RESUMO
Antimicrobial peptides (AMPs) have attracted attention in the field of food preservatives due to their favorable biosafety and potential antimicrobial activity. However, high synthetic cost, systemic toxicity, a narrow antimicrobial spectrum, and poor antimicrobial activity become the main bottlenecks for their practical applications. To address these questions, a set of derived nonapeptides were designed based on a previously discovered ultra-short peptide sequence template (RXRXRXRXL-NH2) and screened to identify an optimal peptide-based food preservative with excellent antimicrobial properties. Among these nonapeptides, the designed peptides 3IW (RIRIRIRWL-NH2) and W2IW (RWRIRIRWL-NH2) presented a membrane-disruptive and reactive oxygen species (ROS) accumulation mechanism to execute potent and rapid broad-spectrum antimicrobial activity without observed cytotoxicity. Moreover, they exhibited favorable antimicrobial stability regardless of high ionic strength, heat, and excessive acid-base conditions, retaining potent antimicrobial effects for chicken meat preservation. Collectively, their ultra-short sequence length and potent broad-spectrum antimicrobial capacity may be beneficial for the further development of green and safe peptide-based food preservatives.
Assuntos
Anti-Infecciosos , Conservantes de Alimentos , Conservantes de Alimentos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Antibacterianos/farmacologia , Antibacterianos/química , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Sequência de Aminoácidos , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: Antimicrobial peptides (AMPs) are considered as the most potential alternatives to antibiotics, but they have several drawbacks, including high cost, medium antimicrobial efficacy, poor cell selectivity, which limit clinical application. To overcome the above problems, combination therapy of AMPs with adjuvants might maximize the effectiveness of AMPs. We found that citronellal can substantially potentiate the ZY4R peptide efficacy against Escherichia coli ATCC25922. However, it is unclear whether ZY4R/citronellal combination poses synergistic antimicrobial effects against most bacteria, and their synergy mechanism has not been elucidated. PURPOSE: To investigate synergistic antimicrobial efficacies, biosafety, and synergy mechanism of ZY4R/citronellal combination. METHOD: Checkerboard, time-kill curves, cytotoxicity assays, and in vivo animal models were conducted to assess synergistic antimicrobial effects and biosafety of the ZY4R/citronellal combination. To evaluate their synergy mechanism, a series of cell-based assays and transcriptome analysis were performed. RESULTS: ZY4R/citronellal combination exhibited synergistic antimicrobial effects against 20 clinically significant pathogens, with the fractional inhibitory concentration index (FICI) ranging from 0.313 to 0.047. Meanwhile, ZY4R/citronellal combination enhanced antimicrobial efficacies without compromising cell selectivity, contributing to decreasing drug dosage and improving biosafety. Compared with ZY4R (4 mg/kg) and citronellal (25 mg/kg) alone, ZY4R (4 mg/kg)/citronellal (25 mg/kg) combination significantly decreased the bacterial load in peritoneal fluid, liver, and kidney (P < 0.05) and alleviated pathological damage of the organs of mice. Mechanistic studies showed that ZY4R allowed citronellal to pass through the outer membrane rapidly and acted on the inner membrane together with citronellal, causing more potent membrane damage. The membrane damage prompted the continuous accumulation of citronellal in cells, and citronellal further induced energy breakdown and inhibited exopolysaccharide (EPS) production, which aggravated ZY4R-induced outer membrane damage, thereby resulting in bacterial death. CONCLUSIONS: ZY4R/citronellal combination exhibited broad-spectrum synergy with a low resistance development and high biosafety. Their synergy mechanism acted on two important cellular targets (energy metabolism and membrane integrity). Combination therapy of ZY4R with citronellal may be a promising mixture to combat bacterial infections facing an antibiotic-resistance crisis.
Assuntos
Adjuvantes Imunológicos , Peptídeos Antimicrobianos , Animais , Camundongos , Monoterpenos Acíclicos/farmacologia , Resistência a Múltiplos MedicamentosRESUMO
Post-translational modification (PTM) has a significant impact on cellular signaling and function regulation. In pancreatic ß cells, PTMs are involved in insulin secretion, cell development, and viability. The dysregulation of PTM in ß cells is clinically associated with the development of diabetes mellitus. Here, we summarized current findings on major PTMs occurring in ß cells and their roles in insulin secretion. Our work provides comprehensive insight into understanding the mechanisms of insulin secretion and potential therapeutic targets for diabetes from the perspective of protein PTMs.
RESUMO
Recently, much emphasis has been placed on solving the intrinsic defects of antimicrobial peptides (AMPs), especially their susceptibility to protease digestion for the systemic application of antibacterial biomaterials. Although many strategies have increased the protease stability of AMPs, antimicrobial activity was severely compromised, thereby substantially weakening their therapeutic effect. To address this issue, we introduced hydrophobic group modifications at the N-terminus of proteolysis-resistant AMPs D1 (AArIIlrWrFR) through end-tagging with stretches of natural amino acids (W and I), unnatural amino acid (Nal) and fatty acids. Of these peptides, N1 tagged with a Nal at N-terminus showed the highest selectivity index (GMSI=19.59), with a 6.73-fold improvement over D1. In addition to potent broad-spectrum antimicrobial activity, N1 also exhibited high antimicrobial stability toward salts, serum and proteases in vitro and ideal biocompatibility and therapeutic efficacy in vivo. Furthermore, N1 killed bacteria through multiple mechanisms, involving disruption of bacterial membranes and inhibition of bacterial energy metabolism. Indeed, appropriate terminal hydrophobicity modification opens up new avenues for developing and applying high-stability peptide-based antibacterial biomaterials. STATEMENT OF SIGNIFICANCE: To improve the potency and stability of proteolysis-resistant antimicrobial peptides (AMPs) without increasing toxicity, we constructed a convenient and tunable platform based on different compositions and lengths of hydrophobic end modifications. By tagging an Nal at the N-terminal, the obtained target compound N1 exhibited strong antimicrobial activity and desirable stability under multifarious environments in vitro (proteases, salts and serum), and also showed favorable biocompatibility and therapeutic efficacy in vivo. Notably, N1 exerted its bactericidal effect by damaging bacterial cell membranes and inhibiting bacterial energy metabolism in a dual mode. The findings provide a potential method for designing or optimizing proteolysis-resistant AMPs thus promoting the development and application of peptide-based antibacterial biomaterial.
Assuntos
Anti-Infecciosos , Peptídeos Antimicrobianos , Proteólise , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Sais , Anti-Infecciosos/farmacologia , Bactérias , Antibacterianos/farmacologia , Antibacterianos/química , Peptídeo Hidrolases/farmacologia , Aminoácidos , Testes de Sensibilidade MicrobianaRESUMO
The biggest application bottleneck of antimicrobial peptides (AMPs) is the low oral bioavailability caused by the poor stability of digestive enzymes in the gastrointestinal tract. However, the research methods and evaluation criteria of available studies about anti-proteolytic strategies are not uniform and far from the actual environment in vivo. Here, we developed a research system and evaluation criteria for proteolytic resistance and systematically evaluated the effectiveness of different strategies for improving the protease stability of AMPs on the same platform for the first time. After a comprehensive analysis, Dab modification is identified as the most effective strategy to improve the trypsin stability of AMPs. By further modulating the proteolytic resistance optimization motif (DabW)n, U1-2WD is obtained with ideal stability and antimicrobial properties in vivo and in vitro. Notably, U1-2WD has a unique antibacterial mechanism, which forms amorphous aggregates in the bacteria environment to trigger the agglutination of bacterial cells to prevent bacterial escape. It then kills bacteria by disrupting bacterial membranes and inhibiting bacterial energy metabolism. Overall, our work has led to a new understanding of the effectiveness of proteolytic resistance strategies and accelerated the development of anti-proteolytic AMPs to combat multidrug-resistant bacterial infections. STATEMENT OF SIGNIFICANCE: We developed research system and evaluation criteria for proteolytic resistance and systematically evaluated the effectiveness of different strategies for improving protease stability of AMPs on the same platform for the first time. we found effective strategies to resist trypsin hydrolysis: modification with backbone (ß-Arg), D-enantiomer (D-Arg) and L-2,4-diaminobutanoic acid (Dab). Further, the proteolytic resistance optimization motif (DabW)n was designed. When n=3, derivative U1-2WD was obtained with desirable stability and antimicrobial properties in vivo and in vitro. Notably, U1-2WD has a unique antibacterial mechanism, which can self-aggregate into amorphous aggregates in the bacteria environment to mediate the agglutination and sedimentation of bacterial cells to prevent bacterial escape, and then kill bacteria by destroying bacterial membranes and inhibiting bacterial energy metabolism.
Assuntos
Anti-Infecciosos , Peptídeos Catiônicos Antimicrobianos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Tripsina/farmacologia , Bactérias , Antibacterianos/farmacologia , Antibacterianos/química , Anti-Infecciosos/farmacologia , Peptídeo Hidrolases/farmacologia , Aglutinação , Testes de Sensibilidade MicrobianaRESUMO
In recent years, peptide self-assembly has received much attention because of its ability to form regular and ordered structures with diverse functions. Self-assembled peptides can form aggregates with defined structures under specific conditions. They show different characteristics and advantages (e.g., good biocompatibility and high stability) compared with monomeric peptides, which form the basis for potential application in the fields of drug delivery, tissue engineering, and antiseptics. In this paper, the molecular mechanisms, types and influencing factors of forming self-assembled peptides were reviewed, followed by introducing the latest advances on fibrous peptide hydrogels and self-assembled antimicrobial peptides. Furthermore, the challenges and perspectives for peptide self-assembly technology were discussed.
Assuntos
Hidrogéis , Peptídeos , Sistemas de Liberação de Medicamentos , Engenharia TecidualRESUMO
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.
RESUMO
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 ß-sheet structures in the anionic membrane-mimetic environment, we found that high ß-sheet amphipathic was accompanied by strong antimicrobial activity. Of such peptides, W5 ([RW]4W) and L5 ([RL]4L) with a nicely amphipathic ß-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 ß-sheet structure is an essential factor that significantly affects the antimicrobial properties.
RESUMO
Commensalism coinfection of pathogens has seriously jeopardized human health. Currently, Kunitzin-RE, as an amphibian-derived bioactivity peptide, is regarded as a potential antimicrobial candidate. However, its antimicrobial properties were unsatisfactory. In this study, a set of shortened variants of Kunitzin-RE was developed by the interception of a peptide fragment and single-site mutation to investigate the effect of chain length, positive charge, hydrophobicity, amphipathicity, and secondary structure on antimicrobial properties. Among them, W8 (AARIILRWRFR) significantly broadened the antimicrobial spectrum and showed the highest antimicrobial activity (GMall = 2.48 µM) against all the fungi and bacteria tested. Additionally, W8 showed high cell selectivity and salt tolerance in vitro, whereas it showed high effectiveness against mice keratitis cause by infection by C. albicans 2.2086. Additionally, it also had obviously lipopolysaccharide-binding ability and a potent membrane-disruptive mechanism. Overall, these findings contributed to the design of short antimicrobial peptides and to combat the serious threat of commensalism coinfection of pathogens.
Assuntos
Antibacterianos/uso terapêutico , Antifúngicos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Antibacterianos/toxicidade , Antifúngicos/farmacologia , Antifúngicos/toxicidade , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/toxicidade , Candida albicans/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Coinfecção/tratamento farmacológico , Desenho de Fármacos , Escherichia coli/efeitos dos fármacos , Infecções Oculares Fúngicas/tratamento farmacológico , Ceratite/tratamento farmacológico , Camundongos , Testes de Sensibilidade Microbiana , Conformação Proteica em alfa-Hélice , Engenharia de Proteínas , Células RAW 264.7 , Salmonella typhimurium/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacosRESUMO
Poor proteolytic resistance is an urgent problem to be solved in the clinical application of antimicrobial peptides (AMPs), yet common solutions, such as complicated chemical modifications and utilization of d-amino acids, greatly increase the difficulty and cost of producing AMPs. In this work, a set of novel peptides was synthesized based on an antitrypsin/antichymotrypsin hydrolytic peptide structure unit (XYPX) n (X represents I, L, and V; Y represents R and K), which was designed using a systematic natural amino acid arrangement. Of these peptides, 16 with seven repeat units had the highest average selectivity index (GMSI = 99.07) for all of the Gram-negative bacteria tested and remained highly effective in combating Escherichia coli infection in vivo. Importantly, 16 also had dramatic resistance to a high concentration of trypsin/chymotrypsin hydrolysis and exerted bactericidal activity through a membrane-disruptive mechanism. Overall, these findings provide new approaches for the development of antiprotease hydrolytic peptides that target Gram-negative bacteria.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Materiais Biocompatíveis , Bactérias Gram-Negativas/ultraestrutura , Células HEK293 , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Proteólise , Células RAW 264.7 , Espectrometria de FluorescênciaRESUMO
Antimicrobial peptides are an important weapon against invading pathogens and are potential candidates as novel antibacterial agents, but their antifungal activities are not fully developed. In this study, a set of imperfectly amphipathic peptides was developed based on the imperfectly amphipathic palindromic structure R n(XRXXXRX)R n ( n = 1, 2; X represents L, I, F, or W), and the engineered peptides exhibited high antimicrobial activities against all fungi and bacteria tested (including fluconazole-resistant Candida albicans), with geometric mean (GM) MICs ranging from 2.2 to 6.62 µM. Of such peptides, 13 (I6) (RRIRIIIRIRR-NH2) that was Ile rich in its hydrophobic face had the highest antifungal activity (GMfungi = 1.64 µM) while showing low toxicity and high salt and serum tolerance. It also had dramatic LPS-neutralizing propensity and a potent membrane-disruptive mechanism against microbial cells. In summary, these findings were useful for short AMPs design to combat the growing threat of drug-resistant fungal and bacterial infections.