Rathi Saravanan

Antimicrobial functionalization of silicone surfaces with engineered short peptides having broad spectrum antimicrobial and salt-resistant properties.

Catheter-associated urinary tract infections (CAUTIs) are often preceded by pathogen colonization on catheter surfaces and are a major health threat facing hospitals worldwide. Antimicrobial peptides (AMPs) are a class of new antibiotics that hold promise in curbing CAUTIs caused by antibiotic-resistant pathogens. This study aims to systematically evaluate the feasibility of immobilizing two newly engineered arginine/lysine/tryptophan-rich AMPs with broad antimicrobial spectra and salt-tolerant properties on silicone surfaces to address CAUTIs. The peptides were successfully immobilized on polydimethylsiloxane and urinary catheter surfaces via an allyl glycidyl ether (AGE) polymer brush interlayer, as confirmed by X-ray photoelectron spectroscopy and water contact angle analyses. The peptide-coated silicone surfaces exhibited excellent microbial killing activity towards bacteria and fungi in urine and in phosphate-buffered saline. Although both the soluble and immobilized peptides demonstrated membrane disruption capabilities, the latter showed a slower rate of kill, presumably due to reduced diffusivity and flexibility resulting from conjugation to the polymer brush. The synergistic effects of the AGE polymer brush and AMPs prevented biofilm formation by repelling cell adhesion. The peptide-coated surface showed no toxicity towards smooth muscle cells. The findings of this study clearly indicate the potential for the development of AMP-based coating platforms to prevent CAUTIs.

NMR Structures and Interactions of Temporin-1Tl and Temporin-1Tb with Lipopolysaccharide Micelles MECHANISTIC INSIGHTS INTO OUTER MEMBRANE PERMEABILIZATION AND SYNERGISTIC ACTIVITY

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 31P 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.

Model of a Putative Pore: The Pentameric α-Helical Bundle of SARS Coronavirus E Protein in Lipid Bilayers

Abstract 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 α-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 α-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 α-helical bundle may explain some of the permabilizing properties of protein E, and should be the basis of mutagenesis efforts in future functional studies.

Immobilization Studies of an Engineered Arginine–Tryptophan-Rich Peptide on a Silicone Surface with Antimicrobial and Antibiofilm Activity

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.

Antimicrobial macromolecules: synthesis methods and future applications

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.

Design of short membrane selective antimicrobial peptides containing tryptophan and arginine residues for improved activity, salt‐resistance, and biocompatibility

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 × 108 and 2.6 × 108 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. Biotechnol. Bioeng. 2014;111: 37–49.

Site specific immobilization of a potent antimicrobial peptide onto silicone catheters: evaluation against urinary tract infection pathogens

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 cysteine-directed 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 ± 0.89 μg cm-2 by Sulfo-SDTB assay. The AMP-coated 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 post-incubation 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.

Oligomeric structure of a cathelicidin antimicrobial peptide in dodecylphosphocholine micelle determined by NMR spectroscopy.

The broad spectrum of antibacterial activities of host defense cationic antimicrobial peptides (AMPs) arises from their ability to perturb membrane integrity of the microbes. The mechanisms are often thought to require assembly of AMPs on the membrane surface to form pores. However, three dimensional structures in the oligomeric form of AMPs in the context of lipid membranes are largely limited. Here, we demonstrate that a 22-residue antimicrobial peptide, termed VK22, derived from fowlicidin-1, a cathelicidin family of AMP from chicken oligomerizes into a predominantly tetrameric state in zwitterionic dodecylphosphocholine (DPC) micelles. An ensemble of NMR structures of VK22 determined in 200mM perdeuterated DPC, from 755 NOE constrains including 19 inter-helical NOEs, had revealed an assembly of four helices arranged in anti-parallel fashion. Hydrogen bonds, C(α)H-O=C types, and van der Waals interactions among the helical sub-units appear to be involved in the stabilization of the quaternary structures. The central region of the barrel shaped tetrameric bundle is non-polar with clusters of aromatic residues, whereas all the cationic residues are positioned at the termini. Paramagnetic spin labeled NMR experiments indicated that the tetrameric structure is embedded into micelles such that the non-polar region located inside the lipid acyl chains. Structure and micelle localization of a monomeric version, obtained from substitution of two Tyr residues with Ala, of the peptide is also compared. The mutated peptide VK22AA has been found be localized at the surface of the micelles. The tetrameric structure of VK22 delineates a small water pore that can be larger in the higher order oligomers. As these results provide structural insights, at atomic resolution, into the oligomeric states of a helical AMP in lipid environment, the structural details may be further utilized for the design of novel self-assembled membrane protein mimics.

Micelle-bound structures and dynamics of the hinge deleted analog of melittin and its diastereomer: Implications in cell selective lysis by D-amino acid containing antimicrobial peptides

Melittin, the major component of the honey bee venom, is a 26-residue hemolytic and membrane active peptide. Structures of melittin determined either in lipid environments by NMR or by use of X-ray demonstrated two helical regions at the N- and C-termini connected by a hinge or a bend at the middle. Here, we show that deletion of the hinge residues along with two C-terminal terminal Gln residues (Q25 and Q26), yielding a peptide analog of 19-residue or Mel-H, did not affect antibacterial activity but resulted in a somewhat reduced hemolytic activity. A diastereomer of Mel-H or Mel-(d)H containing d-amino acids [(d)V5, (d)V8, (d)L11 and (d)K16] showed further reduction in hemolytic activity without lowering antibacterial activity. We have carried out NMR structures, dynamics (H-D exchange and proton relaxation), membrane localization by spin labeled lipids, pulse-field-gradient (PFG) NMR and isothermal titration calorimetry (ITC) in dodecylphosphocholine (DPC) micelles, as a mimic to eukaryotic membrane, to gain insights into cell selectivity of these melittin analogs. PFG-NMR showed Mel-H and Mel-(d)H both were similarly partitioned into DPC micelles. ITC demonstrated that Mel-H and Mel-(d)H interact with DPC with similar affinity. The micelle-bound structure of Mel-H delineated a straight helical conformation, whereas Mel-(d)H showed multiple beta-turns at the N-terminus and a short helix at the C-terminus. The backbone amide-proton exchange with solvent D(2)O demonstrated a large difference in dynamics between Mel-H and Mel-(d)H, whereby almost all backbone protons of Mel-(d)H showed a much faster rate of exchange as compared to Mel-H. Proton T(1) relaxation had suggested a mobile backbone of Mel-(d)H peptide in DPC micelles. Resonance perturbation by paramagnetic lipids indicated that Mel-H inserted deeper into DPC micelles, whereas Mel-(d)H is largely located at the surface of the micelle. Taken together, results presented in this study demonstrated that the poor hemolytic activity of the d-amino acid containing analogs of antimicrobial peptides may be correlated with their flexible dynamics at the membrane surface.

NMR Structure of Temporin-1 Ta in Lipopolysaccharide Micelles: Mechanistic Insight into Inactivation by Outer Membrane

Background Antimicrobial peptides (AMPs) play important roles in the innate defense mechanism. The broad spectrum of activity of AMPs requires an efficient permeabilization of the bacterial outer and inner membranes. The outer leaflet of the outer membrane of Gram negative bacteria is made of a specialized lipid called lipopolysaccharide (LPS). The LPS layer is an efficient permeability barrier against anti-bacterial agents including AMPs. As a mode of protection, LPS can induce self associations of AMPs rendering them inactive. Temporins are a group of short-sized AMPs isolated from frog skin, and many of them are inactive against Gram negative bacteria as a result of their self-association in the LPS-outer membrane. Principal Findings Using NMR spectroscopy, we have determined atomic resolution structure and characterized localization of temporin-1Ta or TA (FLPLIGRVLSGIL-amide) in LPS micelles. In LPS micelles, TA adopts helical conformation for residues L4-I12, while residues F1-L3 are found to be in extended conformations. The aromatic sidechain of residue F1 is involved in extensive packing interactions with the sidechains of residues P3, L4 and I5. Interestingly, a number of long-range NOE contacts have been detected between the N-terminal residues F1, P3 with the C-terminal residues S10, I12, L13 of TA in LPS micelles. Saturation transfer difference (STD) NMR studies demonstrate close proximity of residues including F1, L2, P3, R7, S10 and L13 with the LPS micelles. Notably, the LPS bound structure of TA shows differences with the structures of TA determined in DPC and SDS detergent micelles. Significance We propose that TA, in LPS lipids, forms helical oligomeric structures employing N- and C-termini residues. Such oligomeric structures may not be translocated across the outer membrane; resulting in the inactivation of the AMP. Importantly, the results of our studies will be useful for the development of antimicrobial agents with a broader spectrum of activity.