The coronavirus responsible for the severe acute respiratory syndrome contains a small envelope protein, E, with putative involvement in host apoptosis and virus morphogenesis. To perform these functions, it has been suggested that protein E can form a membrane destabilizing transmembrane (TM) hairpin, or homooligomerize to form a TM pore. Indeed, in a recent study we reported that the alpha-helical putative transmembrane domain of E protein (ETM) forms several SDS-resistant TM interactions: a dimer, a trimer, and two pentameric forms. Further, these interactions were found to be evolutionarily conserved. Herein, we have studied multiple isotopically labeled ETM peptides reconstituted in model lipid bilayers, using the orientational parameters derived from infrared dichroic data. We show that the topology of ETM is consistent with a regular TM alpha-helix. Further, the orientational parameters obtained unequivocally correspond to a homopentameric model, by comparison with previous predictions. We have independently confirmed that the full polypeptide of E protein can also aggregate as pentamers after expression in Escherichia coli. This interaction must be stabilized, at least partially, at the TM domain. The model we report for this pentameric alpha-helical bundle may explain some of the permabilizing properties of protein E, and should be the basis of mutagenesis efforts in future functional studies.

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With the rapid rise of antibiotic-resistant-device-associated infections, there has been increasing demand for an antimicrobial biomedical surface. Synthetic antimicrobial peptides that have excellent bactericidal potency and negligible cytotoxicity are promising targets for immobilization on these target surfaces. An engineered arginine-tryptophan-rich peptide (CWR11) was developed, which displayed potent antimicrobial activity against a broad spectrum of microbes via membrane disruption, and possessed excellent salt resistance properties. A tethering platform was subsequently developed to tether CWR11 onto a model polymethylsiloxane (PDMS) surface using a simple and robust strategy. Surface characterization assays such as attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), and energy-dispersive X-ray spectroscopy (EDX) confirmed the successful grafting of CWR11 onto the chemically treated PDMS surface. The immobilized peptide concentration was 0.8 ± 0.2 μg/cm(2) as quantitated by sulfosuccinimidyl-4-o-(4,4-dimethoxytrityl) butyrate (sulfo-SDTB) assay. Antimicrobial assay and cytotoxic investigation confirmed that the peptide-immobilized surface has good bactericidal and antibiofilm properties, and is also noncytotoxic to mammalian cells. Tryptophan-arginine-rich antimicrobial peptides have the potential for antimicrobial protection of biomedical surfaces and may have important clinical applications in patients.

Thrombin-derived C-terminal peptides (TCPs) of about 2 kDa are present in wounds, where they exert anti-endotoxic functions. Employing a combination of nuclear magnetic resonance spectroscopy (NMR), biophysical, mass spectrometry and cellular studies combined with in silico multiscale modelling, we here determine the bound conformation of HVF18 (HVFRLKKWIQKVIDQFGE), a TCP generated by neutrophil elastase, in complex with bacterial lipopolysaccharide (LPS) and define a previously undisclosed interaction between TCPs and human CD14. Further, we show that TCPs bind to the LPS-binding hydrophobic pocket of CD14 and identify the peptide region crucial for TCP interaction with LPS and CD14. Taken together, our results demonstrate the role of structural transitions in LPS complex formation and CD14 interaction, providing a molecular explanation for the previously observed therapeutic effects of TCPs in experimental models of bacterial sepsis and endotoxin shock.

Antimicrobial peptides (AMPs) kill microbes by non-specific membrane permeabilization, making them ideal templates for designing novel peptide-based antibiotics that can combat multi-drug resistant pathogens. For maximum efficacy in vivo and in vitro, AMPs must be biocompatible, salt-tolerant and possess broad-spectrum antimicrobial activity. These attributes can be obtained by rational design of peptides guided by good understanding of peptide structure-function. Toward this end, this study investigates the influence of charge and hydrophobicity on the activity of tryptophan and arginine rich decamer peptides engineered from a salt resistant human β-defensin-28 variant. Mechanistic investigations of the decamers with detergents mimicking the composition of bacterial and mammalian membrane, reveal a correlation between improved antibacterial activity and the increase in tryptophan and positive residue content, while keeping hemolysis low. The potent antimicrobial activity and high cell membrane selective behavior of the two most active decamers, D5 and D6, are attributed to an optimum peptide charge to hydrophobic ratio bestowed by systematic arginine and tryptophan substitution. D5 and D6 show surface localization behavior with binding constants of 1.86 × 10(8) and 2.6 × 10(8)  M(-1) , respectively, as determined by isothermal calorimetry measurements. NMR derived structures of D5 and D6 in SDS detergent micelles revealed proximity of Trp and Arg residues in an extended structural scaffold. Such potential cation-π interactions may be critical in cell permeabilization of the AMPs. The fundamental characterization of the engineered decamers provided in this study improves the understanding of structure-activity relationship of short arginine tryptophan rich AMPs, which will pave the way for future de novo design of potent AMPs for therapeutic and biomedical applications.

Temporins are a group of closely related short antimicrobial peptides from frog skin. Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, plays important roles in the activity of temporins. Earlier studies have found that LPS induces oligomerization of temporin-1Tb (TB) thus preventing its translocation across the outer membrane and, as a result, reduces its activity on Gram-negative bacteria. On the other hand, temporin-1Tl (TL) exhibits higher activity, presumably because of lack of such oligomerization. A synergistic mechanism was proposed, involving TL and TB in overcoming the LPS-mediated barrier. Here, to gain insights into interactions of TL and TB within LPS, we investigated the structures and interactions of TL, TB, and TL؉TB in LPS micelles, using NMR and fluorescence spectroscopy. In the context of LPS, TL assumes a novel antiparallel dimeric helical structure sustained by intimate packing between aromatic-aromatic and aromatic-aliphatic residues. By contrast, independent TB has populations of helical and aggregated conformations in LPS. The LPS-induced aggregated states of TB are largely destabilized in the presence of TL. Saturation transfer difference NMR studies have delineated residues of TL and TB in close contact with LPS and enhanced interactions of these two peptides with LPS, when combined together. Fluorescence resonance energy transfer and 31 P NMR have pointed out the proximity of TL and TB in LPS and conformational changes of LPS, respectively. Importantly, these results provide the first structural insights into the mode of action and synergism of antimicrobial peptides at the level of the LPS-outer membrane.

This review article presents important and recent progress in the manufacture and application of antimicrobial macromolecules. Microbial infections continue to endanger human health and pose a great economic burden to society. To resolve this crisis, huge efforts to improve or develop macromolecules that can inhibit pathogens without incurring pathogen resistance are required and actively ongoing. Synthetic antimicrobial macromolecules which include antimicrobial peptides (AMPs), polymers and peptide-polymer hybrids represent a huge class of molecules which can incur effective antimicrobial therapy due to their unique biochemical properties. The use of these antimicrobial macromolecules which target the cytoplasmic membrane of microbes, is a promising approach to lower the propensity of pathogen resistance development. Therefore, huge efforts to synthesize these molecules at scales and purities that enable their structure-function and clinical studies are actively underway. Due to the high cost involved in extracting AMPs from natural sources, biological processes are being developed to economically manufacture AMPs at large scale. Synthetic AMP analogs are also being engineered to further improve antimicrobial potency and lower synthesis cost. Synthetic polymers have also been found to exhibit excellent antimicrobial properties which are comparable to those of natural AMPs. Various antimicrobial polymers have been synthesized based on the amphiphilicity of natural AMPs. Although the facile synthesis of polymers poses no cost problems, numerous synthetic antimicrobial polymers are disadvantaged by high toxicity to mammalian cells due to their non-selectivity. To combine the advantages of AMPs and antimicrobial polymers, peptide-polymer hybrid macromolecules are actively being developed, with a few effective and strongly microbicidal models recently demonstrated. With the advancement of biochemical engineering tools and chemical synthesis methods, these antimicrobial macromolecules can be specifically designed to be highly selective, broad spectrum and biocompatible. In this review, we summarize the recent advances and challenges in the manufacture of these antimicrobial macromolecules. Based on their antimicrobial mechanisms, their applications in addressing challenges associated with infectious disease and antibiotic-resistance are also discussed.

Bacterial colonization of urinary catheters is a common problem leading to Catheter Associated Urinary Tract Infections (CAUTIs) in patients, which result in high treatment costs and associated complications. Due to the advantages of antimicrobial peptides (AMPs) compared to most other antimicrobial molecules, an increasing number of AMP-coated surfaces is being developed but their efficacy is hindered by suboptimal coating methods and loss of peptide activity upon surface tethering. This study aims to address this issue by employing a methodic approach that combines a simple selective chemical immobilization platform developed on a silicone catheter with the choice of a potent AMP, Lasioglossin-III (Lasio-III), to allow site specific immobilization of Lasio-III at an effective surface concentration. The Lasio-III peptide was chemically modified at the N-terminal with a cysteine residue to facilitate cysteinedirected immobilization of the peptide onto a commercial silicone catheter surface via a combination of an allyl glycidyl ether (AGE) brush and polyethylene glycol (PEG) based chemical coupling. The amount of immobilized peptide was determined to be 6.59 AE 0.89 mg cm À2 by Sulfo-SDTB assay. The AMPcoated catheter showed good antimicrobial activity against both Gram positive and negative bacteria. The antimicrobial properties of the AMP-coated catheter were sustained for at least 4 days postincubation in a physiologically relevant environment and artificial urine and prevented the biofilm growth of E. coli and E. faecalis. Adenosine tri-phosphate leakage and propidium iodide fluorescence studies further confirmed the membranolytic mode of action of the immobilized peptide. To the best of our knowledge, this is the first proof-of-concept study that reports the efficacy of AMP immobilization by sulfhydryl coupling on a real catheter surface.