Control of cell selectivity of antimicrobial peptides

Antimicrobial peptides (AMPs) are promising novel antibiotics, because they exhibit broad antimicrobial spectra and do not easily induce resistance. For clinical applications, it is important to develop potent AMPs with less toxicity against host cells. This review article summarizes the molecular basis for the cell selectivity (bacteria versus host cells) of AMPs and various attempts to control it, including the optimization of physicochemical parameters of peptides, the introduction of d-, fluorinated, and unusual amino acids into peptides, the constraining of peptide conformations, and the modification of peptides by polymers. Pros and cons of these approaches are discussed.     

Tryptophan end-tagging of antimicrobial peptides for increased potency against Pseudomonas aeruginosa

Background

Due to increasing antibiotics resistance, antimicrobial peptides (AMPs) are receiving increased attention. Pseudomonas aeruginosa is a major pathogen in this context, involved, e.g., in keratitis and wound infections. Novel bactericidal agents against this pathogen are therefore needed.

Methods

Bactericidal potency was monitored by radial diffusion, viable count, and minimal inhibitory concentration assays, while toxicity was probed by hemolysis. Mechanistic information was obtained from assays on peptide-induced vesicle disruption and lipopolysaccharide binding.

Results

End-tagging by hydrophobic amino acids yields increased potency of AMPs against P. aeruginosa, irrespective of bacterial proteinase production. Exemplifying this by two peptides from kininogen, GKHKNKGKKNGKHNGWK and KNKGKKNGKH, potency increased with tag length, correlating to more efficient bacterial wall and vesicle rupture, and to more pronounced P. aeruginosa lipopolysaccharide binding. End-tag effects remained at high electrolyte concentration and in the presence of plasma or anionic macromolecular scavengers. The tagged peptides displayed stability against P. aeruginosa elastase, and were potent ex vivo, both in a contact lens model and in a skin wound model.

General significance

End-tagging, without need for post-peptide synthesis modification, may be employed to enhance AMP potency against P. aeruginosa at maintained limited toxicity.     

N-terminal amphipathic helix as a trigger of hemolytic activity in antimicrobial peptides: A case study in latarcins

In silico structural analyses of sets of α-helical antimicrobial peptides (AMPs) are performed. Differences between hemolytic and non-hemolytic AMPs are revealed in organization of their N-terminal region. A parameter related to hydrophobicity of the N-terminal part is proposed as a measure of the peptide propensity to exhibit hemolytic and other unwanted cytotoxic activities. Based on the information acquired, a rational approach for selective removal of these properties in AMPs is suggested. A proof of concept is gained through engineering specific mutations that resulted in elimination of the hemolytic activity of AMPs (latarcins) while leaving the beneficial antimicrobial effect intact.     

Improved potency and reduced toxicity of the antifungal peptoid AEC5 through submonomer modification

As proteolytically stable peptidomimetics, peptoids could serve as antifungal agents to supplement a therapeutic field wrought with toxicity issues. We report the improvement of an antifungal peptoid, AEC5, through an iterative structure-activity relationship study. A sarcosine scan was used to first identify the most pharmacophorically important peptoid building blocks of AEC5, followed by sequential optimization of each building block. The optimized antifungal peptoid from this study, β-5, has improved potency towards Cryptococcus neoformans and decreased toxicity towards mammalian cells. For example, the selectivity ratio for C. neoformans over mammalian fibroblasts was improved from 8 for AEC5 to 37 for β-5.     

Lysine substitutions convert a bacterial-agglutinating peptide into a bactericidal peptide that retains anti-lipopolysaccharide activity and low hemolytic activity

GL13NH2 is a bacteria-agglutinating peptide derived from the sequence of the salivary protein parotid secretory protein (PSP, BPIFA2, SPLUNC2, C20orf70). The peptide agglutinates both Gram negative and Gram positive bacteria, and shows anti-lipopolysaccharide activity in vitro and in vivo. However, GL13NH2 does not exhibit bactericidal activity. To generate a more cationic peptide with potential bactericidal activity, three amino acid residues were replaced with lysine residues to generate the peptide GL13K. In this report, the antibacterial and anti-inflammatory activities of GL13K were characterized. GL13K had lost the ability to agglutinate bacteria but gained bactericidal activity. Substitution of individual amino acids in GL13K with alanine did not restore bacterial agglutination. GL13K was bactericidal against Pseudomonas aeruginosa, Streptococcus gordonii and Escherichia coli but not Porphyromonas gingivalis. Unlike the agglutinating activity of GL13NH2, the bactericidal activity of GL13K against P. aeruginosa was retained in the presence of saliva. Both GL13NH2 and GL13K exhibited anti-lipopolysaccharide activity. In GL13K, this activity appeared to depend on a serine hydroxyl group. GL13K protected mice from lipopolysaccharide-induced sepsis and the peptide exhibited a low level of hemolysis, suggesting that it may be suitable for in vivo application.     

Serovar distribution and antimicrobial susceptibility of swine Salmonella isolates from clinically ill pigs in diagnostic submissions from Indiana in the United States

Aims:  To determine serovar distribution and levels of antimicrobial susceptibility of Salmonella isolated from clinically ill pigs in diagnostic submissions.
Methods and Results:  A total of 197 Salmonella isolates were obtained by the Indiana Animal Disease Diagnostic Laboratory from 2003 to 2005. Minimal inhibitory concentrations (MICs) were determined using the standard microbroth dilution method. The top four serovars identified were Salm. enterica serovar Typhimurium variant Copenhagen, Salm . Derby, Salm . Choleraesuis var. Kunzendorf and Salm . Typhimurium. All isolates were susceptible to the fluoroquinolones tested except that eight isolates were intermediate to difloxacin. The isolates showed a low prevalence of resistance to trimethoprim/sulphadiazine (Sxt), gentamicin (G), ceftiofur (Cf) and cephalothin (Cp) with low MIC₅₀ value of ≤0·5, 0·5, 1 and 4  μ g ml⁻¹, respectively. They showed a high prevalence of resistance to tetracycline (T; 83·8%), and a moderate prevalence to ampicillin (55·8%), spectinomycin (42·6%), ticarcillin (41·6%) and florfenicol (41·1%). There were more isolates of Salm . Typhimurium, including var. Copenhagen and Salm . Agona, that possessed multiple antimicrobial resistance to amoxicillin/clavulanic acid, ampicillin, ceftiofur and cephalothin (AxApCfCp) than the other serovars.
Conclusions:  The swine Salmonella isolates were susceptible to the fluoroquinolones, Sxt, G, Cf and Cp, but resistant to T.
Significance and Impact of the Study:  These findings provided useful information regarding antimicrobial susceptibility and resistance in dealing with clinical salmonellosis in pig herds.     

The effects of charge and lipophilicity on the antibacterial activity of undecapeptides derived from bovine lactoferricin.

We have investigated the effects of charge and lipophilicity on the antibacterial activity of an undecapeptide (FKCRRWQWRMK) derived from the sequence of bovine lactoferricin. We prepared ten analogues that were modified by the incorporation of Ala, Tyr, Trp, Met and Arg residues, which are amino acids known to be important for the antibacterial activity of longer derivatives of lactoferricins. All undecapeptides contained the native Trp residues in positions 6 and 8, and the Arg residues in positions 5 and 9. Generally, the Gram-positive bacterium Staphylococcus aureus was more susceptible to these undecapeptides than the Gram-negative bacteria, and a higher antibacterial activity was observed against Escherichia coli than against Pseudomonas aeruginosa. The only exception was the peptide Undeca 9 (RRWYRWAWRMR-NH2), which was almost equally active against all three test strains, displaying minimal inhibitory concentrations of 10 microg/ml (5.8 microM), 7.5 microg/ml (4.4 microM) and 5 microg/ml (2.9 microM) against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, respectively. The peptides Undeca 6 (YRAWRWAWRWR-NH2) and Undeca 7 (YRMWRWAWRWR-NH2) were the two most active undecapeptides against Staphylococcus aureus, both displaying a minimal inhibitory concentration of 2.5 microg/ml (1.5 microM). The study showed that a level was reached in which undecapeptides having a net charge above +4 and containing three or four Trp residues all displayed a high antibacterial activity. All undecapeptides prepared were essentially non-haemolytic, but undecapeptides containing more than three Trp residues displayed 50% haemolysis of human red blood cells at concentrations above 400 microg/ml (>230 microM).     

Antimicrobial activity of the Naja atra cathelicidin and related small peptides

We have identified an 11-residue pattern (KR(F/A)KKFFKK(L/P)K), which we have named the ATRA motif, within the sequence of the Chinese cobra (Naja atra) cathelicidin. A series of 11-residue peptides (ATRA-1, -2, -1A and -1P) were designed to probe the significance of the conserved residues within the ATRA motif, and their contributions to antimicrobial performance. The antimicrobial activities of the peptides were assessed against Escherichia coli K12 strain and Aggregatibacter actinomycetemcomitans Y4. ATRA-1 and -1A, demonstrated potencies comparable to that of N. atra cathelicidin. Structural examination by circular dichroism of the four short peptides suggested the significance of specific amino acid positions within the motif by their contribution to helicity. The results of these studies indicate that short peptides derived from the repeated ATRA motif from the N. atra cathelicidin can demonstrate both low toxicity against host cells and high antimicrobial activity against the gram-negative bacteria used in this study. They constitute novel, effective antimicrobial peptides that are much shorter (and thus less expensive to produce) than the natural cathelicidins, and they may represent new templates for therapeutic drug development.     

Effect of antimicrobial peptides on ATPase activity and proton pumping in plasma membrane vesicles obtained from mycobacteria

The potential usefulness of antimicrobial peptides (AMPs) as antimycobacterial compounds has not been extensively explored. Although a myriad of studies on AMPs from different sources have been done, some of its mechanisms of action are still unknown. Maganins are of particular interest since they do not lyse non-dividing mammalian cells. In this work, AMPs with well-recognized activity against bacteria were synthesized, characterized, purified and their antimycobacterial activity and influence on ATPase activity in mycobacterial plasma membrane vesicles were assessed. Using bioinformatics tools, a magainin-I analog peptide (MIAP) with improved antimicrobial activity was designed. The influence of MIAP on proton (H(+)) pumping mediated by F(1)F(0)-ATPase in plasma membrane vesicles obtained from Mycobacterium tuberculosis was evaluated. We observed that the antimycobacterial activity of AMPs was low and variable. However, the activity of the designed peptide MIAP against M. tuberculosis was 2-fold higher in comparison to magainin-I. The basal ATPase activity of mycobacterial plasma membrane vesicles decreased approximately 24-30% in the presence of AMPs. On the other hand, the MIAP peptide completely abolished the F(1)F(0)-ATPase activity involved in H(+) pumping across M. tuberculosis plasma membranes vesicles at levels similar to the specific inhibitor N,N' dicyclohexylcarbodiimide. These finding suggest that AMPs can inhibit the H(+) pumping F(1)F(0)-ATPase of mycobacterial plasma membrane that potentially interferes the internal pH and viability of mycobacteria.     

High level expression of human enteropeptidase light chain in Pichia pastoris

Human enterokinase (enteropeptidase, rhEP), a serine protease expressed in the proximal part of the small intestine, converts the inactive form of trypsinogen to active trypsin by endoproteolytic cleavage. The high specificity of the target site makes enterokinase an ideal tool for cleaving fusion proteins at defined cleavage sites. The mature active enzyme is comprised of two disulfide-linked polypeptide chains. The heavy chain anchors the enzyme in the intestinal brush border membrane, whereas the light chain represents the catalytic enzyme subunit. The synthetic gene encoding human enteropeptidase light chain with His-tag added at the C-terminus to facilitate protein purification was cloned into Pichia pastoris expression plasmids under the control of an inducible AOX1 or constitutive promoters GAP and AAC. Cultivation media and conditions were optimized as well as isolation and purification of the target protein. Up to 4 mg/L of rhEP was obtained in shake-flask experiments and the expression level of about 60-70 mg/L was achieved when cultivating in lab-scale fermentors. The constitutively expressing strains proved more efficient and less labor-demanding than the inducible ones. The rhEP was immobilized on AV 100 sorbent (Iontosorb) to allow repeated use of enterokinase, showing specific activity of 4 U/mL of wet matrix.     

Purification and structural characterization of a novel antibacterial peptide from Bellamya bengalensis: activity against ampicillin and chloramphenicol resistant Staphylococcus epidermidis

Increasing tendency of clinical bacterial strains resistant to conventional antibiotics has being a great challenge to the public's health. Antimicrobial peptides, a new class of antibiotics is known to have the activity against a wide range of bacteria resistant to conventional antibiotics. An antimicrobial peptide of 1676 Da was purified from Bellamya bengalensis, a fresh water snail, using ultrafiltration and reversed phase liquid chromatography. The effect of this peptide on Staphylococcus epidermidis resistant to ampicillin and chloramphenicol was investigated; the MIC and MBC values were 8 μg/ml and 16 μg/ml, respectively. Complete sequence of the peptide was determined by tandem mass spectrometry (MS/MS). Further, peptide net charge, hydrophobicity and molecular modeling were evaluated in silico for better understanding the probable mechanisms of action. The peptide showed the specificity to bacterial membranes. Hence, this reported peptide revealed a promising candidate to contribute in the development of therapeutic agent for Staphylococcal infections.     

Different modes in antibiotic action of tritrpticin analogs, cathelicidin-derived Trp-rich and Pro/Arg-rich peptides

The cathelicidin-derived antimicrobial tritrpticin could be classified as either Trp-rich or Pro/Arg-rich peptide. We recently found that the sequence modification of tritrpticin focused on Trp and Pro residues led to considerable change in structure and antimicrobial potency and selectivity, but their mechanisms of microbial killing action were still unclear. Here, to better understand the bactericidal mechanisms of tritrpticin and its two analogs, TPA and TWF, we studied their effect on the viability of Gram-positive S. aureus and Gram-negative E. coli in relation to their membrane depolarization. Although TWF more effectively inhibited growth of S. aureus and E. coli than TPA, only a 30 min exposure to TPA was sufficient to kill both bacteria and TWF required a lag period of about 3-6 h for bactericidal activity. Their different bactericidal kinetics was associated with membrane permeabilization, i.e., TWF showed negligible ability to depolarize the cytoplasmic membrane potential of target cell membrane, whereas we observed significant membrane depolarization for TPA. In addition, while TPA caused rapid and large dye leakage from negatively charged model vesicles, TWF showed very little membrane-disrupting activity. Interestingly, we have looked for a synergism among the three peptides against E. coli, supporting that they are working with different modes of action. Collectively, our results suggest that TPA disrupts the ion gradients across the membrane, causing depolarization and a loss of microbial viability. By contrast, TWF more likely translocates across the cytoplasmic membrane without depolarization and then acts against one or more intracellular targets. Tritrpticin exhibits intermediate properties and appears to act via membrane depolarization coupled to secondary intracellular targeting.     

Temporins and their synergism against Gram-negative bacteria and in lipopolysaccharide detoxification

Ribosomally synthesized antimicrobial peptides (AMPs) represent an essential component of the ancient and non-specific innate immune system in all forms of life, with the primary role of killing infectious microorganisms. Amphibian skin is one of the richest storehouses for them. Each frog species produces its own set of peptides with up to 10 isoforms, as in the case of the species Rana temporaria. Nowadays, human health is facing two major threats: (i) the increasing emergence of resistant pathogens to one or more available drugs, and (ii) the onset of septic shock, which is associated with the release of lipopolysaccharide (LPS) from the cell walls of Gram-negative bacteria, particularly upon antibiotic treatment. AMPs are considered as potential new anti-infective compounds with a novel mode of action, because many of them can kill bacteria and, at the same time, neutralize the toxic effects of LPS. Recent studies have suggested that the production of large number of structurally similar AMPs within the same animal is a strategy used by nature to increase the spectrum of antimicrobial activities, by using combinations of the peptide's isoforms. The biological rationale for their coexistence within the same organism is discussed. In addition, the distinctive and attractive synergistic effects of temporins in both antimicrobial and anti-endotoxin activities are reviewed, along with their plausible underlying molecular mechanism.     

Antimicrobial peptide mimics for improved therapeutic properties

The relatively recent recognition of the major role played by antimicrobial peptides (AMPs) in sustaining an effective host response to immune challenges was greatly influenced by studies of amphibian peptides. AMPs are also widely regarded as a potential source of future antibiotics owing to a remarkable set of advantageous properties ranging from molecular simplicity to low-resistance swift-kill of a broad range of microbial cells. However, the peptide formula per se, represents less than ideal drug candidates, namely because of poor bioavailability issues, potential immunogenicity, optional toxicity and high production costs. To address these issues, synthetic peptides have been designed, reproducing the critical peptide biophysical characteristic in unnatural sequence-specific oligomers. Thus, the use of peptidomimetics to overcome the limitations inherent to peptides physical characteristics is becoming an important and promising approach for improving the therapeutic potential of AMPs. Here, we review most recent advances in the design strategies and the biophysical properties of the main classes of mimics to natural AMPs, emphasizing the importance of structure-activity relationship studies in fine-tuning of their physicochemical attributes for improved antimicrobial properties.     

Cloning,expression, and purification of a new antimicrobial peptide gene from Musca domestica larva

Musca domestica (Diptera: Muscidae), the housefly, exhibits unique immune defences and can produce antimicrobial peptides upon stimulation with bacteria. Based on the cDNA library constructed using the suppression subtractive hybridization (SSH) method, a 198-bp antimicrobial peptide gene, which we named MDAP-2, was amplified by rapid amplification of cDNA ends (RACE) from M. domestica larvae stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In the present study, the full-length MDAP-2 gene was cloned and inserted into a His-tagged Escherichia coli prokaryotic expression system to enable production of the recombinant peptide. The recombinant MDAP-2 peptide was purified using Ni-NTA HisTrap FF crude column chromatography. The bacteriostatic activity of the recombinant purified MDAP-2 protein was assessed. The results indicated that MDAP-2 had in vitro antibacterial activity against all of the tested Gram − bacteria from clinical isolates, including E. coli (Enterobacteriaceae: Escherichia), one strain of S. pullorum (Enterobacteriaceae: Salmonella), and one strain of Pasteurella multocida. DNA sequencing and BLAST analysis showed that the MDAP-2 antimicrobial peptide gene was not homologous to any other antimicrobial peptide genes in GenBank. The antibacterial mechanisms of the newly discovered MDAP-2 peptide warrant further study.     

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.     

Inhibition of mRNA synthesis in rabbit bone marrow nuclei in vitro by quinone metabolites of benzene

mRNA synthesis by rabbit bone marrow nuclei has been shown to be inhibited by the quinone metabolites of benzene, hydroquinone and p-benzoquinone, in a concentration-dependent manner with 50% inhibitory concentration (IC50[M]) for both compounds of 6 X 10(-6) M. Catechol and 1,2,4-benzenetriol also showed a concentration-dependent inhibition of synthesis, however, 50% inhibition was not reached by 10(-4) M. Phenol did not inhibit mRNA synthesis even at 10(-3) M. It is possible that myelotoxicity from benzene might result from such an inhibition of mRNA synthesis by quinone metabolites in pluripotent and/or committed bone marrow stem cells.