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Medical Microbiology and Immunology Dec 2019In the past few years the increasing incidence of hospital infections with Acinetobacter baumannii, especially in immunocompromised patients, and its proneness to...
In the past few years the increasing incidence of hospital infections with Acinetobacter baumannii, especially in immunocompromised patients, and its proneness to develop multidrug resistance have been raising considerable concern. This study examines the antimicrobial and antibiofilm activity of protegrin 1 (PG-1), an antimicrobial peptide from porcine leukocytes, against A. baumannii strains isolated from surgical wounds. PG-1 was tested both alone and combined with the antibiotics commonly used in clinical settings. Its antimicrobial activity was evaluated by determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), checkerboard assays, and time-kill experiments. Its effects on biofilm inhibition/eradication were tested with crystal violet staining. The strains were grown in subinhibitory or increasing PG-1 concentrations to test the development of resistance. Mammalian cell toxicity was tested by XTT assays. PG-1 MICs and MBCs ranged from 2 to 8 µg/ml. PG-1 was most active and demonstrated a synergistic interaction with colistin, a last resort antibiotic. Interestingly, antagonism was never observed. In time-kill experiments, incubation with 2 × MIC for 30 min suppressed all viable cells. PG-1 did not select resistant strains and showed a limited effect on cell viability, but it did exert a strong activity against multidrug-resistant A. baumannii. In contrast, in our experimental conditions it had no effect on biofilm inhibition/eradication. PG-1 thus seems to be a promising antimicrobial agent against multidrug-resistant Gram-negative infections.
Topics: Acinetobacter baumannii; Anti-Infective Agents; Antimicrobial Cationic Peptides; Biofilms; Cell Survival; Drug Interactions; Epithelial Cells; HeLa Cells; Humans; Microbial Sensitivity Tests; Staining and Labeling; Surgical Wound
PubMed: 31214759
DOI: 10.1007/s00430-019-00624-7 -
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 -
Journal of Peptide Science : An... Mar 2019Protegrin-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...
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 β 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.
Topics: Animals; Antimicrobial Cationic Peptides; Bacillus subtilis; Caco-2 Cells; Dose-Response Relationship, Drug; HEK293 Cells; Humans; Microbial Sensitivity Tests; Microbial Viability; Protein Aggregates; Protein Aggregation, Pathological; Sus scrofa
PubMed: 30714272
DOI: 10.1002/psc.3151 -
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 -
Journal of Biomolecular Structure &... Jun 2019Achieving both, nontoxicity and stability in antimicrobial peptides (AMP) is a challenge. This study predicts a structurally stable, nontoxic scaffold among the...
Achieving both, nontoxicity and stability in antimicrobial peptides (AMP) is a challenge. This study predicts a structurally stable, nontoxic scaffold among the protegrin family, for future therapeutic peptide analogs. Protegrins (PG) are a class of pharmaceutically approved, in demand AMPs, which require further improvement in terms of nontoxicity and stability. Out of five protegrins viz., PG1, PG2, PG3, PG4 and PG5, PG1 has been predicted as best scaffold. Prediction was based upon sequential elimination of other protegrins, using computational methods to assess the extracellular bacterial membrane penetrability, nontoxicity and structural stability by geometric observables. Initially, PG2 and PG4 showing the lowest membrane penetrability and highest toxicity respectively, were screened out. Among the remaining three protegrins, PG1 displayed both lowest root mean square deviation and radius of gyration, with a considerable occupancy of seven H-bonds and established uniform secondary structure profile throughout its ensembles. Therefore, the authors claim the superiority of PG1 as a nontoxic stable scaffold among its family. Communicated by Ramaswamy H. Sarma.
Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Bacteria; Cell Membrane; Hydrogen Bonding; Protein Conformation; Protein Isoforms; Sequence Homology, Amino Acid
PubMed: 30047844
DOI: 10.1080/07391102.2018.1491418 -
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