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Robert I. Lehrer - One of the best experts on this subject based on the ideXlab platform.
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assessing pore forming capability of Protegrin 1 in lipid bilayers of varying cholesterol content
Biophysical Journal, 2011Co-Authors: Eddie Maldonado, Robert I. Lehrer, Michael Henderson, Alan J. WaringAbstract:Pore formation affects the structural integrity of the lipid membrane, compromising its ability to act as a semi-permeable barrier and eventually leading to cell death. We present results obtained via atomic force microscopy (AFM) when lipid bilayer patches made dimyristoylphosphatidylcholine (DMPC) with increasing cholesterol content were subjected to the action of an cationic antimicrobial peptide (AMP), Protegrin-1 (PG-1). The cholesterol content was varied from 0% to 30%, where the lower and the upper bounds represent the cholesterol content in bacterial and mammalian cells, respectively. In the absence of cholesterol, PG-1 penetrated into the bilayer, leading to pore formation.
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killing of fusobacterium nucleaturn porphyromonas gingivalis and prevotella intermedia by Protegrins
Journal of Periodontal Research, 2010Co-Authors: Kenneth T Miyasaki, Rina Iofel, Ami Oren, Thuc Huynh, Robert I. LehrerAbstract:Protegrins are broad spectrum antibiotic peptides isolated from porcine leukocytes. In this study, we (i) examine the sensitivity of Gram-negative, anaerobic periodontal pathogens to synthetic Protegrins; (ii) determine the relative potencies of Protegrin congeners against these bacteria; and (iii) compare the potency of Protegrins with other antibiotic peptides, including magainin MSI-78, tachyplesin I, cecropin P1, human defensins HNP-1-3, and clavanin A. Synthetic L- and D-enantiomers of Protegrin 1 (PG-1 and D-PG-1, respectively), and L-enantiomers of Protegrins 2, 3 and 5 (PG-2, PG-3 and PG-5) were tested against Fusobacterium nucleatum, and black-pigmented organisms including Porphyromonas gingivalis and Prevotella intermedia. Strains of both F. nucleatum and the black-pigmented organisms were sensitive to PG-I, and exhibited mean ED 99 of 2.2-2.3 μg/ml and 3.4-9.9 μg/ml, respectively. The D-form was statistically more potent than the L-form against these oral anaerobes, and although this difference in potency is unlikely to be of decisive therapeutic significance, the D-form may be of value given ability to resist microbial and host-derived proteases. PG-1 was more potent than magainin, tachyplesin, cecropin, defensins and clavanin under test conditions. Hypertonic salt concentrations and heat-inactivated serum were found to be inhibitory to the bactericidal activity of PG-1. PG-1 was found to induce morphologic alterations in the ultrastructural appearance of F. nucleatum consistent with damage to the bacterial membranes. We conclude that Protegrins may be useful antimicrobial agents in therapy against Gram-negative anaerobic bacteria believed to be involved in chronic, adult forms of periodontal infections.
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characterizing the effects of membrane fluidity and lipid chain length on the antimicrobial activity of Protegrin 1
Biophysical Journal, 2010Co-Authors: Oliver S Shafaat, Alan J. Waring, Matthew R Chapman, Robert I. LehrerAbstract:Antimicrobial peptides are naturally occurring short amphipathic proteins, innate to the immune system and shown to induce selective lytic activity towards microbial pathogens. Protegrin-1 is an 18-residue, cationic, β-sheet antimicrobial peptide stabilized by two disulfide bonds. Concentration-dependent structural transformations of supported lipid bilayer patches as a result of peptide-membrane interactions have been visualized through the use of atomic force microscopy. A three-stage concentration-dependent transformation has been characterized, which begins with edge instability, followed by pore formation and worm-like micelle formation. This suggests that Protegrin-1 acts to lower the line-energy at the edge of the bilayer. Membrane and lipid characteristics, including fluidity, charge and acyl chain length, can alter the activity of antimicrobial peptides. To identify the importance of both acyl-chain length and fluidity on the activity of Protegrin-1, these two variables were decoupled. When the bilayers are examined at the same relative fluidity levels, they demonstrate the three-stage transformation observed on a fluid control bilayer, in contrast to the structural transformations that were observed in the gel phase bilayers. This suggests that fluidity exhibits a large influence on the transformations that occur as a result of Protegrin-1. To examine the importance of acyl-chain length, the activity of antimicrobial peptides was studied using unsaturated bilayers. Our results indicate that the longer chain bilayers are less susceptible to disruption. This could be due to the hydrophobic mismatch between Protegrin-1 and the thicker hydrophobic portion of longer chain lipid bilayers . These results highlight the importance of subtle membrane characteristics in the activity of antimicrobial peptides towards bacterial cells. Lipid bilayers with cholesterol are more accurate eukaryotic cell mimics and will allow examination of the selective preference of antimicrobial peptide activity.
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antimicrobial mechanism of pore forming Protegrin peptides 100 pores to kill e coli
Peptides, 2010Co-Authors: Dan S Bolintineanu, Robert I. Lehrer, Ehsan Hazrati, Ted H Davis, Yiannis N KaznessisAbstract:Antimicrobial peptides (AMPs), important effector molecules of the innate immune system, also provide templates for designing novel antibiotics. Protegrin, an especially potent AMP found in porcine leukocytes, was recently shown to form octameric transmembrane pores. We have employed a combination of experiments and models spanning length scales from the atomistic to the cellular level in order to elucidate the microbicidal mechanism of Protegrin. Comparison of the modeling and experimental data suggests that approximately 10-100 Protegrin pores are necessary to explain the observed rates of potassium leakage and Escherichia coli death in exponential-phase bacteria. The kinetics of viability loss suggest that bacterial death results largely from uncontrolled ion exchange processes and decay of transmembrane potential. However, ion exchange processes alone cannot account for the experimentally observed cell swelling and osmotic lysis—a redundant ''overkill'' mechanism most likely to occur in locales with high Protegrin concentrations. Although our study is limited to Protegrin and E. coli, the timeline of events described herein is likely shared by other AMPs that act primarily by permeabilizing microbial membranes. This work provides many of the missing links in describing antimicrobial action, as well as providing a quantitative connection between several previous experimental and simulation studies of Protegrin.
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correlation between simulated physicochemical properties and hemolycity of Protegrin like antimicrobial peptides predicting experimental toxicity
Peptides, 2008Co-Authors: Allison A Langham, Alan J. Waring, Robert I. Lehrer, Himanshu Khandelia, Benjamin S Schuster, Yiannis N KaznessisAbstract:The therapeutic, antibiotic potential of antimicrobial peptides can be prohibitively diminished because of the cytotoxicity and hemolytic profiles they exhibit. Quantifying and predicting antimicrobial peptide toxicity against host cells is thus an important goal of AMP related research. In this work, we present quantitative structure activity relationships for toxicity of Protegrin-like antimicrobial peptides against human cells (epithelial and red blood cells) based on physicochemical properties, such as interaction energies and radius of gyration, calculated from molecular dynamics simulations of the peptides in aqueous solvent. The hypothesis is that physicochemical properties of peptides, as manifest by their structure and interactions in a solvent and as captured by atomistic simulations, are responsible for their toxicity against human cells. Protegrins are β-hairpin peptides with high activity against a wide variety of microbial species, but in their native state are toxic to human cells. Sixty peptides with experimentally determined toxicities were used to develop the models. We test the resulting relationships to determine their ability to predict the toxicity of several Protegrin-like peptides. The developed QSARs provide insight into the mechanism of cytotoxic action of antimicrobial peptides. In a subsequent blind test, the QSAR correctly ranked four of five Protegrin analogues newly synthesized and tested for toxicity.
Yiannis N Kaznessis - One of the best experts on this subject based on the ideXlab platform.
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insights into membrane translocation of Protegrin antimicrobial peptides by multistep molecular dynamics simulations
ACS omega, 2018Co-Authors: Yiannis N KaznessisAbstract: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 membran...
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multiscale models of the antimicrobial peptide Protegrin 1 on gram negative bacteria membranes
International Journal of Molecular Sciences, 2012Co-Authors: Dan S Bolintineanu, Victor Vivcharuk, Yiannis N KaznessisAbstract:Antimicrobial peptides (AMPs) are naturally-occurring molecules that exhibit strong antibiotic properties against numerous infectious bacterial strains. Because of their unique mechanism of action, they have been touted as a potential source for novel antibiotic drugs. We present a summary of computational investigations in our lab aimed at understanding this unique mechanism of action, in particular the development of models that provide a quantitative connection between molecular-level biophysical phenomena and relevant biological effects. Our work is focused on Protegrins, a potent class of AMPs that attack bacteria by associating with the bacterial membrane and forming transmembrane pores that facilitate the unrestricted transport of ions. Using fully atomistic molecular dynamics simulations, we have computed the thermodynamics of peptide-membrane association and insertion, as well as peptide aggregation. We also present a multi-scale analysis of the ion transport properties of Protegrin pores, ranging from atomistic molecular dynamics simulations to mesoscale continuum models of single-pore electrodiffusion to models of transient ion transport from bacterial cells. Overall, this work provides a quantitative mechanistic description of the mechanism of action of Protegrin antimicrobial peptides across multiple length and time scales.
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thermodynamic analysis of Protegrin 1 insertion and permeation through a lipid bilayer
Journal of Physical Chemistry B, 2011Co-Authors: Victor Vivcharuk, Yiannis N KaznessisAbstract:Molecular dynamics (MD) simulations are used to study the pathway for the insertion of the cationic antimicrobial peptide Protegrin-1 (PG1) into mixed anionic lipid bilayers composed of palmitoyl-oleoyl-phosphatidylglycerol (POPG) and palmitoyl-oleoyl-phosphatidylethanolamine (POPE) in a 1:3 ratio (POPG/POPE). We calculate the potential of mean force (PMF) during the transfer of the peptide from the bulk aqueous phase to the transmembrane (TM) configuration using the adaptive biasing force (ABF) method. We find that the PMF has two energy minima separated by an energy barrier. One minimum corresponds to the fully transmembrane inserted state, with a free energy of −20.1 kcal/mol. The second PMF minimum, which corresponds to adsorption to the membrane surface, has a value of −2.5 kcal/mol. The PMF also shows the existence of a free energy barrier of +6.3 kcal/mol for the insertion process. Using the Kramers theory Langevin equation and the Grote–Hynes theory generalized Langevin equation, we calculated the...
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computational studies of Protegrin antimicrobial peptides a review
Peptides, 2011Co-Authors: Dan S Bolintineanu, Yiannis N KaznessisAbstract:Antimicrobial peptides (AMPs) are small, naturally occurring peptides that exhibit strong antibacterial properties generally believed to be a result of selective bacterial membrane disruption. As a result, there has been significant interest in the development of therapeutic antibiotics based on AMPs; however, the poor understanding of the fundamental mechanism of action of these peptides has largely hampered such efforts. We present a summary of computational and theoretical investigations of Protegrin, a particularly potent peptide that is both an excellent model for the mechanism of action of AMPs and a promising therapeutic candidate. Experimental investigations have shed light on many of the key steps in the action of Protegrin: Protegrin monomers are known to dimerize in various lipid environments; Protegrin peptides interact strongly with lipid bilayer membranes, particularly anionic lipids; Protegrins have been shown to form pores in lipid bilayers, which results in uncontrolled ion transport and may be a key factor in bacterial death. In this work, we present a comprehensive review of the computational and theoretical studies that have complemented and extended the information obtained from experimental work with Protegrins, as well as a brief survey of the experimental biophysical studies that are most pertinent to such computational work. We show that a consistent, mechanistic description of the bactericidal mechanism of action of Protegrins is emerging, and briefly outline areas where the current understanding is deficient. We hope that the research reviewed herein offers compelling evidence of the benefits of computational investigations of Protegrins and other AMPs, as well as providing a useful guide to future work in this area.
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dimerization of Protegrin 1 in different environments
International Journal of Molecular Sciences, 2010Co-Authors: Victor Vivcharuk, Yiannis N KaznessisAbstract:The dimerization of the cationic β-hairpin antimicrobial peptide Protegrin-1 (PG1) is investigated in three different environments: water, the surface of a lipid bilayer membrane, and the core of the membrane. PG1 is known to kill bacteria by forming oligomeric membrane pores, which permeabilize the cells. PG1 dimers are found in two distinct, parallel and antiparallel, conformations, known as important intermediate structural units of the active pore oligomers. What is not clear is the sequence of events from PG1 monomers in solution to pores inside membranes. The step we focus on in this work is the dimerization of PG1. In particular, we are interested in determining where PG1 dimerization is most favorable. We use extensive molecular dynamics simulations to determine the potential of mean force as a function of distance between two PG1 monomers in the aqueous subphase, the surface of model lipid bilayers and the interior of these bilayers. We investigate the two known distinct modes of dimerization that result in either a parallel or an antiparallel β-sheet orientation. The model bilayer membranes are composed of anionic palmitoyl-oleoyl-phosphatidylglycerol (POPG) and palmitoyl-oleoyl-phosphatidylethanolamine (POPE) in a 1:3 ratio (POPG:POPE). We find the parallel PG1 dimer association to be more favorable than the antiparallel one in water and inside the membrane. However, we observe that the antiparallel PG1 β-sheet dimer conformation is somewhat more stable than the parallel dimer association at the surface of the membrane. We explore the role of hydrogen bonds and ionic bridges in peptide dimerization in the three environments. Detailed knowledge of how networks of ionic bridges and hydrogen bonds contribute to peptide stability is essential for the purpose of understanding the mechanism of action for membrane-active peptides as well as for designing peptides which can modulate membrane properties. The findings are suggestive of the dominant pathways leading from individual PG1 molecules in solution to functional pores in bacterial membranes.
Alan J. Waring - One of the best experts on this subject based on the ideXlab platform.
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assessing pore forming capability of Protegrin 1 in lipid bilayers of varying cholesterol content
Biophysical Journal, 2011Co-Authors: Eddie Maldonado, Robert I. Lehrer, Michael Henderson, Alan J. WaringAbstract:Pore formation affects the structural integrity of the lipid membrane, compromising its ability to act as a semi-permeable barrier and eventually leading to cell death. We present results obtained via atomic force microscopy (AFM) when lipid bilayer patches made dimyristoylphosphatidylcholine (DMPC) with increasing cholesterol content were subjected to the action of an cationic antimicrobial peptide (AMP), Protegrin-1 (PG-1). The cholesterol content was varied from 0% to 30%, where the lower and the upper bounds represent the cholesterol content in bacterial and mammalian cells, respectively. In the absence of cholesterol, PG-1 penetrated into the bilayer, leading to pore formation.
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characterizing the effects of membrane fluidity and lipid chain length on the antimicrobial activity of Protegrin 1
Biophysical Journal, 2010Co-Authors: Oliver S Shafaat, Alan J. Waring, Matthew R Chapman, Robert I. LehrerAbstract:Antimicrobial peptides are naturally occurring short amphipathic proteins, innate to the immune system and shown to induce selective lytic activity towards microbial pathogens. Protegrin-1 is an 18-residue, cationic, β-sheet antimicrobial peptide stabilized by two disulfide bonds. Concentration-dependent structural transformations of supported lipid bilayer patches as a result of peptide-membrane interactions have been visualized through the use of atomic force microscopy. A three-stage concentration-dependent transformation has been characterized, which begins with edge instability, followed by pore formation and worm-like micelle formation. This suggests that Protegrin-1 acts to lower the line-energy at the edge of the bilayer. Membrane and lipid characteristics, including fluidity, charge and acyl chain length, can alter the activity of antimicrobial peptides. To identify the importance of both acyl-chain length and fluidity on the activity of Protegrin-1, these two variables were decoupled. When the bilayers are examined at the same relative fluidity levels, they demonstrate the three-stage transformation observed on a fluid control bilayer, in contrast to the structural transformations that were observed in the gel phase bilayers. This suggests that fluidity exhibits a large influence on the transformations that occur as a result of Protegrin-1. To examine the importance of acyl-chain length, the activity of antimicrobial peptides was studied using unsaturated bilayers. Our results indicate that the longer chain bilayers are less susceptible to disruption. This could be due to the hydrophobic mismatch between Protegrin-1 and the thicker hydrophobic portion of longer chain lipid bilayers . These results highlight the importance of subtle membrane characteristics in the activity of antimicrobial peptides towards bacterial cells. Lipid bilayers with cholesterol are more accurate eukaryotic cell mimics and will allow examination of the selective preference of antimicrobial peptide activity.
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correlation between simulated physicochemical properties and hemolycity of Protegrin like antimicrobial peptides predicting experimental toxicity
Peptides, 2008Co-Authors: Allison A Langham, Alan J. Waring, Robert I. Lehrer, Himanshu Khandelia, Benjamin S Schuster, Yiannis N KaznessisAbstract:The therapeutic, antibiotic potential of antimicrobial peptides can be prohibitively diminished because of the cytotoxicity and hemolytic profiles they exhibit. Quantifying and predicting antimicrobial peptide toxicity against host cells is thus an important goal of AMP related research. In this work, we present quantitative structure activity relationships for toxicity of Protegrin-like antimicrobial peptides against human cells (epithelial and red blood cells) based on physicochemical properties, such as interaction energies and radius of gyration, calculated from molecular dynamics simulations of the peptides in aqueous solvent. The hypothesis is that physicochemical properties of peptides, as manifest by their structure and interactions in a solvent and as captured by atomistic simulations, are responsible for their toxicity against human cells. Protegrins are β-hairpin peptides with high activity against a wide variety of microbial species, but in their native state are toxic to human cells. Sixty peptides with experimentally determined toxicities were used to develop the models. We test the resulting relationships to determine their ability to predict the toxicity of several Protegrin-like peptides. The developed QSARs provide insight into the mechanism of cytotoxic action of antimicrobial peptides. In a subsequent blind test, the QSAR correctly ranked four of five Protegrin analogues newly synthesized and tested for toxicity.
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comparison of interactions between beta hairpin decapeptides and sds dpc micelles from experimental and simulation data
BMC Biochemistry, 2007Co-Authors: Allison A Langham, Alan J. Waring, Yiannis N KaznessisAbstract:Background We applied a combined experimental and computational approach to ascertain how peptides interact with host and microbial membrane surrogates, in order to validate simulation methodology we hope will enable the development of insights applicable to the design of novel antimicrobial peptides. We studied the interactions of two truncated versions of the potent, but cytotoxic, antimicrobial octadecapeptide Protegrin-1, PC-72 [LCYCRRRFCVC] and PC-73 [CYCRRRFCVC].
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mechanism of supported membrane disruption by antimicrobial peptide Protegrin 1
Journal of Physical Chemistry B, 2006Co-Authors: Yuji Ishitsuka, Alan J. Waring, Yishan Cheng, Karen B Chien, Robert I. LehrerAbstract:While pore formation has been suggested as an important step in the membrane disruption process induced by antimicrobial peptides, membrane pore formation has never been directly visualized. We report on the dynamics of membrane disruption by antimicrobial peptide Protegrin-1 (PG-1) on dimyristoyl-sn-glycero-phosphocholine-supported bilayer patches obtained via atomic force microscopy. The action of PG-1 is found to be concentration-dependent. At low PG-1 concentrations (1 < [PG-1] < 4 μg/mL), the peptide destabilizes the edge of the membrane to form fingerlike structures. At higher concentrations, PG-1 induces the formation of a sievelike nanoporous structure in the membrane. The highest degree of disruption is attained at concentrations ≥20 μg/mL, at which PG-1 disrupts the entire membrane, transforming it into stripelike structures with a well-defined and uniform stripe width. This first direct visualization of these membrane structural transformations helps elucidate the PG-1-induced membrane disrupti...
Julang Li - One of the best experts on this subject based on the ideXlab platform.
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protective effects of Protegrin in dextran sodium sulfate induced murine colitis
Frontiers in Pharmacology, 2019Co-Authors: Evanna Huynh, Julang Li, Jenna Penney, Jeff L CaswellAbstract: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 sulfate sodium (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- form) in attenuating significant body weight loss associated with DSS-induced colitis (p<0.05). PG-1 treatment improved histological scores (P<0.05) and influenced the gene expression of inflammatory mediators and tissue repair factors such as trefoil factor (TFF3) and mucin (MUC-2). Protegrin treatment also altered the metabolite profile, returning the metabolite levels back 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.
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Protective Effects of Protegrin in Dextran Sodium Sulfate-Induced Murine Colitis
Frontiers in Pharmacology, 2019Co-Authors: Evanna Huynh, Jenna Penney, Jeff L Caswell, Julang LiAbstract: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 sulfate sodium (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- form) in attenuating significant body weight loss associated with DSS-induced colitis (p
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Protegrin 1 enhances innate cellular defense via the insulin like growth factor 1 receptor pathway
Frontiers in Cellular and Infection Microbiology, 2018Co-Authors: Julang Li, Jenna PenneyAbstract:: 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.
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efficient production of recombinant Protegrin 1 from pichia pastoris and its antimicrobial and in vitro cell migration activity
Frontiers in Microbiology, 2018Co-Authors: Evanna Huynh, Nadeem Akhtar, Julang LiAbstract:Protegrin (PG) belongs to the antimicrobial peptide (AMP) 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 Pichia pastoris to express antmicrobially active recombinant Protegrin (rPG-1). Additionally, the effect of PG-1 on cell migration and proliferation was also examined in vitro 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 Escherichia coli DH5α at the same level as chemically synthesized PG-1. Enhanced cell migration was observed (p< 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 P. pastoris suggesting potential feasibility for future therapeutic applications.
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enhanced resistance to bacterial infection in Protegrin 1 transgenic mice
Antimicrobial Agents and Chemotherapy, 2008Co-Authors: Queenie C K Cheung, Patricia V Turner, Cheng Song, De Wu, Janet I Macinnes, Julang LiAbstract:Antibiotic-resistant bacteria have become a public health concern. It was suggested that one source of resistant pathogens may be food-producing animals. Alternative approaches are therefore needed to enhance the resistance of farm animals to bacterial infection. Protegrin-1 (PG-1) is a neutrophil-derived antimicrobial peptide that possesses activity against a wide range of bacteria and enveloped viruses. Here we report on the production of transgenic mice that ectopically expressed PG-1 and compare their susceptibilities to Actinobacillus suis infection with those of their wild-type (WT) littermates. Of the 126 mice that were challenged with A. suis, 87% of the transgenic mice survived, whereas 31% of their WT littermates survived. The PG-1 transgenic mice had significantly lower bacterial loads in their lungs and reduced numbers of pulmonary pathological lesions. The antimicrobial function of PG-1 was confirmed in vitro by using fibroblast cells isolated from the transgenic mice but not the WT mice. Moreover, differential blood cell counts in bronchoalveolar lavage fluid indicated greater number of neutrophils in PG-1 transgenic mice than in their WT littermates after bacterial challenge. Our data suggest that the ectopic expression of PG-1 in mice confers enhanced resistance to bacterial infection, laying the foundation for the development of livestock with improved resistance to infection.
Mei Hong - One of the best experts on this subject based on the ideXlab platform.
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intermolecular packing and alignment in an ordered beta hairpin antimicrobial peptide aggregate from 2d solid state nmr
Journal of the American Chemical Society, 2005Co-Authors: Ming Tang, And Alan J Waring, Mei HongAbstract:The aggregation and packing of a membrane-disruptive β-hairpin antimicrobial peptide, Protegrin-1 (PG-1), in the solid state are investigated to understand its oligomerization and hydrogen-bonding propensity. Incubation of PG-1 in phosphate buffer saline produced well-ordered nanometer-scale aggregates, as indicated by 13C and 15N NMR line widths, chemical shifts, and electron microscopy. Two-dimensional 13C and 1H spin diffusion experiments using C-terminus strand and N-terminus strand labeled peptides indicate that the β-hairpin molecules in these ordered aggregates are oriented parallel to each other with like strands lining the intermolecular interface. In comparison, disordered and lyophilized peptide samples are randomly packed with both parallel and antiparallel alignments. The PG-1 aggregates show significant immobilization of the Phe ring near the β-turn, further supporting the structural ordering. The intermolecular packing of PG-1 found in the solid state is consistent with its oligomerization ...
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immobilization and aggregation of the antimicrobial peptide Protegrin 1 in lipid bilayers investigated by solid state nmr
Biochemistry, 2003Co-Authors: Jarrod J Buffy, Alan J. Waring, Robert I. Lehrer, Mei HongAbstract:The dynamics and aggregation of a β-sheet antimicrobial peptide, Protegrin-1 (PG-1), are investigated using solid-state NMR spectroscopy. Chemical shift anisotropies of F12 and V16 carbonyl carbons are uniaxially averaged in 1,2-dilauryl-sn-glycero-3-phosphatidylcholine (DLPC) bilayers but approach rigid-limit values in the thicker 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (POPC) bilayers. The Cα−Hα dipolar coupling of L5 is scaled by a factor of 0.16 in DLPC bilayers but has a near-unity order parameter of 0.96 in POPC bilayers. The larger couplings of PG-1 in POPC bilayers indicate immobilization of the peptide, suggesting that PG-1 forms oligomeric aggregates at the biologically relevant bilayer thickness. Exchange NMR experiments on F12 13CO-labeled PG-1 show that the peptide undergoes slow reorientation with a correlation time of 0.7 ± 0.2 s in POPC bilayers. This long correlation time suggests that in addition to aggregation, geometric constraints in the membrane may also contribute to ...
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solid state nmr investigations of peptide lipid interaction and orientation of a β sheet antimicrobial peptide Protegrin
Biochemistry, 2002Co-Authors: Satoru Yamaguchi, Alan J. Waring, Robert I. Lehrer, Teresa Hong, Mei HongAbstract:Protegrin-1 (PG-1) is a broad-spectrum β-sheet antimicrobial peptide found in porcine leukocytes. The mechanism of action and the orientation of PG-1 in lipid bilayers are here investigated using 2H, 31P, 13C, and 15N solid-state NMR spectroscopy. 2H spectra of mechanically aligned and chain-perdeuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers indicate that PG-1 at high concentrations destroys the orientational order of the aligned lamellar bilayer. The conformation of the lipid headgroups in the unoriented region is significantly altered, as seen from the 31P spectra of POPC and the 2H spectra of headgroup-deuterated 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine. These observations indicate that PG-1 disrupts microbial membranes by breaking the extended bilayer into smaller disks, where a significant fraction of lipids is located in the edges of the disks with a distribution of orientations. These edges allow the lipid bilayer to bend back on itself as in toroidal pores....
