Outer membrane protein I of Pseudomonas aeruginosa is a target of cationic antimicrobial peptide/protein.
ABSTRACT: Cationic antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms against invading microorganisms. Here we demonstrate that OprI (outer membrane protein I) of Pseudomonas aeruginosa is responsible for its susceptibility to human ribonuclease 7 (hRNase 7) and alpha-helical cationic AMPs, instead of surface lipopolysaccharide, which is the initial binding site of cationic AMPs. The antimicrobial activities of hRNase 7 and alpha-helical cationic AMPs against P. aeruginosa were inhibited by the addition of exogenous OprI or anti-OprI antibody. On modification and internalization of OprI by hRNase 7 into cytosol, the bacterial membrane became permeable to metabolites. The lipoprotein was predicted to consist of an extended loop at the N terminus for hRNase 7/lipopolysaccharide binding, a trimeric alpha-helix, and a lysine residue at the C terminus for cell wall anchoring. Our findings highlight a novel mechanism of antimicrobial activity and document a previously unexplored target of alpha-helical cationic AMPs, which may be used for screening drugs to treat antibiotic-resistant bacterial infection.
Project description:Antimicrobial peptides (AMPs) are important components of the host innate defense mechanism against invading pathogens. Our previous studies have shown that the outer membrane protein, OprI from Pseudomonas aeruginosa or its homologue, plays a vital role in the susceptibility of Gram-negative bacteria to cationic ?-helical AMPs (Y. M. Lin, S. J. Wu, T. W. Chang, C. F. Wang, C. S. Suen, M. J. Hwang, M. D. Chang, Y. T. Chen, Y. D. Liao, J Biol Chem 285:8985-8994, 2010, http://dx.doi.org/10.1074/jbc.M109.078725; T. W. Chang, Y. M. Lin, C. F. Wang, Y. D. Liao, J Biol Chem 287:418-428, 2012, http://dx.doi.org/10.1074/jbc.M111.290361). Here, we obtained two forms of recombinant OprI: rOprI-F, a hexamer composed of three disulfide-bridged dimers, was active in AMP binding, while rOprI-R, a trimer, was not. All the subunits predominantly consisted of ?-helices and exhibited rigid structures with a melting point centered around 76°C. Interestingly, OprI tagged with Escherichia coli signal peptide was expressed in a hexamer, which was anchored on the surface of E. coli, possibly through lipid acids added at the N terminus of OprI and involved in the binding and susceptibility to AMP as native P. aeruginosa OprI. Deletion and mutation studies showed that Cys1 and Asp27 played a key role in hexamer formation and AMP binding, respectively. The increase of OprI hydrophobicity upon AMP binding revealed that it undergoes conformational changes for membrane fusion. Our results showed that OprI on bacterial surfaces is responsible for the recruitment and susceptibility to amphipathic ?-helical AMPs and may be used to screen antimicrobials.
Project description:Antimicrobial peptides are important components of the host innate defense mechanism against invading pathogens, especially for drug-resistant bacteria. In addition to bactericidal activity, the 25 residue peptide TP4 isolated from Nile tilapia also stimulates cell proliferation and regulates the innate immune system in mice. In this report, TP4 hyperpolarized and depolarized the membrane potential of Pseudomonas aeruginosa at sub-lethal and lethal concentrations. It also inhibited and eradicated biofilm formation. The in vitro binding of TP4 to bacterial outer membrane target protein, OprI, was markedly enhanced by a membrane-like surfactant sarkosyl and lipopolysaccharide, which converted TP4 into an ?-helix. The solution structure of TP4 in dodecylphosphocholine was solved by NMR analyses. It contained a typical ?-helix at residues Phe10-Arg22 and a distorted helical segment at Ile6-Phe10, as well as a hydrophobic core at the N-terminus and a cationic patch at the C-terminus. Residues Ile16, Leu19 and Ile20 in the hydrophobic face of the main helix were critical for the integrity of amphipathic structure, other hydrophobic residues played important roles in hemolytic and bactericidal activities. A model for the assembly of helical TP4 embedded in sarkosyl vesicle is proposed. This study may provide valuable insight for engineering AMPs to have potent bactericidal activity but low hemolytic activity.
Project description:The emergence of antibiotic-resistant microbial strains has become a public health issue and there is an urgent need to develop new anti-infective molecules. Although natural antimicrobial peptides (AMPs) can exert bactericidal activities, they have not shown clinical efficacy. The limitations of native peptides may be overcome with rational design and synthesis. Here, we provide evidence that the bactericidal activity of a synthetic peptide, GW-Q6, against Pseudomonas aeruginosa is mediated through outer membrane protein OprI. Hyperpolarization/depolarization of membrane potential and increase of membrane permeability were observed after GW-Q6 treatment. Helical structure as well as hydrophobicity was induced by an amphipathic surfactant, sarkosyl, for binding to OprI and possible to membrane. NMR studies demonstrated GW-Q6 is an amphipathic ?-helical structure in DPC micelles. The paramagnetic relaxation enhancement (PRE) approach revealed that GW-Q6 orients its ?-helix segment (K7-K17) into DPC micelles. Additionally, this ?-helix segment is critical for membrane permeabilization and antimicrobial activity. Moreover, residues K3, K7, and K14 could be critical for helical formation and membrane binding while residues Y19 and W20 for directing the C-terminus of the peptide to the surface of micelle. Taken together, our study provides mechanistic insights into the mode of action of the GW-Q6 peptide and suggests its applicability in modifying and developing potent AMPs as therapeutic agents.
Project description:Many organisms rely on antimicrobial peptides (AMPs) as a first line of defense against pathogens. In general, most AMPs are thought to kill bacteria by binding to and disrupting cell membranes. However, certain AMPs instead appear to inhibit biomacromolecule synthesis, while causing less membrane damage. Despite an unclear understanding of mechanism(s), there is considerable interest in mimicking AMPs with stable, synthetic molecules. Antimicrobial N-substituted glycine (peptoid) oligomers ("ampetoids") are structural, functional and mechanistic analogs of helical, cationic AMPs, which offer broad-spectrum antibacterial activity and better therapeutic potential than peptides. Here, we show through quantitative studies of membrane permeabilization, electron microscopy, and soft X-ray tomography that both AMPs and ampetoids trigger extensive and rapid non-specific aggregation of intracellular biomacromolecules that correlates with microbial death. We present data demonstrating that ampetoids are "fast killers", which rapidly aggregate bacterial ribosomes in vitro and in vivo. We suggest intracellular biomass flocculation is a key mechanism of killing for cationic, amphipathic AMPs, which may explain why most AMPs require micromolar concentrations for activity, show significant selectivity for killing bacteria over mammalian cells, and finally, why development of resistance to AMPs is less prevalent than developed resistance to conventional antibiotics.
Project description:Antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms. Here we demonstrate that the outer membrane lipoprotein, Lpp, of Enterobacteriaceae interacts with and promotes susceptibility to the bactericidal activities of AMPs. The oligomeric Lpp was specifically recognized by several cationic ?-helical AMPs, including SMAP-29, CAP-18, and LL-37; AMP-mediated bactericidal activities were blocked by anti-Lpp antibody blocking. Blebbing of the outer membrane and increase in membrane permeability occurred in association with the coordinate internalization of Lpp and AMP. Interestingly, the specific binding of AMP to Lpp was resistant to divalent cations and salts, which were able to inhibit the bactericidal activities of some AMPs. Furthermore, using His-tagged Lpp as a ligand, we retrieved several characterized AMPs, including SMAP-29 and hRNase 7, from a peptide library containing crude mammalian cell lysates. Overall, this study explores a new mechanism and target of antimicrobial activity and provides a novel method for screening of antimicrobials for use against drug-resistant bacteria.
Project description:Antimicrobial peptides (AMPs) represent a promising therapeutic alternative for the treatment of antibiotic-resistant bacterial infections. The present study investigates the antimicrobial activity of new, rationally-designed derivatives of a short ?-helical peptide, RR. From the peptides designed, RR4 and its D-enantiomer, D-RR4, emerged as the most potent analogues with a more than 32-fold improvement in antimicrobial activity observed against multidrug-resistant strains of Pseudomonas aeruginosa and Acinetobacter baumannii. Remarkably, D-RR4 demonstrated potent activity against colistin-resistant strains of P. aeruginosa (isolated from cystic fibrosis patients) indicating a potential therapeutic advantage of this peptide over several AMPs. In contrast to many natural AMPs, D-RR4 retained its activity under challenging physiological conditions (high salts, serum, and acidic pH). Furthermore, D-RR4 was more capable of disrupting P. aeruginosa and A. baumannii biofilms when compared to conventional antibiotics. Of note, D-RR4 was able to bind to lipopolysaccharide to reduce the endotoxin-induced proinflammatory cytokine response in macrophages. Finally, D-RR4 protected Caenorhabditis elegans from lethal infections of P. aeruginosa and A. baumannii and enhanced the activity of colistin in vivo against colistin-resistant P. aeruginosa.
Project description:Antimicrobial peptides (AMPs) are one of the most important defense mechanisms against bacterial infections in insects, plants, non-mammalian vertebrates, and mammals. In the present study, a class of synthetic AMPs was evaluated for anti-inflammatory activity. One cationic AMP, GW-A2, demonstrated the ability to inhibit the expression levels of nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-activated macrophages. GW-A2 reduced LPS-induced increases in the phosphorylation of mitogen-activated protein kinase and protein kinase C-α/δ and the activation of NF-κB. GW-A2 also inhibited NLRP3 inflammasome activation induced by LPS and ATP. Furthermore, in the mice injected with LPS, GW-A2 reduced (1) the concentration of IL-1β, IL-6 and TNF-α in the serum; (2) the concentration of TNF-α in the peritoneal lavage; (3) the expression levels of iNOS, COX-2 and NLRP3 in the liver and lung; (4) the infiltration of polymorphonuclear neutrophils in the liver and lung. The underlying mechanisms for the anti-inflammatory activity of GW-A2 were found to be partially due to LPS and ATP neutralization. These results provide insights into how GW-A2 inhibits inflammation and the NLRP3 inflammasome and provide a foundation for the design of rational therapeutics for inflammation-related diseases.
Project description:Cationic antimicrobial peptides are ubiquitous immune effectors of multicellular organisms. We previously reported, that in contrast to most of the classic antibiotics, cationic antimicrobial peptides (AMPs) do not increase mutation rates in E. coli Here, we provide new evidence showing that AMPs do not stimulate or enhance bacterial DNA recombination in the surviving fractions. Recombination accelerates evolution of antibiotic resistance. Our findings have implications for our understanding of host-microbe interactions, the evolution of innate immune defences, and shed new light on the dynamic of antimicrobial-resistance evolution.
Project description:Antimicrobial proteins and peptides (AMPs) are essential effectors of innate immunity, acting as a first line of defense against bacterial infections. Many AMPs exhibit high affinity for cell wall structures such as lipopolysaccharide (LPS), a potent endotoxin able to induce sepsis. Hence, understanding how AMPs can interact with and neutralize LPS endotoxin is of special relevance for human health. Eosinophil cationic protein (ECP) is an eosinophil secreted protein with high activity against both Gram-negative and Gram-positive bacteria. ECP has a remarkable affinity for LPS and a distinctive agglutinating activity. By using a battery of LPS-truncated E. coli mutant strains, we demonstrate that the polysaccharide moiety of LPS is essential for ECP-mediated bacterial agglutination, thereby modulating its antimicrobial action. The mechanism of action of ECP at the bacterial surface is drastically affected by the LPS structure and in particular by its polysaccharide moiety. We have also analyzed an N-terminal fragment that retains the whole protein activity and displays similar cell agglutination behavior. Conversely, a fragment with further minimization of the antimicrobial domain, though retaining the antimicrobial capacity, significantly loses its agglutinating activity, exhibiting a different mechanism of action which is not dependent on the LPS composition. The results highlight the correlation between the protein's antimicrobial activity and its ability to interact with the LPS outer layer and promote bacterial agglutination.
Project description:Antimicrobial peptides (AMPs) have been an area of great interest, due to the high selectivity of these molecules toward bacterial targets over host cells and the limited development of bacterial resistance to these molecules throughout evolution. The peptide C18G has been shown to be a selective, broad spectrum AMP with a net +8 cationic charge from seven lysine residues in the sequence. In this work, the cationic Lys residues were replaced with other natural or non-proteinogenic cationic amino acids: arginine, histidine, ornithine, or diaminopropionic acid. These changes vary in the structure of the amino acid side chain, the identity of the cationic moiety, and the pKa of the cationic group. Using a combination of spectroscopic and microbiological methods, the influence of these cationic groups on membrane binding, secondary structure, and antibacterial activity was investigated. The replacement of Lys with most other cationic residues had, at most, 2-fold effects on minimal inhibitory concentration against a variety of Gram-positive and Gram-negative bacteria. However, the peptide containing His as the cationic group showed dramatically reduced activity. All peptide variants retained the ability to bind lipid vesicles and showed clear preference for binding vesicles that contained anionic lipids. Similarly, all peptides adopted a helical conformation when bound to lipids or membrane mimetics, although the peptide containing diaminopropionic acid exhibited a decreased helicity. The peptides exhibited a wider variety of activity in the permeabilization of bacterial membranes, with peptides containing Lys, Arg, or Orn being the most broadly active. In all, the antibacterial activity of the C18G peptide is generally tolerant to changes in the structure and identity of the cationic amino acids, yielding new possibilities for design and development of AMPs that may be less susceptible to immune and bacterial recognition or in vivo degradation.