Bioactive peptides: signaling the future.

Natural processes within the body are modulated almost exclusively by the interaction of specific amino acid sequences, either as peptides or as subsections of proteins. With respect to skin, proteins and peptides are involved in the modulation of cell proliferation, cell migration, inflammation, angiogenesis, melanogenesis, and protein synthesis and regulation. The creation of therapeutic or bioactive peptide analogs of specific interactive sequences has opened the door to a diverse new field of pharmaceutical and active cosmetic ingredients for the skincare industry. Here, we describe the origin of such sequences, their role in nature, their application to dermatology, as well as the advantages and challenges posed by this new technology.

Identification of Proteus mirabilis mutants with increased sensitivity to antimicrobial peptides.

Antimicrobial peptides (APs) are important components of the innate defenses of animals, plants, and microorganisms. However, some bacterial pathogens are resistant to the action of APs. For example, Proteus mirabilis is highly resistant to the action of APs, such as polymyxin B (PM), protegrin, and the synthetic protegrin analog IB-367. To better understand this resistance, a transposon mutagenesis approach was used to generate P. mirabilis mutants sensitive to APs. Four unique PM-sensitive mutants of P. mirabilis were identified (these mutants were >2 to >128 times more sensitive than the wild type). Two of these mutants were also sensitive to IB-367 (16 and 128 times more sensitive than the wild type). Lipopolysaccharide (LPS) profiles of the PM- and protegrin-sensitive mutants demonstrated marked differences in both the lipid A and O-antigen regions, while the PM-sensitive mutants appeared to have alterations of either lipid A or O antigen. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of the wild-type and PM-sensitive mutant lipid A showed species with one or two aminoarabinose groups, while lipid A from the PM- and protegrin-sensitive mutants was devoid of aminoarabinose. When the mutants were streaked on an agar-containing medium, the swarming motility of the PM- and protegrin-sensitive mutants was completely inhibited and the swarming motility of the mutants sensitive to only PM was markedly decreased. DNA sequence analysis of the mutagenized loci revealed similarities to an O-acetyltransferase (PM and protegrin sensitive) and ATP synthase and sap loci (PM sensitive). These data further support the role of LPS modifications as an elaborate mechanism in the resistance of certain bacterial species to APs and suggest that LPS surface charge alterations may play a role in P. mirabilis swarming motility.

Development of protegrins for the treatment and prevention of oral mucositis: structure-activity relationships of synthetic protegrin analogues.

Protegrin antimicrobial peptides possess activity against gram-positive and gram-negative bacteria and yeasts. An extensive structure-activity relationship (SAR) study was conducted on several hundred protegrin analogues to gain understanding of the relationship between the primary and secondary structure of the protegrins and their antimicrobial activities, and to identify a protegrin analogue for clinical development. Native sequence protegrins are cationic, amphiphilic peptides that are characterized by the presence of a beta-sheet structure that is maintained by two disulfide bridges. The presence of the beta-sheet is key to the stability of the protegrin structure; linearized analogues or analogues that have amino acid substitutions that eliminate hydrogen bonding across the beta-sheet have reduced activity, especially in the presence of physiological concentrations of NaCl. Also, maintaining amphiphilicity of the beta-sheet is key; analogues with substitutions of polar amino acids in the hydrophobic face have reduced activity. Analogues with reduced positive charge tend to be less active, an observation that is more marked for gram-negative than gram-positive bacteria, and may implicate binding to lipopolysaccharide as a key mechanistic step in the killing of gram-negative bacteria. A very large number of amino acid substitutions are tolerated by the protegrin structure, implying that overall structural features such as amphiphilicity, charge, and shape are more important to activity than the presence of specific amino acids. This lack of importance of specific stereochemistry is supported by the fact that completely D-amino acid substituted protegrins are fully potent. Based on the SAR studies, and on the microbiological data from an animal model, one protegrin analogue, IB-367, was selected for clinical development as a topical agent to prevent the oral mucositis associated with cancer therapy.

Dynamics of meningococcal long-term carriage among university students and their implications for mass vaccination.

In the 1997-98 academic year, we conducted a longitudinal study of meningococcal carriage and acquisition among first-year students at Nottingham University, Nottingham, United Kingdom. We examined the dynamics of long-term meningococcal carriage with detailed characterization of the isolates. Pharyngeal swabs were obtained from 2,453 first-year students at the start of the academic year (October), later on during the autumn term, and again in March. Swabs were immediately cultured on selective media, and meningococci were identified and serologically characterized. Nongroupable strains were genetically grouped using a PCR-based assay. Pulsed-field gel electrophoresis was used to determine the link between sequential isolates. Of the carriers initially identified in October, 44.1% (98 of 222) were still positive later on in the autumn (November or December); 57.1% of these remained persistent carriers at 6 months. Of the index carriers who lost carriage during the autumn, 16% were recolonized at 6 months. Of 344 index noncarriers followed up, 22.1% acquired carriage during the autumn term and another 13.7% acquired carriage by March. Overall, 43.9% (397 of 904) of the isolates were noncapsulated (serologically nongroupable); by PCR-based genogrouping, a quarter of these belonged to the capsular groups B and C. The ratio of capsulated to noncapsulated forms for group B and C strains was 2.9 and 0.95, respectively. Sequential isolates of persistent carriers revealed that individuals may carry the same or entirely different organisms at different times. We identified three strains that clearly switched their capsular expression on and off at different times in vivo. One student developed invasive meningococcal disease after carrying the same organism for over 7 weeks. The study revealed a high rate of turnover of meningococcal carriage among students. Noncapsulated organisms are capable of switching their capsular expression on and off (both ways) in the nasopharynx, and group C strains are more likely to be noncapsulated than group B strains. Carriage of a particular meningococcal strain does not necessarily protect against colonization or invasion by a homologous or heterologous strain.

IB-367, a protegrin peptide with in vitro and in vivo activities against the microflora associated with oral mucositis.

Although the microflora associated with oral mucositis initiated by cytotoxic therapy is not well characterized, several studies suggest that reduction of the microbial load in the oral cavity has some clinical benefit. The MICs of IB-367, a synthetic protegrin analog, ranged from 0.13 to 64 microgram/ml for gram-positive bacteria (Streptococcus mitis, Streptococcus sanguis, Streptococcus salivarius, and Staphylococcus aureus) and from 0.06 to 8 microgram/ml for gram-negative species (Klebsiella, Escherichia, and Pseudomonas). IB-367 exhibited rapid, microbicidal activity against both log- and stationary-phase cultures of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. At concentrations near the MICs for these two organisms (4 and 2 microgram/ml, respectively), IB-367 reduced viability by more than 3 logs in less than 16 min. Similarly, IB-367 effected a 4-log reduction of the endogenous microflora in pooled human saliva within 2 min at 250 microgram/ml, a concentration readily attained by local delivery. After nine serial transfers at 0.5x the MIC, the MIC of IB-367 for MRSA and P. aeruginosa increased only two to four times. In a phase I clinical study with healthy volunteers, IB-367 was well tolerated, with no detectable systemic absorption. One hour after treatment with 9 mg of IB-367, the prevalence of gram-negative bacteria and yeast was reduced, and the density of the predominant gram-positive oral flora was decreased 1,000 times. IB-367's properties (speed of killing, breadth of spectrum, and lack of resistance) make the compound a strong candidate for the prophylaxis of oral mucositis. Phase II clinical trials with IB-367 are under way for this indication in immunocompromised subjects.

Epidemiological studies of large resistance plasmids in Haemophilus.

The distribution of large conjugative Haemophilus influenzae plasmids in the nasopharyngeal haemophili of a group of people and in a large collection of 541 H. influenzae type b (Hib) isolates was studied. A newly developed PCR-based assay was used to detect the plasmids. The target sequences were chosen from sequence analysis of part of p1056, a large multiresistance plasmid isolated from a clinical Hib isolate, 1056. Fifty-nine per cent of people were found to carry beta-lactamase-positive (beta-lac(+)), ampicillin-resistant (ampR) haemophili with detectable plasmid sequences. Of these, 83% were in Haemophilus parainfluenzae and 17% were in H. influenzae. In the collection of 541 Hib, antibiotic resistance [beta-lac(+)ampR, beta-lac(+)ampR plus tetracycline resistance (tetR) or tetR] was highly correlated with large plasmids. It was found that 2.3% of the isolates contained large cryptic plasmids (i.e. these isolates were susceptible to antibiotics). The distribution of plasmids between invasive and carried Hib did not differ significantly (25 of 245 and 23 of 276, respectively). Isolates with large plasmids occur at high frequency in the nasopharynx of the normal human population and consist of two populations in Hib, one associated with specific antibiotic resistance traits and the other cryptic. These plasmids do not appear to influence the invasiveness of Hib.

Expression of the Staphylococcus aureus UDP-N-acetylmuramoyl- L-alanyl-D-glutamate:L-lysine ligase in Escherichia coli and effects on peptidoglycan biosynthesis and cell growth.

The monomer units in the Escherichia coli and Staphylococcus aureus cell wall peptidoglycans differ in the nature of the third amino acid in the L-alanyl-gamma-D-glutamyl-X-D-alanyl-D-alanine side chain, where X is meso-diaminopimelic acid or L-lysine, respectively. The murE gene from S. aureus encoding the UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: L-lysine ligase was identified and cloned into plasmid vectors. Induction of its overexpression in E. coli rapidly results in abnormal morphological changes and subsequent cell lysis. A reduction of 28% in the peptidoglycan content was observed in induced cells, and analysis of the peptidoglycan composition and structure showed that ca. 50% of the meso-diaminopimelic acid residues were replaced by L-lysine. Lysine was detected in both monomer and dimer fragments, but the acceptor units from the latter contained exclusively meso-diaminopimelic acid, suggesting that no transpeptidation could occur between the epsilon-amino group of L-lysine and the alpha-carboxyl group of D-alanine. The overall cross-linking of the macromolecule was only slightly decreased. Detection and analysis of meso-diaminopimelic acid- and L-lysine-containing peptidoglycan precursors confirmed the presence of L-lysine in precursors containing amino acids added after the reaction catalyzed by the MurE ligase and provided additional information about the specificity of the enzymes involved in these latter processes.

Joint tolerance to beta-lactam and fluoroquinolone antibiotics in Escherichia coli results from overexpression of hipA.

The basis of joint tolerance to beta-lactam and fluoroquinolone antibiotics in Escherichia coli mediated by hipA was examined. An antibiotic tolerance phenotype was produced by overexpression of hipA under conditions that did not affect the growth rate of the organism. Overexpressing hipA probably decreases the period in which bacteria are susceptible to the antibiotics by temporarily affecting some aspect of chromosome replication or cell division.

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.

Improved activity of a synthetic indolicidin analog.

A novel cationic peptide, CP-11, based on the structure of the bovine neutrophil peptide indolicidin, was designed to increase the number of positively charged residues, maintain the short length (13 amino acids), and enhance the amphipathicity relative to those of indolicidin. CP-11, and especially its carboxymethylated derivative, CP-11C, demonstrated improved activity against gram-negative bacteria and Candida albicans, while it maintained the activity of indolicidin against staphylococci and demonstrated a reduced ability to lyse erythrocytes. In Escherichia coli, CP-11 was better able than indolicidin to permeabilize both the outer membrane, as indicated by the enhancement of uptake of 1-N-phenylnaphthylamine, and the inner membrane, as determined by the unmasking of cytoplasmic beta-galactosidase, providing an explanation for its improved activity.

Mode of action of the antimicrobial peptide indolicidin.

Indolicidin is a cationic antimicrobial peptide isolated from bovine neutrophils. It consists of only 13 amino acids, has the highest tryptophan content of any known protein, and is amidated at the carboxyl terminus in nature. By circular dichroism spectroscopy a weak poly-L-proline II extended helix structure was observed that became substantially more pronounced upon interaction with liposomes. Indolicidin bound purified surface lipopolysaccharide with high affinity and permeabilized the outer membrane of Escherichia coli to the small hydrophobic molecule 1-N-phenylnapthylamine (Mr 200), results consistent with indolicidin crossing the outer membrane via the self-promoted uptake pathway. The methyl esterification of indolicidin's carboxyl terminus increased its activity for Gram-negative and Gram-positive bacteria. In Gram-negative bacteria this was associated with an increased binding to lipopolysaccharide and increased permeabilization of the outer membrane. The cytoplasmic membrane was the site of action of indolicidin as assayed in E. coli by the unmasking of cytoplasmic beta-galactosidase due to membrane permeabilization. The mechanism for this activity was shown to be the ability of the peptide to cause an increase in the transmembrane current of planar lipid bilayers. This current increase was activated by transmembrane potentials in excess of -70 to -80 mV. Consistent with this, there was a substantial decrease in indolicidin-mediated bacterial killing and permeabilization of the cytoplasmic membrane of E. coli that had been pretreated with the uncoupler carbonyl cyanide-m-chlorophenyl hydrazone. In planar bilayers, indolicidin induced the formation of discrete channels, which ranged in conductance from 0.05-0.15 nS. Thus despite the small size and unique composition of indolicidin, it was capable of killing Gram-negative bacteria by crossing the outer membrane and causing disruption of the cytoplasmic membrane by channel formation.

The elucidation of novel capsular genotypes of Haemophilus influenzae type b with the polymerase chain reaction.

Molecular characterization is an important pre-requisite for post-vaccine studies of Haemophilus influenzae type b (Hib). Three capsular genotyping patterns, b(S), b(G) and b(V), have been described in the major phylogenetic lineage of Hib. However, in a recent series of prospective studies, three new hybridisation patterns were observed among 425 strains of Hib. Four pairs of polymerase chain reaction (PCR) primers were used to identify the capsular gene (cap) structure of these Hib strains. This showed that the strains possessed simple DNA re-arrangements. In two instances a change in restriction enzyme recognition site was the most likely cause of the new hybridisation pattern. The third strain possessed a cap b locus consisting of intact tandem repeats of cap b in a b(S) background. It was reasoned that a similar cap b locus would not be readily recognised by hybridisation in a b(G) background, and b(G) strains were therefore characterized by the PCR method. This showed one of 35 b(G) strains to possess a cap locus with intact tandem repeat copies of cap b. The novel capsular genotypes described here are rare, but can be detected rapidly and accurately by a combination of PCR and capsular genotyping hybridisation patterns.

PCR for capsular typing of Haemophilus influenzae.

A PCR method for the unequivocal assignment of Haemophilus influenzae capsular type (types a to f) was developed. PCR primers were designed from capsule type-specific DNA sequences cloned from the capsular gene cluster of each of the six capsular types. PCR product was amplified only from the capsular type for which the primers were designed. Product was confirmed by using either an internal oligonucleotide or restriction endonuclease digestion. A total of 172 H. influenzae strains of known capsular type (determined genetically) comprising all capsular types and noncapsulate strains were tested by PCR capsular typing. In all cases the PCR capsular type corresponded to the capsular genotype determined by restriction fragment length polymorphism analysis of the cap region. When used in conjunction with PCR primers derived from the capsular gene bexA, capsulate, noncapsulate, and capsule-deficient type b mutant strains could be differentiated. PCR capsular typing overcomes the problems of cross-reaction and autoagglutination associated with the serotyping of H. influenzae strains. The rapid and unequivocal capsular typing method that is described will be particularly important for typing invasive H. influenzae strains isolated from recipients of H. influenzae type b vaccine.

Population-based study of non-typable Haemophilus influenzae invasive disease in children and neonates.

The extent of non-capsulate, non-serotypable Haemophilus influenzae (NST) as a cause of serious invasive disease in children has not been fully defined. We describe the epidemiology of these childhood infections from cases identified during a continuing prospective survey of invasive H influenzae disease in the Oxford region, UK. 408 strains of H influenzae were isolated from cases of invasive disease. 383 (94%) were H influenzae type b (Hib), 24 (6%) were NST strains, and 1 was a type f strain. 3 of the NST strains were non-capsulate type b mutants (b-), but the remaining 21 strains were from the phylogenetically distinct and heterogeneous population of non-capsulate H influenzae (NC). 10 of the NC strains were isolated from neonates with sepsis; crude mortality rate was 40%, with an incidence of 4.6 cases per 100,000 livebirths. 11 NC strains were isolated from children after the neonatal period and under 10 years of age, 4 (36%) of which had severe, unrelated, predisposing conditions. The incidence of NC invasive diseases in these children was 0.5 per 100,000 per year. The attributable mortality for these infections was 10%. Infections due to these H influenzae strains are, after the implementation of Hib vaccines, likely to persist and represent a substantial proportion of the serious infections caused by this species.