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Biomedical subjects

R E Hancock

Publications and source records attributed to R E Hancock.

At least 19 recordsLinked to original sources
PubMed1999

Dissociation of antimicrobial and hemolytic activities in cyclic peptide diastereomers by systematic alterations in amphipathicity.

We have investigated the role of amphipathicity in a homologous series of head-to-tail cyclic antimicrobial peptides in efforts to delineate features resulting in high antimicrobial activity coupled with low hemolytic activity (i.e. a high therapeutic index). The peptide GS14, cyclo(VKLKVd-YPLKVKLd-YP), designed on the basis of gramicidin S (GS), exists in a preformed highly amphipathic beta-sheet conformation and was used as the base compound for this study. Fourteen diastereomers of GS14 were synthesized; each contained a different single enantiomeric substitution within the framework of GS14. The beta-sheet structure of all GS14 diastereomers was disrupted as determined by CD and NMR spectroscopy under aqueous conditions; however, all diastereomers exhibited differential structure inducibility in hydrophobic environments. Because the diastereomers all have the same composition, sequence, and intrinsic hydrophobicity, the amphipathicity of the diastereomers could be ranked based upon retention time from reversed-phase high performance liquid chromatography. There was a clear correlation showing that high amphipathicity resulted in high hemolytic activity and low antimicrobial activity in the diastereomers. The latter may be the result of increased affinity of highly amphipathic peptides to outer membrane components of Gram-negative microorganisms. The diastereomers possessing the most favorable therapeutic indices possessed some of the lowest amphipathicities, although there was a threshold value below which antimicrobial activity decreased. The best diastereomer exhibited 130-fold less hemolytic activity compared with GS14, as well as greatly increased antimicrobial activities, resulting in improvement in therapeutic indices of between 1,000- and 10,000-fold for a number of microorganisms. The therapeutic indices of this peptide were between 16- and 32-fold greater than GS for Gram-negative microorganisms and represents a significant improvement in specificity over GS. Our findings show that a highly amphipathic nature is not desirable in the design of constrained cyclic antimicrobial peptides and that an optimum amphipathicity can be defined by systematic enantiomeric substitutions.

Amino Acid Sequence↗
PubMed1999

Interaction of the cyclic antimicrobial cationic peptide bactenecin with the outer and cytoplasmic membrane.

Bactenecin, a 12-amino acid cationic antimicrobial peptide from bovine neutrophils, has two cysteine residues, which form one disulfide bond, making it a cyclic molecule. To study the importance of the disulfide bond, a linear derivative Bac2S was made and the reduced form (linear bactenecin) was also included in this study. Circular dichroism spectroscopy showed that bactenecin existed as a type I beta-turn structure regardless of its environment, while the reduced form and linear bactenecin adopted different conformations according to the lipophilicity of the environment. Bactenecin was more active against the Gram-negative wild type bacteria Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium than its linear derivative and reduced form, while all three peptides were equally active against the outer membrane barrier-defective mutants of the first two bacteria. Only the two linear peptides showed activity against the Gram-positive bacteria Staphylococcus epidermidis and Enterococcus facaelis. Bactenecin interacted well with the outer membrane and its higher affinity for E. coli UB1005 lipopolysaccharide and improved ability to permeabilize the outer membrane seemed to account for its better antimicrobial activity against Gram-negative bacteria. The interaction of bactenecin with the cytoplasmic membrane was determined by its ability to dissipate the membrane potential by using the fluorescence probe 3, 3-dipropylthiacarbocyanine and an outer membrane barrier-defective mutant E. coli DC2. It was shown that the linear derivative and reduced form were able to dissipate the membrane potential at much lower concentrations than bactenecin despite the similar minimal inhibitory concentrations of all three against this barrier-defective mutant.

Anti-Bacterial Agents↗
PubMed1999

Negative regulation of the Pseudomonas aeruginosa outer membrane porin OprD selective for imipenem and basic amino acids.

Pseudomonas aeruginosa OprD is a specific porin which facilitates the uptake of basic amino acids and imipenem, a carbapenem antibiotic. Resistance to imipenem due to the loss of OprD is an important mechanism for the loss of clinical effectiveness. To investigate the negative regulatory mechanisms influencing oprD expression, a gene upstream of the coregulated mexEF-oprN efflux operon, designated mexT, was cloned. The predicted 304-amino-acid mature MexT protein showed strong homology to LysR-type regulators. When overexpressed it induced the expression of the mexEF-oprN efflux operon while decreasing the level of expression of OprD. The use of an oprD::xylE transcriptional fusion indicated that it acted by repressing the transcription of oprD. Salicylate, a weak aromatic acid known to reduce porin expression and induce low levels of multiple antibiotic resistance in Escherichia coli, was able to induce imipenem resistance and reduce the expression of OprD but not multiple antibiotic resistance or OprN expression in P. aeruginosa. This was also demonstrated to occur at the level of transcription. Acetyl salicylate and benzoate, but not catechol, were also able to reduce the levels of OprD in the P. aeruginosa outer membranes. These OprD-suppressing compounds increased imipenem resistance even in a mexT-overexpressing and nfxC mutant backgrounds, suggesting that such resistance is independent of the MexT repressor and that oprD is influenced by more than a single mechanism of repression.

Amino Acids↗
PubMed1999

Improved derivatives of bactenecin, a cyclic dodecameric antimicrobial cationic peptide.

Both linear and cyclic derivatives of the cyclic 12-amino-acid antimicrobial peptide bactenecin were designed based on optimization of amphipathicity and charge location. In general, increasing the number of positive charges at the N and C termini and adding an extra tryptophan residue in the loop not only increased the activities against both gram-positive and gram-negative bacteria but also broadened the antimicrobial spectrum.

Amino Acid Sequence↗
PubMed1999

Biological properties of structurally related alpha-helical cationic antimicrobial peptides.

A series of alpha-helical cationic antimicrobial peptide variants with small amino acid changes was designed. Alterations in the charge, hydrophobicity, or length of the variant peptides did not improve the antimicrobial activity, and there was no statistically significant correlation between any of these factors and the MIC for Pseudomonas aeruginosa, Escherichia coli, or Salmonella typhimurium. Individual peptides demonstrated synergy with conventional antibiotics against antibiotic-resistant strains of P. aeruginosa. The peptides varied considerably in the ability to bind E. coli O111:B4 lipopolysaccharide (LPS), and this correlated significantly with their antimicrobial activity and ability to block LPS-stimulated tumor necrosis factor and interleukin-6 production. In general, the peptides studied here demonstrated a broad range of activities, including antimicrobial, antiendotoxin, and enhancer activities.

Amino Acid Sequence↗
PubMed1999

Host defence (cationic) peptides: what is their future clinical potential?

Host defence, cationic antimicrobial peptides are now recognised as an important component, in most species, of the early innate and induced defences against invading microbes. They are small (12 to 35 amino acids), cationic due to the presence of an excess of arginine and lysine over acidic amino acids, and able to fold into a variety of different secondary structures. They have highly desirable properties, such as the ability to kill rapidly a broad spectrum of microorganisms including drug resistant bacteria and often fungi at around the minimal inhibitory concentration, a low level of resistance development in vitro, the ability to protect animals against both topical and systemic infections and the capability to neutralise endotoxin and demonstrated synergy with conventional antibiotics. In addition, given the 20 building blocks (amino acids) for these peptides. even a small peptide offers enormous diversity and potential for design of improved variants. For this reason such peptides have entered clinical trials, largely as agents for topical therapy of polymicrobial infections and are considered to have excellent potential for being a novel antibiotic class.

Animals↗
PubMed1998

Determinants of recombinant production of antimicrobial cationic peptides and creation of peptide variants in bacteria.

Cationic peptides possessing antibacterial activity are virtually ubiquitous in nature, and offer exciting prospects as new therapeutic agents. We had previously demonstrated that such peptides could be produced by fusion protein technology in bacteria and several carrier proteins had been tested as fusion partners including glutathione-S-transferase, S. aureus protein A, IgG binding protein and P. aeruginosa outer membrane protein OprF. However these fusion partners, while successfully employed in peptide expression, were not optimized for high level production of cationic peptides (Piers, K., Brow, M. L., and Hancock, R. E. W. 1993, Gene 137, 7-13). In this paper we took advantage of a small replication protein RepA from E. coli and used its truncated version to construct fusion partners. The minimal elements required for high level expression of cationic peptide were defined as a DNA sequence encoding a fusion protein comprising, from the N-terminus, a 68 amino acid carrier region, an anionic prepro domain, a single methionine and the peptide of interest. The 68 amino acid carrier region was a block of three polypeptides consisting of a truncated RepA, a synthetic cellulose binding domain and a hexa histidine domain. The improved system showed high level expression and simplified downstream purification. The active peptide could be yielded by CNBr cleavage of the fusion protein. This novel vector was used to express three classes of cationic peptides including the alpha-helical peptide CEMA, the looped peptide bactenecin and the extended peptide indolicidin. In addition, mutagenesis of the peptide gene to produce peptide variants of CEMA and indolicidin using the improved vector system was shown to be successful.

Amino Acid Sequence↗
PubMed1998

Structural variations in nisin associated with different membrane mimicking and pH environments.

Nisin is a membrane active antimicrobial peptide containing unusual dehydrated amino acid residues. The secondary structure of nisin in aqueous solution, membrane mimicking solvents and at various pH values was investigated using circular dichroism. In aqueous solution nisin is largely randomly coiled. In liposomes and at pH 6 and above, however, the presence of a maximum at 195 nm and a minimum at 190 nm was notable and indicative of beta-turn formation in these environments. This change in structure was speculated to result in an increasing unavailability of the site for initial reaction of peptide and membrane at higher pH.

Anti-Bacterial Agents↗
PubMed1998

Cationic peptides: a new source of antibiotics.

Antimicrobial cationic peptides are an important component of the innate defenses of all species of life. Different peptides may have antibacterial, antiendotoxic, antibiotic-potentiating or antifungal properties, and so they are being developed for use as a novel class of antimicrobial agents and as the basis for making transgenic disease-resistant plants and animals.

Amino Acid Sequence↗
PubMed1998

Resistance mechanisms in Pseudomonas aeruginosa and other nonfermentative gram-negative bacteria.

Nonfermentative gram-negative bacilli are still a major concern in compromised individuals. By far the most important of these organisms is Pseudomonas aeruginosa, although Acinetobacter baumannii (previously Acinetobacter calcoaceticus), Stenotrophomonas maltophilia (previously Pseudomonas and Xanthomonas maltophilia), and Burkholderia cepacia (previously Pseudomonas cepacia) are also of substantative concern because of their similar high intrinsic resistances to antibiotics. The basis for the high intrinsic resistance of these organisms is the lower outer-membrane permeability of these species, coupled with secondary resistance mechanisms such as an inducible cephalosporinase or antibiotic efflux pumps, which take advantage of low outer-membrane permeability. Even a small change in antibiotic susceptibility of these organisms can result in an increase in the MIC of a drug to a level that is greater than the clinically achievable level. In this review, the major mechanisms of resistance observed in the laboratory and clinic are summarized.

Drug Resistance, Microbial↗
PubMed1998

Influence of preformed alpha-helix and alpha-helix induction on the activity of cationic antimicrobial peptides.

One prominent class of cationic antibacterial peptides comprises the alpha-helical class, which is unstructured in free solution but folds into an amphipathic alpha-helix upon insertion into the membranes of target cells. To investigate the importance of alpha-helicity and its induction on interaction with membranes, a series of peptides was constructed based on a hybrid of moth cecropin (amino acids 1-8) and bee melittin (amino acids 1-18) peptides. The new peptides were predicted to have a high tendency to form alpha-helices or to have preformed alpha-helices by virtue of construction of a lactam bridge between glutamate and lysine side-chains at positions i and i + 4 at various locations along the primary sequence. In two examples where the use of lactam bridge constraints induced and stabilized alpha-helical structure in benign (aqueous buffer) and/or hydrophobic medium, there was a decrease in antibacterial activity relative to the linear counterparts. Thus the preformation of alpha-helix in solution was not necessarily beneficial to antimicrobial activity. In the one case where the lactam bridge did result in increased antibacterial activity (lower minimal inhibitory concentration values) it did not increase alpha-helical content in benign or hydrophobic medium. Broadly speaking, good activity of the peptides against Pseudomonas aeruginosa correlated best (r2 = 0.88) with a helican parameter which was calculated as the induction of alpha-helix in a membrane-mimicking environment divided by the alpha-helix formation under benign conditions. Interestingly, the activity of the lactam bridge peptide constructs correlated in part with alterations in bacterial outer or cytoplasmic membrane permeability.

Amino Acid Sequence↗
PubMed1998

Influence of culture conditions on expression of the 40-kilodalton porin protein of Vibrio anguillarum serotype O2.

Vibrio anguillarum serotype O2 strains express a 40-kDa outer membrane porin protein. Immunoblot analysis revealed that antigenic determinants of the V. anguillarum O2 40-kDa porin were conserved within bacterial species of the genus Vibrio. The relative amounts of the V. anguillarum O2 40-kDa porin were enhanced by growth of V. anguillarum O2 in CM9 medium containing 5 to 10% sucrose or 0.1 to 0.5 M NaCl. In contrast, the levels of the porin were significantly reduced when cells were grown at 37 degrees C, and a novel 60-kDa protein was also observed. However, the osmolarity or ionic concentration of the growth medium did not influence expression of the 60-kDa protein. Growth in medium containing greater than 0.6 mM EDTA reduced production of the V. anguillarum O2 40-kDa porin and enhanced levels of a novel 19-kDa protein. Thus, expression of the V. anguillarum O2 40-kDa porin was osmoregulated and possibly coregulated by temperature. The N-terminal amino acid sequence of the V. anguillarum O2 40-kDa protein and the effect of environmental factors on the cellular levels of the porin suggested that the V. anguillarum O2 40-kDa porin was functionally similar to the OmpC porin of Escherichia coli. However, pore conductance assays revealed that the V. anguillarum O2 40-kDa porin was a general diffusion porin with a pore size in the range of that of the OmpF porin of E. coli.

Amino Acid Sequence↗
PubMed1998

Roles of the carboxy-terminal half of Pseudomonas aeruginosa major outer membrane protein OprF in cell shape, growth in low-osmolarity medium, and peptidoglycan association.

OprF, the major outer membrane protein of Pseudomonas aeruginosa, is multifunctional in that it can act as a nonspecific porin, plays a role in the maintenance of cell shape, and is required for growth in a low-osmolarity environment. The latter two structural roles of OprF, and OprF's association with the peptidoglycan, have been proposed to be localized in the carboxy terminus of the protein, based on this region's similarity to members of the OmpA family of proteins. To determine if this is correct, we constructed a series of C-terminally truncated OprF derivatives and examined their effects on P. aeruginosa cell length and growth in low-osmolarity medium. While the C terminus of OprF was required for wild-type cell length and growth in low-osmolarity medium, expression of the N terminus (first 163 amino acids [aa]) also influenced these phenotypes (compared with OprF deficiency). The first 154 to 164 aa of OprF seemed required for stable protein expression, consistent with the existence of a beta-barrel domain in the N terminus of OprF. Greater than 215 aa of the protein were required for strong peptidoglycan association, confirming that residues in the C-terminal end of OprF are required for peptidoglycan binding. OprF deficiency did not affect the in vivo growth of an OprF-deficient strain in a mouse chamber model. Collectively, these data suggest that the C terminus of OprF plays a role in cell length, growth of P. aeruginosa in low-osmolarity media (but not in vivo), and peptidoglycan association, while the N terminus has an influence on the first two characteristics and is additionally important for stable protein expression.

Culture Media↗
PubMed1997

Utilization of alkaline phosphatase fusions to identify secreted proteins, including potential efflux proteins and virulence factors from Helicobacter pylori.

The targeted genomic strategy of random fusions to a partial gene encoding a signal sequence-deficient fragment of bacterial alkaline phosphatase was utilized to screen for secreted proteins in Helicobacter pylori. The rationale for targeting extracytoplasmic proteins was based on the hypothesis that most virulence factors and vaccine candidates are secreted or exported proteins. In addition, extracytosolic proteins represent good potential targets for drug intervention since they are in general more accessible to drugs than are cytoplasmically localized proteins. The application of this strategy to H. pylori allowed the identification of putative virulence factors and novel targets for drug intervention including four putative antibiotic efflux genes. The strategy used here is rapid and technically simple, relatively inexpensive, adaptable to a wide variety of microbes and genetic systems, and selects for expressed and accessible proteins.

Alkaline Phosphatase↗
PubMed1997

Peptide antibiotics.

The era of the "classical antibiotic" may be over. The emergence of resistance has seen to that. Yet no truly novel class of antibacterial agent has come on the market in the past 30 years. Currently there is great interest in peptide antibiotics, especially the cationic peptides. Thousands of such molecules have been synthesised and just a few are entering clinical trials. Because they kill bacteria quickly by the physical disruption of cell membranes, peptide antibiotics may not face the rapid emergence of resistance.

Animals↗
PubMed1997

The bacterial outer membrane as a drug barrier.

The outer membranes of Gram-negative bacteria constitute a semi-permeable barrier, as indicated by the corresponding alterations in outer membrane permeability and in antibiotic susceptibility resulting from mutation or polycation action. Restricted outer membrane permeability works in synergy with co-determinant resistance mechanisms, such as the periplasmic enzyme beta-lactamase or active efflux mechanisms, bringing about antibiotic resistance.

Anti-Bacterial Agents↗
PubMed1997

Antimycobacterial polyynes of Devil's Club (Oplopanax horridus), a North American native medicinal plant.

Two new (3 and 5), as well as three known (1, 2, and 4), polyynes were isolated from Devil's Club (Oplopanax horridus; Araliaceae), a medicinal plant of North America. The structures were established by 1H and 13C NMR. The absolute configurations of 2 and 5 were determined by application of Mosher's method. All the polyynes exhibited significant anti-Candida, antibacterial, and antimycobacterial activity, with an ability to kill Mycobacterium tuberculosis and isoniazid-resistant Mycobacterium avium at 10 micrograms/disk in a disk diffusion assay.

Anti-Bacterial Agents↗