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Computational Biology↗
Biomedical subjects
R E Hancock
Publications and source records attributed to R E Hancock.
Diastereoisomeric analogues of gramicidin S: structure, biologicalactivity and interaction with lipid bilayers.
Analogues of a structurally equivalent version of theantimicrobial decameric cyclic peptide gramicidin S, GS10 [cyclo-(Val-Lys-Leu-d-Tyr-Pro)(2)], were designed to study theeffect of distortion in the beta-sheet/beta-turn structure of thecyclic peptide on its biological activity. In one approach, thehydrophobic nature of GS10 was conserved, and single amino acids in itsbackbone were replaced systematically with their correspondingenantiomers to give five diastereoisomeric analogues. In a relatedapproach, a more basic and hydrophilic analogue of GS10 [cyclo-(Lys-Val-Lys-d-Tyr-Pro(5)-Lys-Leu-Lys-d-Tyr-Pro(10))], together with two of itsmonosubstituted diastereoisomeric analogues (featuring d-Lys(1) or d-Val(2) respectively), weresynthesized. CD spectra were measured in a variety of environments,i.e. aqueous, aqueous trifluoroethanol and those containing SDSmicelles or phospholipid vesicles. In comparison with GS10 spectra, CDspectra of both groups of analogues in these environments exhibitedstructural distortion. Moreover, compared with GS10, antimicrobial andhaemolytic activities of the analogues were drastically decreased, implying the existence of a threshold minimum amphipathicity foreffective biological activity. However, in both groups of analogues,there was a correlation between amphipathicity and antimicrobial andhaemolytic activities. In the second group of analogues, bothelectrostatic and hydrophobic factors were related to theirantimicrobial and haemolytic activities. In order to gain an insightinto the nature of the biological activity of the two classes of cyclicpeptides, the relationship of their structure to interaction with lipidmembranes, and the implied mechanisms, were analysed in some detail inthe present study.
Amino Acid Sequence↗
The role of antimicrobial peptides in animal defenses.
It is becoming clear that the cationic antimicrobial peptides are an important component of the innate defenses of all species of life. Such peptides can be constitutively expressed or induced by bacteria or their products. The best peptides have good activities vs. a broad range of bacterial strains, including antibiotic-resistant isolates. They kill very rapidly, do not easily select resistant mutants, are synergistic with conventional antibiotics, other peptides, and lysozyme, and are able to kill bacteria in animal models. It is known that bacterial infections, especially when treated with antibiotics, can lead to the release of bacterial products such as lipopolysaccharide (LPS) and lipoteichoic acid, resulting in potentially lethal sepsis. In contrast to antibiotics, the peptides actually prevent cytokine induction by bacterial products in tissue culture and human blood, and they block the onset of sepsis in mouse models of endotoxemia. Consistent with this, transcriptional gene array experiments using a macrophage cell line demonstrated that a model peptide, CEMA, blocks the expression of many genes whose transcription was induced by LPS. The peptides do this in part by blocking LPS interaction with the serum protein LBP. In addition, CEMA itself has a direct effect on macrophage gene expression. Because cationic antimicrobial peptides are induced by LPS and are able to dampen the septic response of animal cells to LPS, we propose that, in addition to their role in direct and lysozyme-assisted killing of microbes, they have a role in feedback regulation of cytokine responses. We are currently developing variant peptides as therapeutics against antibiotic-resistant infections.
Amino Acid Sequence↗
Salmonella typhimurium infection and lipopolysaccharide stimulation induce similar changes in macrophage gene expression.
Changes in macrophage phenotype induced during infection result from the recognition of bacterial products as well as the action of bacterial virulence factors. We used the unprecedented opportunity provided by gene arrays to simultaneously study the expression of hundreds of genes during Salmonella typhimurium infection of macrophages and to assess the contribution of the bacterial virulence factor, LPS, in initiating the host responses to Salmonella. We found that S. typhimurium infection caused significant changes in the expression of numerous genes encoding chemokines, cell surface receptors, signaling molecules, and transcriptional activators at 4 h postinfection of the RAW 264.7 murine macrophage cell line. Our results revealed changes in the expression of several genes that had not been previously implicated in the host responses to S. typhimurium infection, as well as changes in the expression of several genes previously shown to be regulated by S. typhimurium infection. An overlapping spectrum of genes was expressed in response to virulent S. typhimurium and purified S. typhimurium LPS, reinforcing the major role of this surface molecule in stimulating the early response of macrophages to bacterial infection. The macrophage gene expression profile was further altered by activation with IFN-gamma, indicating that host cell responses depend on the activation state of the cell.
Animals↗
Cutting edge: cationic antimicrobial peptides block the binding of lipopolysaccharide (LPS) to LPS binding protein.
We investigated the mechanism by which cationic antimicrobial peptides block the activation of macrophages by LPS. The initial step in LPS signaling is the transfer of LPS to CD14 by LPS binding protein (LBP). Because many cationic antimicrobial peptides bind LPS, we asked whether these peptides block the binding of LPS to LBP. Using an assay that measures the binding of LPS to immobilized LBP, we show for the first time that a variety of structurally diverse cationic antimicrobial peptides block the interaction of LPS with LBP. The relative ability of different cationic peptides to block the binding of LPS to LBP correlated with their ability to block LPS-induced TNF-alpha production by the RAW 264.7 macrophage cell line.
Acute-Phase Proteins↗
The role of cationic antimicrobial peptides in innate host defences.
Cationic antimicrobial peptides are found in all living species. A single animal can contain >24 different antimicrobial peptides, which fall into four structural classes. These peptides are produced in large quantities at sites of infection and/or inflammation and can have broad-spectrum antibacterial, antifungal, antiviral, antiprotozoan and antisepsis properties. In addition, they interact directly with host cells to modulate the inflammatory process and innate defences.
Amino Acid Sequence↗
Transgenic plants expressing cationic peptide chimeras exhibit broad-spectrum resistance to phytopathogens.
Here we describe a strategy for engineering transgenic plants with broad-spectrum resistance to bacterial and fungal phytopathogens. We expressed a synthetic gene encoding a N terminus-modified, cecropin-melittin cationic peptide chimera (MsrA1), with broad-spectrum antimicrobial activity. The synthetic gene was introduced into two potato (Solanum tuberosum L.) cultivars, Desiree and Russet Burbank, stable incorporation was confirmed by PCR and DNA sequencing, and expression confirmed by reverse transcription (RT)-PCR and recovery of the biologically active peptide. The morphology and yield of transgenic Desiree plants and tubers was unaffected. Highly stringent challenges with bacterial or fungal phytopathogens demonstrated powerful resistance. Tubers retained their resistance to infectious challenge for more than a year, and did not appear to be harmful when fed to mice. Expression of msrA1 in the cultivar Russet Burbank caused a striking lesion-mimic phenotype during leaf and tuber development, indicating its utility may be cultivar specific. Given the ubiquity of antimicrobial cationic peptides as well as their inherent capacity for recombinant and combinatorial variants, this approach may potentially be used to engineer a range of disease-resistant plants.
Amino Acid Sequence↗
Interactions of bacterial cationic peptide antibiotics with outer and cytoplasmic membranes of Pseudomonas aeruginosa.
Polymyxins B and E1 and gramicidin S are bacterium-derived cationic antimicrobial peptides. The polymyxins were more potent than gramicidin S against Pseudomonas aeruginosa, with MICs of 0.125 to 0. 25 and 8 microg/ml, respectively. These peptides differed in their affinities for binding to lipopolysaccharide, but all were able to permeabilize the outer membrane of wild-type P. aeruginosa PAO1 strain H103, suggesting differences in their mechanisms of self-promoted uptake. Gramicidin S caused rapid depolarization of the bacterial cytoplasmic membrane at concentrations at which no killing was observed within 30 min, whereas, conversely, the concentrations of the polymyxins that resulted in rapid killing resulted in minimal depolarization. These data indicate that the depolarization of the cytoplasmic membrane by these peptides did not correlate with bacterial cell lethality.
Anti-Bacterial Agents↗
Helicobacter pylori uptake and efflux: basis for intrinsic susceptibility to antibiotics in vitro.
We previously demonstrated (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995) that Helicobacter pylori has at least one nonspecific porin, HopE, which has a low abundance in the outer membrane but forms large channels. H. pylori is relatively susceptible to most antimicrobial agents but less susceptible to the polycationic antibiotic polymyxin B. We demonstrate here that H. pylori is able to take up higher basal levels of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN) than Pseudomonas aeruginosa or Escherichia coli, consistent with its enhanced susceptibility to hydrophobic agents. Addition of polymyxin B led to a further increase in NPN uptake, indicative of a self-promoted uptake pathway, but it required a much higher amount of polymyxin B to yield a 50% increase in NPN uptake in H. pylori (6 to 8 microg/ml) than in P. aeruginosa or E. coli (0.3 to 0.5 microg/ml), suggesting that H. pylori has a less efficient self-promoted uptake pathway. Since intrinsic resistance involves the collaboration of restricted outer membrane permeability and secondary defense mechanisms, such as periplasmic beta-lactamase (which H. pylori lacks) or efflux, we examined the possible role of efflux in antibiotic susceptibility. We had previously identified in H. pylori 11637 the presence of portions of three genes with homology to potential restriction-nodulation-division (RND) efflux systems. It was confirmed that H. pylori contained only these three putative RND efflux systems, named here hefABC, hefDEF, and hefGHI, and that the hefGHI system was expressed only in vivo while the two other RND systems were expressed both in vivo and in vitro. In uptake studies, there was no observable energy-dependent tetracycline, chloramphenicol, or NPN efflux activity in H. pylori. Independent mutagenesis of the three putative RND efflux operons in the chromosome of H. pylori had no effect on the in vitro susceptibility of H. pylori to 19 antibiotics. These results, in contrast to what is observed in E. coli, P. aeruginosa, and other clinically important gram-negative bacteria, suggest that active efflux does not play a role in the intrinsic resistance of H. pylori to antibiotics.
1-Naphthylamine↗
Role of putative loops 2 and 3 in imipenem passage through the specific porin OprD of Pseudomonas aeruginosa.
Mutant proteins with eight amino acid deletions in putative surface loops 2 and 3 of the imipenem-specific porin OprD of Pseudomonas aeruginosa failed to reconstitute imipenem susceptibility in an oprD-deficient background. The loop 3 deletion prevented the ability of imipenem to inhibit KCl conductance through the OprD channel, as previously shown for a loop 2 deletion. This suggests that both loops 2 and 3 have a role in imipenem binding to the OprD channel.
Amino Acid Sequence↗
Antibacterial action of structurally diverse cationic peptides on gram-positive bacteria.
Antimicrobial cationic peptides are ubiquitous in nature and are thought to be a component of the first line of defense against infectious agents. It is widely believed that the killing mechanism of these peptides on bacteria involves an interaction with the cytoplasmic membrane. Cationic peptides from different structural classes were used in experiments with Staphylococcus aureus and other medically important gram-positive bacteria to gain insight into the mechanism of action. The membrane potential-sensitive fluorophore dipropylthiacarbocyanine was used to assess the interactions of selected antimicrobial peptides with the cytoplasmic membrane of S. aureus. Study of the kinetics of killing and membrane depolarization showed that, at early time points, membrane depolarization was incomplete, even when 90% or more of the bacteria had been killed. CP26, a 26-amino-acid alpha-helical peptide with a high MIC against S. aureus, still had the ability to permeabilize the membrane. Cytoplasmic-membrane permeabilization was a widespread ability and an action that may be necessary for reaching an intracellular target but in itself did not appear to be the killing mechanism. Transmission electron microscopy of S. aureus and Staphylococcus epidermidis treated with CP29 (a 26-amino-acid alpha-helical peptide), CP11CN (a 13-amino-acid, proline- and tryptophan-rich peptide), and Bac2A-NH(2) (a linearized version of the 12-amino-acid loop peptide bactenecin) showed variability in effects on bacterial structure. Mesosome-like structures were seen to develop in S. aureus, whereas cell wall effects and mesosomes were seen with S. epidermidis. Nuclear condensation and abherrent septation were occasionally seen in S. epidermidis. Our experiments indicated that these peptides vary in their mechanisms of action and that the mechanism of action likely does not solely involve cytoplasmic-membrane permeabilization.
Amino Acid Sequence↗
Antimicrobial peptides protect coho salmon from Vibrio anguillarum infections.
Fish losses from infectious diseases are a significant problem in aquaculture worldwide. Therefore, we investigated the ability of cationic antimicrobial peptides to protect against infection caused by the fish pathogen Vibrio anguillarum. To identify effective peptides for fish, the MICs of certain antimicrobial peptides against fish pathogens were determined in vitro. Two of the most effective antimicrobial peptides, CEME, a cecropin-melittin hybrid peptide, and pleurocidin amide, a C-terminally amidated form of the natural flounder peptide, were selected for in vivo studies. A single intraperitoneal injection of CEME did not affect mortality rates in juvenile coho salmon infected with V. anguillarum, the causative agent of vibriosis. Therefore, the peptides were delivered continuously using miniosmotic pumps placed in the peritoneal cavity. Twelve days after pump implantation, the fish received intraperitoneal injections of V. anguillarum at a dose that would kill 50 to 90% of the population. Fish receiving 200 microg of CEME per day survived longer and had significantly lower accumulated mortalities (13%) than the control groups (50 to 58%). Fish receiving pleurocidin amide at 250 microg per day also survived longer and had significantly lower accumulated mortalities (5%) than the control groups (67 to 75%). This clearly shows the potential for antimicrobial peptides to protect fish against infections and indicates that the strategy of overexpressing the peptides in transgenic fish may provide a method of decreasing bacterial disease problems.
Amino Acid Sequence↗
Comparative genomics of Helicobacter pylori: analysis of the outer membrane protein families.
The two complete genomic sequences of Helicobacter pylori J99 and 26695 were used to compare the paralogous families (related genes within one genome, likely to have related function) of genes predicted to encode outer membrane proteins which were present in each strain. We identified five paralogous gene families ranging in size from 3 to 33 members; two of these families contained members specific for either H. pylori J99 or H. pylori 26695. Most orthologous protein pairs (equivalent genes between two genomes, same function) shared considerable identity between the two strains. The unusual set of outer membrane proteins and the specialized outer membrane may be a reflection of the adaptation of H. pylori to the unique gastric environment where it is found. One subfamily of proteins, which contains both channel-forming and adhesin molecules, is extremely highly related at the sequence level and has likely arisen due to ancestral gene duplication. In addition, the largest paralogous family contained two essentially identical pairs of genes in both strains. The presence and genomic organization of these two pairs of duplicated genes were analyzed in a panel of independent H. pylori isolates. While one pair was present in every strain examined, one allele of the other pair appeared partially deleted in several isolates.
Alleles↗
The amino terminus of Pseudomonas aeruginosa outer membrane protein OprF forms channels in lipid bilayer membranes: correlation with a three-dimensional model.
Pseudomonas aeruginosa OprF forms 0.36-nS channels and, rarely, 2- to 5-nS channels in lipid bilayer membranes. We show that a protein comprising only the N-terminal 162-amino-acid domain of OprF formed the smaller, but not the larger, channels in lipid bilayers. Circular dichroism spectroscopy indicated that this protein folds into a beta-sheet-rich structure, and three-dimensional comparative modeling revealed that it shares significant structural similarity with the amino terminus of the orthologous protein Escherichia coli OmpA, which has been shown to form a beta-barrel. OprF and OmpA share only 15% identity in this domain, yet these results support the utility of modeling such widely divergent beta-barrel domains in three dimensions in order to reveal similarities not readily apparent through primary sequence comparisons. The model is used to further hypothesize why porin activity differs for the N-terminal domains of OprF and OmpA.
Amino Acid Sequence↗
Functional expression in Escherichia coli and membrane topology of porin HopE, a member of a large family of conserved proteins in Helicobacter pylori.
HopE is one of the smallest members of a family of 31 outer membrane proteins in Helicobacter pylori and has been shown to function as a porin. In this study it was cloned into Escherichia coli where it was expressed in the outer membrane, as confirmed by indirect immunofluorescence using HopE-specific antibodies. HopE purified from E. coli reconstituted channels in planar bilayer membranes that were the same size as those formed by HopE purified from H. pylori. A model of the membrane topology of HopE was constructed and indicated that this protein formed a beta-barrel with 16 transmembrane amphipathic beta-strands. The accuracy of this model was tested by linker insertion mutagenesis, assuming that, like other porins, amino acid insertions were not tolerated in the transmembrane beta-strands but were tolerated in the adjoining loop regions. Generally, the results obtained with a series of 12 insertions of the sequence RSKDV and two substitutions were consistent with the topological model. The preponderance of amino acids that were conserved in the extended family of HopE paralogs were predicted to be within the membrane and comprised 45% of all residues in the membrane.
Amino Acid Sequence↗
Insertion mutagenesis and membrane topology model of the Pseudomonas aeruginosa outer membrane protein OprM.
Pseudomonas aeruginosa OprM is a protein involved in multiple-antibiotic resistance as the outer membrane component for the MexA-MexB-OprM efflux system. Planar lipid bilayer experiments showed that OprM had channel-forming activity with an average single-channel conductance of only about 80 pS in 1 M KCl. The gene encoding OprM was subjected to insertion mutagenesis by cloning of a foreign epitope from the circumsporozoite form of the malarial parasite Plasmodium falciparum into 11 sites. In Escherichia coli, 8 of the 11 insertion mutant genes expressed proteins at levels comparable to those obtained with the wild-type gene and the inserted malarial epitopes were surface accessible as assessed by indirect immunofluorescence. When moved to a P. aeruginosa OprM-deficient strain, seven of the insertion mutant genes expressed proteins at variable levels comparable to that of wild-type OprM and three of these reconstituted MIC profiles resembling those of the wild-type protein, while the other mutant forms showed variable MIC results. Utilizing the data from these experiments, in conjunction with multiple sequence alignments and structure predictions, an OprM topology model with 16 beta strands was proposed.
Amino Acid Sequence↗
Cationic antimicrobial peptides: towards clinical applications.
Cationic antimicrobial peptides are important components of the innate immune defences of all species of life. Variants of these natural molecules have a broad range of antibiotic, antifungal, antiviral and anti-endotoxic activity. Two of these cationic peptides have shown signs of efficacy in early clinical trials of oral mucositis and the sterilisation of central venous catheters, respectively and are currently proceeding through Phase III clinical trials. Thus, cationic antimicrobial peptides are currently being investigated as topical agents. In addition, the cationic protein rBPI 21 has recently completed Phase III clinical trials of parenteral use for meningococcaemia.
Animals↗
In vitro characterization of the anticancer activity of membrane-active cationic peptides. I. Peptide-mediated cytotoxicity and peptide-enhanced cytotoxic activity of doxorubicin against wild-type and p-glycoprotein over-expressing tumor cell lines.
Cationic amphipathic peptides, such as the defensins and cecropins, induce cell death in prokaryotic and eukaryotic cells by increasing membrane permeability. Increased permeability may lead to cell lysis or, alternatively, may produce subtle changes in the membrane's barrier function that promote cell death. The in vitro cytotoxic and lytic activity of short mammalian-derived extended-helical cationic peptides and insect-derived alpha-helical peptides was measured in this study with the objective of establishing the anticancer potential of these agents. Two specific aims were addressed: (i) to assess the activity of peptides against non-malignant cells (sheep erythrocytes and human umbilical vein endothelial cells) versus tumor cells; and (ii) to characterize the cytotoxic activity using multidrug-resistant tumor cell lines in the presence and absence of the anthracycline doxorubicin. Cell lysis assays demonstrated that the lytic activity of the peptides tested was 2->50 times more cytotoxic to tumor cells than to non-malignant cells. Further, the cytotoxic activity of these peptides was equivalent when tested against sensitive and multidrug-resistant cell lines. In addition to their inherent cytotoxic activity, these membrane-active peptides can also augment the in vitro cytotoxic activity of doxorubicin against multidrug-resistant tumor cells.
ATP Binding Cassette Transporter, Subfamily B, Mem↗