Antimicrobial peptides (AMPs) have been studied for three decades, and yet a molecular understanding of their mechanism of action is still lacking. Here we summarize current knowledge for both synthetic vesicle experiments and microbe experiments, with a focus on comparisons between the two. Microbial experiments are done at peptide to lipid ratios that are at least 4 orders of magnitude higher than vesicle-based experiments. To close the gap between the two concentration regimes, we propose an “interfacial activity model”, which is based on an experimentally testable molecular image of AMP–membrane interactions. The interfacial activity model may be useful in driving engineering and design of novel AMPs.

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Describing the mechanism of antimicrobial peptide action with the interfacial activity model.

Obtaining the correct balance of secondary structure propensities is a central priority in protein force-field development. Given that current force fields differ significantly in their alpha-helical propensities, a correction to match experimental results would be highly desirable. We have determined simple backbone energy corrections for two force fields to reproduce the fraction of helix measured in short peptides at 300 K. As validation, we show that the optimized force fields produce results in excellent agreement with nuclear magnetic resonance experiments for folded proteins and short peptides not used in the optimization. However, despite the agreement at ambient conditions, the dependence of the helix content on temperature is too weak, a problem shared with other force fields. A fit of the Lifson-Roig helix-coil theory shows that both the enthalpy and entropy of helix formation are too small: the helix extension parameter w agrees well with experiment, but its entropic and enthalpic components are both only about half the respective experimental estimates. Our structural and thermodynamic analyses point toward the physical origins of these shortcomings in current force fields, and suggest ways to address them in future force-field development.

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Optimized molecular dynamics force fields applied to the helix-coil transition of polypeptides.

Many bacteria can infect and persist inside their hosts for long periods of time In this Review, Fisher, Gollan and Helaine discuss recent developments in our understanding of bacterial persisters and their potential implications for the treatment of persistent infections Many bacteria can infect and persist inside their hosts for long periods of time This can be due to immunosuppression of the host, immune evasion by the pathogen and/or ineffective killing by antibiotics Bacteria can survive antibiotic treatment if they are resistant or tolerant to a drug Persisters are a subpopulation of transiently antibiotic-tolerant bacterial cells that are often slow-growing or growth-arrested, and are able to resume growth after a lethal stress The formation of persister cells establishes phenotypic heterogeneity within a bacterial population and has been hypothesized to be important for increasing the chances of successfully adapting to environmental change The presence of persister cells can result in the recalcitrance and relapse of persistent bacterial infections, and it has been linked to an increase in the risk of the emergence of antibiotic resistance during treatment If the mechanisms of the formation and regrowth of these antibiotic-tolerant cells were better understood, it could lead to the development of new approaches for the eradication of persistent bacterial infections In this Review, we discuss recent developments in our understanding of bacterial persisters and their potential implications for the treatment of persistent infections

Persistent bacterial infections and persister cells

Development of the AMOEBA (atomic multipole optimized energetics for biomolecular simulation) force field for proteins is presented. The current version (AMOEBA-2013) utilizes permanent electrostatic multipole moments through the quadrupole at each atom, and explicitly treats polarization effects in various chemical and physical environments. The atomic multipole electrostatic parameters for each amino acid residue type are derived from high-level gas phase quantum mechanical calculations via a consistent and extensible protocol. Molecular polarizability is modeled via a Thole-style damped interactive induction model based upon distributed atomic polarizabilities. Inter- and intramolecular polarization is treated in a consistent fashion via the Thole model. The intramolecular polarization model ensures transferability of electrostatic parameters among different conformations, as demonstrated by the agreement between QM and AMOEBA electrostatic potentials, and dipole moments of dipeptides. The backbone and...

/pdf/polarizable-atomic-multipole-based-amoeba-force-field-for-4od3yyv8c4.pdf

Polarizable Atomic Multipole-Based AMOEBA Force Field for Proteins

Bacterial cells may escape the effects of antibiotics without undergoing genetic change; these cells are known as persisters. Unlike resistant cells that grow in the presence of antibiotics, persister cells do not grow in the presence of antibiotics. These persister cells are a small fraction of exponentially growing cells (due to carryover from the inoculum) but become a significant fraction in the stationary phase and in biofilms (up to 1%). Critically, persister cells may be a major cause of chronic infections. The mechanism of persister cell formation is not well understood, and even the metabolic state of these cells is debated. Here, we review studies relevant to the formation of persister cells and their metabolic state and conclude that the best model for persister cells is still dormancy, with the latest mechanistic studies shedding light on how cells reach this dormant state.

/pdf/bacterial-persister-cell-formation-and-dormancy-2z2w09pn6u.pdf

Bacterial Persister Cell Formation and Dormancy

A class of antimicrobial peptides involved in host defense consists of sequences rich in Arg and Trp-R and -W. Analysis of the pharmacophore in these peptides revealed that chains as short as trimers of sequences such as WRW and RWR have antimicrobial activity (M. B. Strom, B. E. Haug, M. L. Skar, W. Stensen, T. Stiberg, and J. S. Svendsen, J. Med. Chem. 46:1567-1570, 2003). To evaluate the effect of chain length on antimicrobial activity, we synthesized a series of peptides containing simple sequence repeats, (RW)n-NH2 (where n equals 1, 2, 3, 4, or 5), and determined their antimicrobial and hemolytic activity. The antimicrobial activity of the peptides increases with chain length, as does the hemolysis of red blood cells. Within the experimental error, longer peptides (n equals 3, 4, or 5) show similar values for the ratio of hemolytic activity to antibacterial activity, or the hemolytic index. The (RW)3 represents the optimal chain length in terms of the efficacy of synthesis and selectivity as evaluated by the hemolytic index. Circular dichroism spectroscopy indicates that these short peptides appear to be unfolded in aqueous solution but acquire structure in the presence of phospholipids. Interaction of the peptides with model lipid vesicles was examined using tryptophan fluorescence. The (RW)n peptides preferentially interact with bilayers containing the negatively charged headgroup phosphatidylglycerol relative to those containing a zwitterionic headgroup, phosphatidylcholine.

Length Effects in Antimicrobial Peptides of the (RW)n Series

Synthesis and biological evaluation of novel 1,3,5-triazine derivatives as antimicrobial agents

Persister cells are dormant phenotypic variants inherent in a bacterial population. They play important roles in chronic infections and present great challenges to therapy due to extremely enhanced tolerance to antibiotics compared to that of normal cells of the same genotype. In this study, we report that cationic membrane-penetrating peptides containing various numbers of arginine and tryptophan repeats are effective in killing persister cells of Escherichia coli HM22, a hyper-persister producer. The activities of three linear peptides [(RW)(n)-NH(2), where n is 2, 3, or 4] and a dendrimeric peptide, (RW)(4D), in killing bacterial persisters were compared. Although the dendrimeric peptide (RW)(4D) requires a lower threshold to kill planktonic persisters, octameric peptide (RW)(4)-NH(2) is the most effective against planktonic persister cells at high concentrations. For example, treatment with 80 μM (RW)(4)-NH(2) for 60 min led to a 99.7% reduction in the number of viable persister cells. The viability of persister cells residing in surface-attached biofilms was also significantly reduced by (RW)(4)-NH(2) and (RW)(4D). These two peptides were also found to significantly enhance the susceptibility of biofilm cells to ofloxacin. The potency of (RW)(4)-NH(2) was further marked by its ability to disperse and kill preformed biofilms harboring high percentages of persister cells. Interestingly, approximately 70% of the dispersed cells were found to have lost their intrinsic tolerance and become susceptible to ampicillin if not killed directly by this peptide. These results are helpful for better understanding the activities of these peptides and may aid in future development of more effective therapies of chronic infections.

https://aem.asm.org/content/aem/77/14/4878.full.pdf

Control of Bacterial Persister Cells by Trp/Arg-Containing Antimicrobial Peptides

Antimicrobial dendrimer active against Escherichia coli biofilms

Rapid emergence of antibacterial drug resistance poses a critical problem for the treatment of infectious diseases. The number of new classes of antibiotics approved by the FDA has declined in recent years. Since their discovery in the early 1980s, antimicrobial peptides (AMPs, also called host defense peptides) have stimulated interest as prospective antibiotic agents because they rapidly inactivate a wide range of microorganisms including Gram-positive and negative bacteria, fungi, and some viruses. In many cases they are indifferent to current multidrug-resistant (MDR) strains. Naturally occurring AMPs span a wide range of size, sequence, and structure. They generally share only amphiphilicity and positive charge. Different chemical approaches have been pursued in efforts to increase the effectiveness of AMPs, including incorporation of unnatural dor b-amino acids, cyclization of peptides, and screening of synthetic AMP combinatorial libraries. Despite recent progress, the diversity and lack of structural homology among known AMPs make it difficult to predict the activity of a peptide or to design AMPs with desired activities in vivo. In a recent study, a linguistic model was applied to the rational design of AMPs. To date, classical drug R&D approaches have not been successfully applied to derive novel antibiotics from AMPs. The amino acids Arg (R) and Trp (W) occur in natural AMPs that span a range of sizes and secondary structures, including indolicidin and tritrpticin, which have a broad spectrum of antibacterial activity but lyse erythrocytes. QSAR analysis of Rand W-rich peptides suggested that the order of amino acids is not crucial whereas R and W content correlates most strongly with activity. Strom et al. proposed that simple combinations of R and W side chains constitute one type of pharmacophore for AMPs. We recently investigated the length requirement for antimicrobial activity of RW peptides. Even short repeats of RW are active, providing a starting point for de novo design of AMPs. The hypothesis we test in this work is that multi/polyvalent RW peptide display can enhance the effectiveness of antibacterials against MDR bacterial strains. Application of multivalency to cell surface receptors has been demonstrated previously. Our thinking was originally based on the two-state model of AMP action proposed by Huang, which is broadly consistent with other current ideas. The model assumes that AMPs act at the level of the cell membrane via electrostatic interactions with the negatively charged head groups of bacterial lipids. At peptide-to-lipid ratios (P/L) above a threshold value, the peptides form clusters that lead to permeation of the cell membrane. The susceptibility of a cell to an AMP depends on the value of P/L determined by the lipid composition of the cell membrane. Huang ascribes this to an elastic perturbation of the surface. However alternative physical mechanisms can be invoked. A consequence of assembly models is that prenucleating AMP monomers might reduce the critical concentration for cluster formation as AMPs bind to the bacterial membrane. Initial attachment of AMPs to bacterial surfaces or membranes is salt sensitive, consistent with electrostatic interactions. General arguments suggest that covalently tethering a number of weakly interacting ligands can enhance overall avidity for targets such as cell surfaces. We recently applied this strategy to create new antimicrobial agents by linking tetrapeptides RWRW and RRWW to a polydisperse polymer scaffold. These constructs enhanced antimicrobial activity, with an increase of roughly tenfold in potency against both Gram-negative and positive strains. At the same time the hemolytic activity of the polymers increased, but the ratio of antimicrobial to hemolytic activity remained roughly constant. To further explore multivalent AMP designs, we have synthesized a variety of dendrimeric AMPs containing multiple RW dipeptides. Of these the most successful is (RW)4D (Scheme 1). A detailed account of the constructs based on alternative scaffolds will be presented elsewhere. Dendrimeric peptide displays were first developed in the 1980s as multiple antigenic peptides. They consist of a core formed by radially branched lysines or other residues. Peptide sequences are grafted to the core by standard solid-phase chemistry. Herein, we report the antibacterial and hemolytic activity of (RW)4D compared to the natural AMP indolicidin (ILPWKWPWWPWRR-NH2) from bovine

Chunhui Zhou

Papers

Length Effects in Antimicrobial Peptides of the (RW)n Series

Synthesis and biological evaluation of novel 1,3,5-triazine derivatives as antimicrobial agents

Control of Bacterial Persister Cells by Trp/Arg-Containing Antimicrobial Peptides

Antimicrobial dendrimer active against Escherichia coli biofilms

Tuning the membrane selectivity of antimicrobial peptides by using multivalent design.