Host-defense peptides (HDPs) feature evolution-tested potency against life-threatening pathogens. While piscidin 1 (p1) and piscidin 3 (p3) are homologous and potent fish HDPs, only p1 is strongly membranolytic. Here, we hypothesize that another mechanism imparts p3 strong potency. We demonstrate that the N-termini of both peptides coordinate Cu and p3-Cu cleaves isolated DNA at a rate on par with free Cu but significantly faster than p1-Cu. On planktonic bacteria, p1 is more antimicrobial but only p3 features copper-dependent DNA cleavage. On biofilms and persister cells, p3-Cu is more active than p1-Cu, commensurate with stronger peptide-induced DNA damage. Molecular dynamics and NMR show that more DNA-peptide interactions exist with p3 than p1, and the peptides adopt conformations simultaneously poised for metal- and DNA-binding. These results generate several important conclusions. First, homologous HDPs cannot be assumed to have identical mechanisms since p1 and p3 eradicate bacteria through distinct relative contributions of membrane and DNA-disruptive effects. Second, the nuclease and membrane activities of p1 and p3 show that naturally occurring HDPs can inflict not only physicochemical but also covalent damage. Third, strong nuclease activity is essential for biofilm and persister cell eradication, as shown by p3, the homolog more specific toward bacteria and more expressed in vascularized tissues. Fourth, p3 combines several physicochemical properties (e.g., Amino Terminal Copper and Nickel binding motif; numerous arginines; moderate hydrophobicity) that confer low membranolytic effects, robust copper-scavenging capability, strong interactions with DNA, and fast nuclease activity. This new knowledge could help design novel therapeutics active against hard-to-treat persister cells and biofilms.

Piscidins were the first antimicrobial peptides discovered in the mast cells of vertebrates. While two family members, piscidin 1 (p1) and piscidin 3 (p3), have highly similar sequences and α-helical structures when bound to model membranes, p1 generally exhibits stronger antimicrobial and hemolytic activity than p3 for reasons that remain elusive. In this study, we combine activity assays and biophysical methods to investigate the mechanisms underlying the cellular function and differing biological potencies of these peptides, and report findings spanning three major facets. First, added to Gram-positive (Bacillus megaterium) and Gram-negative (Escherichia coli) bacteria at sublethal concentrations and imaged by confocal microscopy, both p1 and p3 translocate across cell membranes and colocalize with nucleoids. In E. coli, translocation is accompanied by nonlethal permeabilization that features more pronounced leakage for p1. Second, p1 is also more disruptive than p3 to bacterial model membranes, as quantified by a dye-leakage assay and (2)H solid-state NMR-monitored lipid acyl chain order parameters. Oriented CD studies in the same bilayers show that, beyond a critical peptide concentration, both peptides transition from a surface-bound state to a tilted orientation. Third, gel retardation experiments and CD-monitored titrations on isolated DNA demonstrate that both peptides bind DNA but p3 has stronger condensing effects. Notably, solid-state NMR reveals that the peptides are α-helical when bound to DNA. Overall, these studies identify two polyreactive piscidin isoforms that bind phosphate-containing targets in a poised amphipathic α-helical conformation, disrupt bacterial membranes, and access the intracellular constituents of target cells. Remarkably, the two isoforms have complementary effects; p1 is more membrane active, while p3 has stronger DNA-condensing effects. Subtle differences in their physicochemical properties are highlighted to help explain their contrasting activities.

Cell delivery or cell killing processes often involve the crossing or disruption of cellular membranes. We review how, by modifying the composition and properties of membranes, membrane oxidation can be exploited to enhance the delivery of macromolecular cargos into live human cells. We also describe how membrane oxidation can be utilized to achieve efficient killing of bacteria by antimicrobial peptides. Finally, we present recent evidence highlighting how membrane oxidation is intimately engaged in natural biological processes such as antigen delivery in dendritic cells and in the killing of bacteria by human macrophages. Overall, the insights that have been recently gained in this area should facilitate the development of more effective delivery technologies and antimicrobial therapeutic approaches.

Tuberculosis now ranks as the leading cause of death in the world due to a single infectious agent. Current standard of care treatment can achieve very high cure rates for drug-sensitive disease but requires a 6-month duration of chemotherapy. Drug-resistant disease requires significantly longer treatment durations with drugs associated with a higher risk of adverse events. Thus, there is a pressing need for a drug regimen that is safer, shorter in duration and superior to current front-line chemotherapy in terms of efficacy. The TB drug pipeline contains several candidates that address one or more of the required attributes of chemotherapeutic regimens that may redefine the standard of care of this disease. Several new drugs have been reported and novel targets have been identified allowing regimens containing new compounds to trickle into clinical studies. Furthermore, a recent paradigm-shift in understanding the pharmacokinetics of anti-tubercular drugs is revolutionizing the way we select compounds for clinical progression.

The emergence of new pathogens and multidrug resistant bacteria is an important public health issue that requires the development of novel classes of antibiotics. Antimicrobial peptides (AMPs) are a promising platform with great potential for the identification of new lead compounds that can combat the aforementioned pathogens due to their broad-spectrum antimicrobial activity and relatively low rate of resistance emergence. AMPs of multicellular organisms made their debut four decades ago thanks to ingenious researchers who asked simple questions about the resistance to bacterial infections of insects. Questions such as "Do fruit flies ever get sick?", combined with pioneering studies, have led to an understanding of AMPs as universal weapons of the immune system. This review focuses on a subclass of AMPs that feature a metal binding motif known as the amino terminal copper and nickel (ATCUN) motif. One of the metal-based strategies of hosts facing a pathogen, it includes wielding the inherent toxicity of copper and deliberately trafficking this metal ion into sites of infection. The sudden increase in the concentration of copper ions in the presence of ATCUN-containing AMPs (ATCUN-AMPs) likely results in a synergistic interaction. Herein, we examine common structural features in ATCUN-AMPs that exist across species, and we highlight unique features that deserve additional attention. We also present the current state of knowledge about the molecular mechanisms behind their antimicrobial activity and the methods available to study this promising class of AMPs.

Translational frameshift errors are often deleterious to the synthesis of functional proteins and could therefore be promoted therapeutically to kill bacteria. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and represents an attractive potential target for the development of new antibiotics. Here, we describe the application of a structure-guided fragment-based drug discovery approach to the design of a new class of inhibitors against TrmD in Mycobacterium abscessus. Fragment library screening, followed by structure-guided chemical elaboration of hits, led to the rapid development of drug-like molecules with potent in vitro TrmD inhibitory activity. Several of these compounds exhibit activity against planktonic M. abscessus and M. tuberculosis as well as against intracellular M. abscessus and M. leprae, indicating their potential as the basis for a novel class of broad-spectrum mycobacterial drugs.

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The biophysical properties of cellular membranes intimately influence the delivery of cargoes into cells by cell-penetrating peptides and the bactericidal activity of antimicrobial peptides. We discuss how lipid oxidation creates important chemical and biophysical changes in membranes and hypothesize about the observed synergy between oxidized membranes and membrane-active peptides.