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Biophysical Journal Jan 2020Multidrug-resistant Gram-negative bacteria have increased the prevalence of a variety of serious diseases in modern times. Polymyxins are used as the last-line...
Multidrug-resistant Gram-negative bacteria have increased the prevalence of a variety of serious diseases in modern times. Polymyxins are used as the last-line therapeutic options for the treatment of infections. However, the mechanism of action of polymyxins remains in dispute. In this work, we used a coarse-grained molecular dynamics simulation to investigate the mechanism of the cationic antimicrobial peptide polymyxin B (PmB) interacting with both the inner and outer membrane models of bacteria. Our results show that the binding of PmB disturbs the outer membrane by displacing the counterions, decreasing the orientation order of the lipopolysaccharide tail, and creating more lipopolysaccharide packing defects. Upon binding onto the inner membrane, in contrast to the traditional killing mechanism that antimicrobial peptides usually use to induce holes in the membrane, PmBs do not permeabilize the inner membrane but stiffen it by filling up the lipid packing defect, increasing the lipid tail order and the membrane bending rigidity as well as restricting the lipid diffusion. PmBs also mediate intermembrane contact and adhesion. These joint effects suggest that PmBs deprive the biological activity of Gram-negative bacteria by sterilizing the cell.
Topics: Antimicrobial Cationic Peptides; Cell Membrane; Lipid Bilayers; Lipopolysaccharides; Melitten; Models, Molecular; Phospholipids; Polymyxin B; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand
PubMed: 31812355
DOI: 10.1016/j.bpj.2019.11.008 -
International Journal of Molecular... Aug 2019Antibacterial peptides (APMs) are a new type of antibacterial substance. The relationship between their structure and function remains indistinct; in particular, there...
Antibacterial peptides (APMs) are a new type of antibacterial substance. The relationship between their structure and function remains indistinct; in particular, there is a lack of a definitive and fixed template for designing new antimicrobial peptides. Previous studies have shown that porcine Protegrin-1 (PG-1) exhibits considerable antimicrobial activity and cytotoxicity. In this study, to reduce cytotoxicity and increase cell selectivity, we designed histidine-rich peptides based on the sequence template RR(XY)XPGX(YX)RR-NH, where X represents I, W, V, and F. The results showed that the peptides form more β-hairpin structures in a lipid-rich environment that mimics cell membranes. Among them, the antimicrobial peptide HV2 showed strong antibacterial activity against Gram-negative strains and almost no toxicity to normal cells. The results of our analysis of its antibacterial mechanism showed that peptide HV2 acts on the bacterial cell membrane to increase its permeability, resulting in cell membrane disruption and death. Furthermore, peptide HV2 inhibited bacterial movement in a concentration-dependent manner and had a more robust anti-inflammatory effect by inhibiting the production of TNF-α. In summary, peptide HV2 exhibits high bactericidal activity and cell selectivity, making it a promising candidate for future use as an antibiotic.
Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Anti-Inflammatory Agents; Antimicrobial Cationic Peptides; Cell Membrane Permeability; Drug Design; Gram-Negative Bacteria; Histidine; Mice; Microbial Sensitivity Tests; Models, Molecular; Molecular Conformation; Peptides; Protein Conformation, beta-Strand; RAW 264.7 Cells
PubMed: 31416220
DOI: 10.3390/ijms20163954 -
Scientific Reports Aug 2019Porcine protegrin-1 (PG-1) is a broad-spectrum antimicrobial peptide (AMP) with potent antimicrobial activities. We produced recombinant PG-1 and evaluated its...
Porcine protegrin-1 (PG-1) is a broad-spectrum antimicrobial peptide (AMP) with potent antimicrobial activities. We produced recombinant PG-1 and evaluated its cytotoxicity toward various types of mammalian cell lines, including embryonic fibroblasts, retinal cells, embryonic kidney cells, neuroblastoma cells, alveolar macrophage cells, and neutrophils. The sensitivity of the different mammalian cells to cytotoxic damage induced by PG-1 differed significantly among the cell types, with retinal neuron cells and neutrophils being the most significantly affected. A circular dichroism analysis showed there was a precise correlation between conformational changes in PG-1 and the magnitude of cytotoxicity among the various cell type. Subsequently, a green fluorescent protein (GFP) penetration assay using positively charged GFPs indicated there was a close correlation between the degree of penetration of charged GFP into cells and the magnitude of PG-1 cytotoxicity. Furthermore, we also showed that inhibition of the synthesis of anionic sulphated proteoglycans on the cell surface decreases the cytotoxic damage induced by PG-1 treatment. Taken together, the observed cytotoxicity of PG-1 towards different membrane surfaces is highly driven by the membrane's anionic properties. Our results reveal a possible mechanism underlying cell-type dependent differences in cytotoxicity of AMPs, such as PG-1, toward mammalian cells.
Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Cell Line; Cell Survival; HEK293 Cells; Humans; Mice; Models, Molecular; NIH 3T3 Cells; Neurons; Protein Conformation; Recombinant Proteins; Retina
PubMed: 31399625
DOI: 10.1038/s41598-019-47955-2 -
Frontiers in Cellular and Infection... 2019Rapidly growing resistance of pathogenic bacteria to conventional antibiotics leads to inefficiency of traditional approaches of countering infections and determines the...
Rapidly growing resistance of pathogenic bacteria to conventional antibiotics leads to inefficiency of traditional approaches of countering infections and determines the urgent need for a search of fundamentally new anti-infective drugs. Antimicrobial peptides (AMPs) of the innate immune system are promising candidates for a role of such novel antibiotics. However, some cytotoxicity of AMPs toward host cells limits their active implementation in medicine and forces attempts to design numerous structural analogs of the peptides with optimized properties. An alternative route for the successful AMPs introduction may be their usage in combination with conventional antibiotics. Synergistic antibacterial effects have been reported for a number of such combinations, however, the molecular mechanisms of the synergy remain poorly understood and little is known whether AMPs cytotoxicy for the host cells increases upon their application with antibiotics. Our study is directed to examination of a combined action of natural AMPs with different structure and mode of action (porcine protegrin 1, caprine bactenecin ChBac3.4, human alpha- and beta-defensins (HNP-1, HNP-4, hBD-2, hBD-3), human cathelicidin LL-37), and egg white lysozyme with varied antibiotic agents (gentamicin, ofloxacin, oxacillin, rifampicin, polymyxin B, silver nanoparticles) toward selected bacteria, including drug-sensitive and drug-resistant strains, as well as toward some mammalian cells (human erythrocytes, PBMC, neutrophils, murine peritoneal macrophages and Ehrlich ascites carcinoma cells). Using "checkerboard titrations" for fractional inhibitory concentration indexes evaluation, it was found that synergy in antibacterial action mainly occurs between highly membrane-active AMPs (e.g., protegrin 1, hBD-3) and antibiotics with intracellular targets (e.g., gentamicin, rifampcin), suggesting bioavailability increase as the main model of such interaction. In some combinations modulation of dynamics of AMP-bacterial membrane interaction in presence of the antibiotic was also shown. Cytotoxic effects of the same combinations toward normal eukaryotic cells were rarely synergistic. The obtained data approve that combined application of antimicrobial peptides with antibiotics or other antimicrobials is a promising strategy for further development of new approach for combating antibiotic-resistant bacteria by usage of AMP-based therapeutics. Revealing the conventional antibiotics that increase the activity of human endogenous AMPs against particular pathogens is also important for cure strategies elaboration.
Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacteria; Cell Line; Cell Survival; Drug Synergism; Humans; Microbial Sensitivity Tests
PubMed: 31114762
DOI: 10.3389/fcimb.2019.00128 -
Biochimica Et Biophysica Acta.... Oct 2019Antimicrobial peptides (AMPs) are a promising class of innate host defense molecules for next-generation antibiotics, as they uniquely target and permeabilize membranes...
Antimicrobial peptides (AMPs) are a promising class of innate host defense molecules for next-generation antibiotics, as they uniquely target and permeabilize membranes of pathogens. This selectivity has been explained by the electrostatic attraction between these predominantly cationic peptides and the bacterial membrane, which is heavily populated with anionic lipids. However, AMP-resistant bacteria have non-electrostatic countermeasures that modulate membrane rigidity and thickness. We explore how variations in physical properties affect the membrane affinity and disruption process of protegrin-1 (PG-1) in phosphatidylcholine (PC) membranes with altered lipid packing densities and thicknesses. From isothermal titration calorimetry and atomic force microscopy, our results showed that PG-1 could no longer insert into membranes of increasing cholesterol amounts nor into monounsaturated PC membranes of increasing thicknesses with similar fluidities. Prevention of PG-1's incorporation consequently made the membranes more resistant to peptide-induced structural transformations like pore formation. Our study provides evidence that AMP affinity and activity are strongly correlated with the fluidity and thickness of the membrane. A basic understanding of how physical mechanisms can regulate cell selectivity and resistance towards AMPs will aid in the development of new antimicrobial agents.
Topics: Anti-Bacterial Agents; Anti-Infective Agents; Antimicrobial Cationic Peptides; Calorimetry; Cell Membrane; Cholesterol; Lipid Bilayers; Membrane Lipids; Microscopy, Atomic Force; Peptides; Phosphatidylcholines; Static Electricity
PubMed: 31077677
DOI: 10.1016/j.bbamem.2019.04.011 -
Frontiers in Pharmacology 2019Cathelicidins, a class of antimicrobial peptides, have been widely studied for their antimicrobial role in innate immune responses during infection and inflammation. At...
Cathelicidins, a class of antimicrobial peptides, have been widely studied for their antimicrobial role in innate immune responses during infection and inflammation. At sub-antimicrobial concentrations, various cathelicidins from different species have been reported to exert chemotactic activity on neutrophils, monocytes, dendritic cells and T-cells, and also enhance angiogenesis and wound healing. To date, the role of the pig cathelicidin, protegrin-1 (PG-1), in immune modulation and tissue repair in the intestinal tract has not been investigated. The aim of the present study was to examine the potential protective effects of recombinant PG-1 in a mouse dextran sodium sulfate (DSS)-induced colitis inflammation model. This is the first report showing the protective effects of PG-1 in its various forms (pro-, cathelin-, and mature-forms) in attenuating significant body weight loss associated with DSS-induced colitis ( < 0.05). PG-1 treatment improved histological scores ( < 0.05) and influenced the gene expression of inflammatory mediators and tissue repair factors such as trefoil factor 3 (TFF3) and mucin (MUC-2). Protegrin treatment also altered the metabolite profile, returning the metabolite levels closer to untreated control levels. These findings lay the foundation for future oral application of recombinant PG-1 to potentially treat intestinal damage and inflammation.
PubMed: 30873029
DOI: 10.3389/fphar.2019.00156 -
Frontiers in Cellular and Infection... 2018Antimicrobial peptides (AMPs) represent a promising area of research to help combat the ever-growing problem of antibiotic resistance. Protegrin-1 is an AMP from the...
Antimicrobial peptides (AMPs) represent a promising area of research to help combat the ever-growing problem of antibiotic resistance. Protegrin-1 is an AMP from the cathelicidin family. It is produced naturally in pigs and its mature form (mPG-1) has potent bactericidal properties and a unique β-hairpin structure that separates it from most AMPs found in mice and humans. While the antibacterial properties of protegrin-1 are well established, the role it plays in immune modulation has yet to be investigated, and our current study sought to explore this alternate role and potential mechanism behind. We found that mPG-1 stimulated intestinal cell migration, this is accompanied with altered expression of genes associated with cell migration, in addition to increased expression of pro-inflammatory cytokines and immune-related factors. Further study suggested that mPG-1 activates insulin-like growth factor 1 receptor (IGF1R) and through this receptor it modulates immune activity as well as cell migration. Our study revealed a novel function of mPG-1, and its associated pathway, suggesting therapeutic potential of the antimicrobial peptide for infection and/or immune disorders, particularly ones affecting the gastrointestinal tract such as inflammatory bowel syndrome.
Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Cells, Cultured; Immunity, Innate; Receptor, IGF Type 1; Swine
PubMed: 30324092
DOI: 10.3389/fcimb.2018.00331 -
Frontiers in Microbiology 2018Protegrin (PG) belongs to the antimicrobial peptide cathelicidin family. To date, five protegrin sequences have been identified in pigs, PG-1 to PG-5. Of these, PG-1...
Protegrin (PG) belongs to the antimicrobial peptide cathelicidin family. To date, five protegrin sequences have been identified in pigs, PG-1 to PG-5. Of these, PG-1 exhibits potent antimicrobial activity against a broad range of antibiotic-resistant microorganisms as well as viruses. However, the other potential role(s) of PG beyond antimicrobial has largely been unexplored. The aim of this study was to use nonpathogenic yeast to express antimicrobially active recombinant protegrin (rPG-1). Additionally, the effect of PG-1 on cell migration and proliferation was also examined using pig intestinal epithelial cells as a model. Highest level of rPG-1 (104 ± 11 μg/mL) was detected at 24 h in fermentation culture medium. Similar to rPG-1, 0.8 ± 0.10 g/L of proform PG-1 (rProPG-1) and 0.2 ± 0.02 g/L of the PG-1 cathelin domain (rCath) was detected in fermentation culture medium. Resulting recombinant PG-1 and cleaved rProPG-1 exerted antimicrobial activity against DH5α at the same level as chemically synthesized PG-1. Enhanced cell migration was observed ( < 0.05) in groups treated with rProPG-1, rCath, and rPG-1 compared to the control. Furthermore, rPG-1 was stable at temperatures ranging from 25°C to 80°C. In summary, biologically active recombinant protegrin in its pro-, cathelin-, and mature- forms were successfully expressed in suggesting potential feasibility for future therapeutic applications.
PubMed: 30319593
DOI: 10.3389/fmicb.2018.02300 -
ACS Omega Jun 2018Protegrin-1 (PG-1) is a cationic arginine-rich antimicrobial peptide. It is widely accepted that PG-1 induces membrane disruption by forming pores that lead to cell...
Protegrin-1 (PG-1) is a cationic arginine-rich antimicrobial peptide. It is widely accepted that PG-1 induces membrane disruption by forming pores that lead to cell death. However, the insertion mechanism for these highly cationic peptides into the hydrophobic membrane environment is still poorly understood at the molecular scale. It has previously been determined that the association of arginine guanidinium and lipid phosphate groups results in strong bidentate bonds that stabilize peptide-lipid complexes. It has also been suggested that arginine residues are able to drag phosphate groups as they insert inside the membrane to form a toroidal pore. However, whether bidentate bonds play a significant role in inducing a pore formation remains unclear. To investigate the role of bidentate complexes in PG-1 translocation, we conducted molecular dynamics simulations. Two computational electroporation methods were implemented to examine the translocation process. We found that PG-1 could insert into the membrane, provided the external electric potential is large enough to first induce a water column or a pore within the lipid bilayer membrane. We also found that the highly charged PG-1 is capable in itself of inducing molecular electroporation. Substitution of arginines with charge-equivalent lysines showed a markedly reduced tendency for insertion. This indicates that the guanidinium group likely facilitates PG-1 translocation. Potential of mean force calculations suggests that peptide insertion inside the hydrophobic environment of the membrane core is not favored. We found that formation of a water column or a pore might be a prerequisite for PG-1 translocation. We also found that PG-1 can stabilize the pore after insertion. We suggest that PG-1 could be a pore inducer and stabilizer. This work sheds some light on PG-1 translocation mechanisms at the molecular level. Methods presented in this study may be extended to other arginine-rich antimicrobial and cell-penetrating peptides.
PubMed: 29978143
DOI: 10.1021/acsomega.8b00483 -
Annals of Agricultural and... Jun 2017The emergence of resistance in microorganisms on a global scale has made it necessary to search for new antimicrobial factors. Antimicrobial peptides (AMPs) seem to meet...
The emergence of resistance in microorganisms on a global scale has made it necessary to search for new antimicrobial factors. Antimicrobial peptides (AMPs) seem to meet these expectations. AMPs are produced by bacteria, viruses, plants, and animals, and may be considered as a new class of drugs intended for the prophylaxis and treatment of both systemic and topical infections. The aim of this study is to review the results of studies on the use of peptides to combat infections in vivo. Antimicrobial peptides may be applied topically and systemically. Among the peptides used topically, a very important area for their application is ophthalmology. AMPs in ophthalmology may be used mainly for the protection of contact lenses from ocular pathogens. Many AMPs are in clinical trials for application in the therapy of local infections. There may be mentioned such preparations as: pexiganan (magainin analogue), MX-226 (based on indolicidin), NEUPREX (isolated from human BPI (bactericidal/permeability-increasing) protein), IB-367 (variant of porcine protegrin), P113 (based on histatin), daptomycin, polymyxins, as well as peptidomimetics. In the combat against systemic infections are used such peptides as: P113D (modified P113 peptide containing D-amino acids), colistin, peptoids, and peptides containing non-typical amino acids or non-peptide elements. AMPs are also used as antiprotozoal, antifungal, antitoxic and immunostimulatory agents. The limitations in the use of peptides in the treatment of infections, such as susceptibility to proteolysis, and resistance of microorganisms to the peptides, are also discussed. AMPs are a promising strategy in the fight against microbial infections.
Topics: Animals; Anti-Bacterial Agents; Bacteria; Bacterial Infections; Bacterial Physiological Phenomena; Humans; Peptides
PubMed: 29936826
DOI: 10.26444/aaem/74471