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Molecules (Basel, Switzerland) Dec 2020Respiratory infections are a real threat for humans, and therefore the pig model is of interest for studies. As one of a case for studies, (APP) caused infections and...
Respiratory infections are a real threat for humans, and therefore the pig model is of interest for studies. As one of a case for studies, (APP) caused infections and still worries many pig breeders around the world. To better understand the influence of pathogenic effect of APP on a respiratory system-lungs and tracheobronchial lymph nodes (TBLN), we aimed to employ matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF MSI). In this study, six pigs were intranasally infected by APP and two were used as non-infected control, and 48 cryosections have been obtained. MALDI-TOF MSI and immunohistochemistry (IHC) were used to study spatial distribution of infectious markers, especially interleukins, in cryosections of porcine tissues of lungs (necrotic area, marginal zone) and tracheobronchial lymph nodes (TBLN) from pigs infected by APP. CD163, interleukin 1β (IL‑1β) and a protegrin-4 precursor were successfully detected based on their tryptic fragments. CD163 and IL‑1β were confirmed also by IHC. The protegrin-4 precursor was identified by MALDI-TOF/TOF directly on the tissue cryosections. CD163, IL‑1β and protegrin‑4 precursor were all significantly ( < 0.001) more expressed in necrotic areas of lungs infected by APP than in marginal zone, TBLN and in control lungs.
Topics: Actinobacillus Infections; Actinobacillus pleuropneumoniae; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antimicrobial Cationic Peptides; Biomarkers; Bronchi; Interleukin-1beta; Lung; Lymph Nodes; Receptors, Cell Surface; Respiratory Tract Infections; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Swine
PubMed: 33287430
DOI: 10.3390/molecules25235723 -
Scientific Reports Sep 2020About 70% of all antibiotics produced in the world are used in the farm animal industry. The massive usage of antibiotics during farm animal production has caused rapid...
About 70% of all antibiotics produced in the world are used in the farm animal industry. The massive usage of antibiotics during farm animal production has caused rapid development of antibiotic resistance in bacteria, which poses a serious risk to human and livestock health when treating bacterial infections. Protegrin-1 (PG-1) is a potent antimicrobial peptide (AMP). It was initially identified in pig leukocytes with a broad-spectrum antibacterial and antiviral activity, and a low rate of inducing bacterial resistance. To develop a genetic approach for reducing the use of antibiotics in farm animal production, we produced transgenic mice carrying a bovine tracheal AMP gene promoter-controlled PG-1 transgene. The PG-1 transgene was specifically expressed in the respiratory tract of transgenic mice upon induction by bacterial infection. These PG-1 transgenic mice exhibited enhanced resistance to nasal bacterial infection as the transgenic mice showed a higher survival rate (79.17% VS. 34.78%), lower bacterial load and milder histological severity than their wild-type control littermates. The improved resistance to bacterial infection in the PG-1 transgenic mice could be resulted from the direct bacteria-killing activities of PG-1, and the immunomodulatory effects of PG-1 via stimulating interleukin 1 beta secretion. The present study provides a promising genetic strategy to prevent airway bacterial infections in farm animals by bacteria-inducible tissue-specific expression of PG-1 transgene. This approach may also be helpful for decreasing the possibility of inducing bacterial resistance during farm animal production.
Topics: Animals; Antimicrobial Cationic Peptides; Bacterial Infections; Disease Models, Animal; Humans; Interleukin-1beta; Mice; Mice, Transgenic; Microbial Sensitivity Tests; Promoter Regions, Genetic; Respiratory System; Respiratory Tract Infections; Survival Analysis
PubMed: 32994542
DOI: 10.1038/s41598-020-73084-2 -
Biomolecules Jul 2020Multi-drug resistant (MDR) bacteria and their biofilms are a concern in veterinary and human medicine. Protegrin-1 (PG-1), a potent antimicrobial peptide (AMP) with...
The Addition of a Synthetic LPS-Targeting Domain Improves Serum Stability While Maintaining Antimicrobial, Antibiofilm, and Cell Stimulating Properties of an Antimicrobial Peptide.
Multi-drug resistant (MDR) bacteria and their biofilms are a concern in veterinary and human medicine. Protegrin-1 (PG-1), a potent antimicrobial peptide (AMP) with antimicrobial and immunomodulatory properties, is considered a potential alternative for conventional antibiotics. AMPs are less stable and lose activity in the presence of physiological fluids, such as serum. To improve stability of PG-1, a hybrid peptide, SynPG-1, was designed. The antimicrobial and antibiofilm properties of PG-1 and the PG-1 hybrid against MDR pathogens was analyzed, and activity after incubation with physiological fluids was compared. The effects of these peptides on the IPEC-J2 cell line was also investigated. While PG-1 maintained some activity in 25% serum for 2 h, SynPG-1 was able to retain activity in the same condition for up to 24 h, representing a 12-fold increase in stability. Both peptides had some antibiofilm activity against and . While both peptides prevented biofilm formation of methicillin-resistant (MRSA), neither could destroy MRSA's pre-formed biofilms. Both peptides maintained activity after incubation with trypsin and porcine gastric fluid, but not intestinal fluid, and stimulated IPEC-J2 cell migration. These findings suggest that SynPG-1 has much better serum stability while maintaining the same antimicrobial potency as PG-1.
Topics: Animals; Antimicrobial Cationic Peptides; Biofilms; Cell Line; Cell Proliferation; Culture Media; Drug Design; Drug Resistance, Multiple, Bacterial; Drug Stability; Escherichia coli; Lipopolysaccharides; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Pore Forming Cytotoxic Proteins; Salmonella typhimurium; Swine
PubMed: 32650576
DOI: 10.3390/biom10071014 -
The Journal of Membrane Biology Aug 2020The original version of the article was published without the Graphic Abstract. Graphic Abstract image of the article is given below.
The original version of the article was published without the Graphic Abstract. Graphic Abstract image of the article is given below.
PubMed: 32601712
DOI: 10.1007/s00232-020-00128-z -
The Journal of Membrane Biology Jun 2020Protegrin-1 (PG-1), an 18-residue β-hairpin stabilized by two disulfide bonds, is a member of a family of powerful antimicrobial peptides which are believed to act...
Protegrin-1 (PG-1), an 18-residue β-hairpin stabilized by two disulfide bonds, is a member of a family of powerful antimicrobial peptides which are believed to act through membrane permeabilization. Here we used a combination of experimental and computational approaches to characterize possible structural arrangements of PG-1 in lipid bilayers mimicking bacterial membranes. We have measured the dose-response function of the PG-1-induced leakage of markers of various sizes from vesicles and found it to be consistent with the formation of pores of two different sizes. The first one allows the release of small dyes and occurs at peptide:lipid ratios < 0.006. Above this ratio, larger pores are observed through which the smallest of dextrans FD4 can be released. In parallel with pore formation, we observe a general large-scale destabilization of vesicles which is probably related to complete rupture of some vesicles. The population of vesicles that are completely ruptured depends linearly on PG-1:lipid ratio. Neither pore size, nor vesicle rupture are influenced by the formation of disulfide bonds. Previous computational work on oxidized protegrin is complemented here by all-atom MD simulations of PG-1 with reduced disulfide bonds both in solution (monomer) and in a bilayer (dimer and octamer). The simulations provide molecular insights into the influence of disulfide bonds on peptide conformation, aggregation, and oligomeric structure.
Topics: Algorithms; Antimicrobial Cationic Peptides; Lipid Bilayers; Models, Molecular; Models, Theoretical; Molecular Conformation; Structure-Activity Relationship
PubMed: 32500172
DOI: 10.1007/s00232-020-00124-3 -
Animals : An Open Access Journal From... Mar 2020The emergence of staphylococcal canine pathogens resistant to multiple antimicrobial agents is a growing and urgent problem in veterinary practice. Antimicrobial...
The emergence of staphylococcal canine pathogens resistant to multiple antimicrobial agents is a growing and urgent problem in veterinary practice. Antimicrobial peptides (AMPs) seem to be a promising alternative to conventional antibiotics. The aim of this in vitro study was to evaluate the antimicrobial activity of selected AMPs against pathogenic staphylococcal strains, including multidrug- and methicillin-resistant strains isolated from canine pyoderma cases. Seven antimicrobial peptides (aurein 1.2, CAMEL, citropin 1.1, protegrin-1, pexiganan, temporin A and uperin 3.6) synthesized by the 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase method were tested. The minimal inhibitory and minimal bactericidal concentrations (MIC and MBC) were determined by the broth microdilution method. The study showed that analyzed AMPs exerted an extensive effect against canine pathogens, with the most active peptide being uperin 3.6. The tested AMPs were equally efficient against both resistant- and susceptible staphylococcal strains and were more efficient against S than against strains. Our findings are particularly interesting from a clinical perspective, as they point to AMPs as potential therapeutic topical agents in canine pyoderma cases associated with antimicrobial resistance of staphylococci.
PubMed: 32168952
DOI: 10.3390/ani10030470 -
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 -
Proteins Mar 2020The cross-strand disulfides (CSDs) found in β-hairpin antimicrobial peptides (β-AMPs) show a unique disulfide geometry that is characterized by unusual torsion angles...
The cross-strand disulfides (CSDs) found in β-hairpin antimicrobial peptides (β-AMPs) show a unique disulfide geometry that is characterized by unusual torsion angles and a short Cα-Cα distance. While the sequence and disulfide bond connectivity of disulfide-rich peptides is well studied, much less is known about the disulfide geometry found in CSDs and their role in the stability of β-AMPs. To address this, we solved the nuclear magnetic resonance (NMR) structure of the β-AMP gomesin (Gm) at 278, 298, and 310 K, examined the disulfide bond geometry of over 800 disulfide-rich peptides, and carried out extensive molecular dynamics (MD) simulation of the peptides Gm and protegrin. The NMR data suggests Cα-Cα distances characteristic for CSDs are independent of temperature. Analysis of disulfide-rich peptides from the Protein Data Bank revealed that right-handed and left-handed rotamers are equally likely in CSDs. The previously reported preference for right-handed rotamers was likely biased by restricting the analysis to peptides and proteins solved using X-ray crystallography. Furthermore, data from MD simulations showed that the short Cα-Cα distance is critical for the stability of these peptides. The unique disulfide geometry of CSDs poses a challenge to biomolecular force fields and to retain the stability of β-hairpin fold over long simulation times, restraints on the torsion angles might be required.
Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Disulfides; Molecular Dynamics Simulation; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Stability; Spiders; Stereoisomerism; Temperature; Thermodynamics
PubMed: 31589791
DOI: 10.1002/prot.25828 -
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