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American Journal of Physiology.... Sep 2021Neonatal antibiotics administered to human infants initiate gut microbiota dysbiosis that may have long-term effects on body weight and metabolism. We examined...
Neonatal antibiotics administered to human infants initiate gut microbiota dysbiosis that may have long-term effects on body weight and metabolism. We examined antibiotic-induced adaptations in pancreatic islets of the piglet, a well-accepted model of human infant microbiota and pancreas development. Neonatal piglets randomized to amoxicillin [30 mg/kg body wt/day; = 7, antibiotic (ANTI)] or placebo [vehicle control; = 7, control (CON)] from ()- were euthanized at , , and . The metabolic phenotype along with functional, immunohistological, and transcriptional phenotypes of the pancreatic islets were studied. The gut microbiome was characterized by 16S rRNA gene sequencing, and microbial metabolites and microbiome-sensitive host molecules were measured. Compared with CON, ANTI piglets had elevated transcripts of genes involved in glucagon-like peptide 1 ((GLP-1) synthesis or signaling in islets ( < 0.05) coinciding with higher plasma GLP-1 ( = 0.11), along with increased tumor necrosis factor α () ( < 0.05) and protegrin 1 () ( < 0.05). Antibiotic-induced relative increases in , , , , and of the ileal microbiome at normalized after antibiotic withdrawal. In ANTI islets at , the expression of key regulators pancreatic and duodenal homeobox 1 (), insulin-like growth factor-2 (), and transcription factor 7-like 2 () was downregulated, preceding a 40% reduction of β-cell area ( < 0.01) and islet insulin content at ( < 0.05). At , a twofold elevated plasma insulin concentration ( = 0.07) was observed in ANTI compared with CON. We conclude that antibiotic treatment of neonatal piglets elicited gut microbial changes accompanied by phasic alterations in key regulatory genes in pancreatic islets at and . By , reduced β-cell area and islet insulin content were accompanied by elevated nonfasted insulin despite normoglycemia, indicative of islet stress.
Topics: Animals; Anti-Bacterial Agents; Gastrointestinal Microbiome; Glucagon; Glucagon-Like Peptide 1; Insulin; Insulin-Secreting Cells; Islets of Langerhans; Swine
PubMed: 34259034
DOI: 10.1152/ajpregu.00090.2021 -
Chemistry (Weinheim An Der Bergstrasse,... Sep 2021The search for novel antimicrobial agents to combat microbial pathogens is intensifying in response to the rapid development of drug resistance to current antibiotic...
The search for novel antimicrobial agents to combat microbial pathogens is intensifying in response to the rapid development of drug resistance to current antibiotic therapeutics. Respiratory failure and septicemia are the leading causes of mortality among hospitalized patients. Here, the development of a novel engineered cyclotide with effective broad-spectrum antibacterial activity against several ESKAPE bacterial strains and clinical isolates is reported. The most active antibacterial cyclotide was extremely stable in serum, showed little hemolytic activity, and provided protection in vivo in a murine model of P. aeruginosa peritonitis. These results highlight the potential of the cyclotide scaffold for the development of novel antimicrobial therapeutic leads for the treatment of bacteremia.
Topics: Animals; Anti-Bacterial Agents; Anti-Infective Agents; Cyclotides; Humans; Mice; Microbial Sensitivity Tests; Pseudomonas aeruginosa
PubMed: 34159664
DOI: 10.1002/chem.202101438 -
Frontiers in Physiology 2021Antimicrobial peptides (AMPs) are traditionally known to be essential components in host defense their broad activities against bacteria, fungi, viruses, and protozoa....
Antimicrobial peptides (AMPs) are traditionally known to be essential components in host defense their broad activities against bacteria, fungi, viruses, and protozoa. Their immunomodulatory properties have also recently received considerable attention in mammalian somatic tissues of various species. However, little is known regarding the role of AMPs in the development and maturation of ovarian follicles. Protegrin-1 (PG-1) is an antimicrobial peptide which is known to have potent antimicrobial activity against both gram positive and negative bacteria. Here we report that the PG-1 is present in the porcine ovarian follicular fluid. Treatment of granulosa cell with PG-1 enhanced granulosa cell proliferation in a dose-dependent manner. This is accompanied by increased expression of cell-cycle progression-related genes such as cyclin D1(), cyclin D2 (), and cyclin B1(). Additionally, Western blot analysis showed that PG-1 increased phosphorylated epidermal growth factor receptor (EGFR), and the phosphorylated-/total extracellular signal-regulated kinase (ERK)1/2 ratio. Pretreatment with either U0126, a specific ERK1/2 phosphorylation inhibitor, or EGFR kinase inhibitor, AG1478, blocked the PG-1 induced proliferation. Moreover, luciferase reporter assay revealed that ETS domain-containing protein-1 (Elk1) C/EBP homologous protein (CHOP), and the transcription activators downstream of the MAPK pathway, were activated by PG-1. These data collectively suggest that PG-1 may regulate pig granulosa cell proliferation via EGFR-MAPK pathway., Hence, our finding offers insights into the role of antimicrobial peptides on follicular development regulation.
PubMed: 34093234
DOI: 10.3389/fphys.2021.673777 -
International Journal of Molecular... Apr 2021The efficiency of existing cell lysis methods to isolate nucleic acids from diverse bacteria varies depending on cell wall structures. This study tested a novel idea of...
The efficiency of existing cell lysis methods to isolate nucleic acids from diverse bacteria varies depending on cell wall structures. This study tested a novel idea of using broad-spectrum antimicrobial peptides to improve the lytic efficiency of hard-to-lyse bacteria and characterized their differences. The lysis conditions of using recombinant porcine myeloid antimicrobial peptide 36 (PMAP-36), a broad-spectrum pig cathelicidin, was optimized, and RNA isolation was performed with cultured pellets of ten bacterial species using various membranolytic proteins. Additionally, three other antimicrobial peptides, protegrin-1 (PG-1), melittin, and nisin, were evaluated for their suitability as the membranolytic agents of bacteria. However, PMAP-36 use resulted in the most successful outcomes in RNA isolation from diverse bacterial species. The amount of total RNA obtained using PMAP-36 increased by ~2-fold compared to lysozyme in . species were refractory to all lytic proteins tested, although the RNA yield from PMAP-36 treatment was slightly higher than that from other methods. PMAP-36 use produced high-quality RNA, and reverse transcription PCR showed the efficient amplification of the 16S rRNA gene from all tested strains. Additionally, the results of genomic DNA isolation were similar to those of RNA isolation. Thus, our findings present an additional option for high quality and unbiased nucleic acid isolation from microbiomes or challenging bacterial strains.
Topics: Antimicrobial Cationic Peptides; Cell Fractionation; DNA, Bacterial; RNA, Bacterial; Staphylococcus aureus
PubMed: 33923762
DOI: 10.3390/ijms22084149 -
International Journal of Molecular... Feb 2021Antimicrobial peptides (AMPs) are of interest as alternatives to antibiotics or immunomodulators. We generated and characterized the phenotypes of transgenic mice...
Antimicrobial peptides (AMPs) are of interest as alternatives to antibiotics or immunomodulators. We generated and characterized the phenotypes of transgenic mice overexpressing protegrin 1 (PG1), a potent porcine cathelicidin. No obvious differences were observed between PG1 transgenic and wild-type mice in terms of growth, development, general behaviour, and the major immune cell population. However, PG1 transgenic mice intranasally infected with resulted in a reduction in microscopic pulmonary injury, improved clearance of bacteria, and lower proinflammatory cytokine secretion, compared to those of wild-type mice. On the other hand, approximately 25% of PG1 transgenic mice ( = 54/215) showed corneal opacity and developed inflammation in the eye, resulting ultimately in phthisis bulbi. Immunohistochemical analyses revealed that PG1 and its activator, neutrophil elastase, localized to the basal cells of the cornea and glands in eyelids, respectively. In addition, apoptosis indicated by a Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive signal was detected from flat cells of the cornea. Our study suggests that the expression regulation or localization of AMPs such as PG1 is important to prevent their adverse effects. However, our results also showed that the cytotoxic effects of PG1 on cells could be tolerated in animals, except for the eyes.
Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Corneal Opacity; Eye Diseases; Female; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mucin-1; Promoter Regions, Genetic; Staphylococcal Infections; Staphylococcus aureus; Swine
PubMed: 33557346
DOI: 10.3390/ijms22041586 -
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 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