide to embed into the bacterial cell membrane, which in turn decreases the AMP antibacterial activity. Our findings provide a route for predicting and controlling the antibacterial activity of AMPs against Gram-negative bacteria via reductions of intermolecular AMP interactions.

Fusion expression of the PGLa-AM1 with native structure and evaluation of its anti-Helicobacter pylori activity.

Helicobacter pylori (H. pylori) shows increasingly enhanced resistance to various antibiotics, and its eradication has become a major problem in medicine. The antimicrobial peptide PGLa-AM1 is a short peptide with 22 amino acids and exhibits strong antibacterial activity. In this study, we investigated whether it has anti-H. pylori activity for the further development of anti-H. pylori drugs to replace existing antibiotics. However, the natural antimicrobial peptide PGLa-AM1 shows a low yield and is difficult to separate, limiting its application. A good strategy to solve this problem is to express the antimicrobial peptide PGLa-AM1 using gene engineering at a high level and low cost. For getting PGLa-AM1 with native structure, in this study, a specific protease cleavage site of tobacco etch virus (TEV) was designed before the PGLa-AM1 peptide. For convenience to purify and identify high-efficiency expression PGLa-AM1, the PGLa-AM1 gene was fused with the polyhedrin gene of Bombyx mori (B. mori), and a 6 × His tag was designed to insert before the amino terminus of the fusion protein. The fusion antibacterial peptide PGLa-AM1 (FAMP) gene codon was optimized, and the gene was synthesized and cloned into the Escherichia coli (E. coli) pET-30a (+) expression vector. The results showed that the FAMP was successfully expressed in E. coli. Its molecular weight was approximately 34 kDa, and its expression level was approximately 30 mg/L. After the FAMP was purified, it was further digested with TEV protease. The acquired recombinant antimicrobial peptide PGLa-AM1 exerted strong anti-H. pylori activity and therapeutic effect in vitro and in vivo.