Tryptophan- and arginine-rich antimicrobial peptides: Anti-infectives with great potential.

With the increasing prevalence of multidrug resistant (MDR) bacteria, there is a need to design synthetic antimicrobial peptides (AMPs) that are effective and selective for bacteria, i.e. non-toxic to mammalian cells. One design strategy, namely the use of tryptophan- and arginine-rich AMPs, is rooted in the study of natural AMPs that are composed mainly of these amino acids, such as lactoferricin, tritrpticin, and puroindoline. A number of important studies on these AMPs by the Vogel group are reviewed here. More recent work on W-/R-rich peptides is also presented. The examples show that these peptides represent anti-infectives with great potential.

Smart thrombosis inhibitors without bleeding side effects via charge tunable ligand design.

Current treatments to prevent thrombosis, namely anticoagulants and platelets antagonists, remain complicated by the persistent risk of bleeding. Improved therapeutic strategies that diminish this risk would have a huge clinical impact. Antithrombotic agents that neutralize and inhibit polyphosphate (polyP) can be a powerful approach towards such a goal. Here, we report a design concept towards polyP inhibition, termed macromolecular polyanion inhibitors (MPI), with high binding affinity and specificity. Lead antithrombotic candidates are identified through a library screening of molecules which possess low charge density at physiological pH but which increase their charge upon binding to polyP, providing a smart way to enhance their activity and selectivity. The lead MPI candidates demonstrates antithrombotic activity in mouse models of thrombosis, does not give rise to bleeding, and is well tolerated in mice even at very high doses. The developed inhibitor is anticipated to open avenues in thrombosis prevention without bleeding risk, a challenge not addressed by current therapies.

Reassessing the Host Defense Peptide Landscape.

Current research has demonstrated that small cationic amphipathic peptides have strong potential not only as antimicrobials, but also as antibiofilm agents, immune modulators, and anti-inflammatories. Although traditionally termed antimicrobial peptides (AMPs) these additional roles have prompted a shift in terminology to use the broader term host defense peptides (HDPs) to capture the multi-functional nature of these molecules. In this review, we critically examined the role of AMPs and HDPs in infectious diseases and inflammation. It is generally accepted that HDPs are multi-faceted mediators of a wide range of biological processes, with individual activities dependent on their polypeptide sequence. In this context, we explore the concept of chemical space as it applies to HDPs and hypothesize that the various functions and activities of this class of molecule exist on independent but overlapping activity landscapes. Finally, we outline several emerging functions and roles of HDPs and highlight how an improved understanding of these processes can potentially be leveraged to more fully realize the therapeutic promise of HDPs.

Mechanisms of Action for Antimicrobial Peptides With Antibacterial and Antibiofilm Functions.

The antibiotic crisis has led to a pressing need for alternatives such as antimicrobial peptides (AMPs). Recent work has shown that these molecules have great potential not only as antimicrobials, but also as antibiofilm agents, immune modulators, anti-cancer agents and anti-inflammatories. A better understanding of the mechanism of action (MOA) of AMPs is an important part of the discovery of more potent and less toxic AMPs. Many models and techniques have been utilized to describe the MOA. This review will examine how biological assays and biophysical methods can be utilized in the context of the specific antibacterial and antibiofilm functions of AMPs.

Aurein-Derived Antimicrobial Peptides Formulated with Pegylated Phospholipid Micelles to Target Methicillin-Resistant Staphylococcus aureus Skin Infections.

Antimicrobial peptides have been the focus of considerable research; however, issues associated with toxicity and aggregation have the potential to limit clinical applications. Here, a derivative of a truncated version of aurein 2.2 (aurein 2.2Δ3), namely peptide 73, was investigated, along with its d-amino acid counterpart (D-73) and a retro-inverso version (RI-73). A version that incorporated a cysteine residue to the C-terminus (73c) was also generated, as this form is required to covalently attach antimicrobial peptides to polymers (e.g., polyethylene glycol (PEG) or hyperbranched polyglycerol (HPG)). The antimicrobial activity of the 73-derived peptides was enhanced 2- to 8-fold, and all the derivatives eradicated preformed Staphylococcus aureus biofilms. Formulation of the peptides with compatible polyethylene glycol (PEG)-modified phospholipid micelles alleviated toxicity toward human cells and reduced aggregation. When evaluated in vivo, the unformulated d-enantiomers aggregated when injected under the skin of mice, but micelle encapsulated peptides were well absorbed. Pegylated micelle formulated peptides were investigated for their potential as therapeutic agents for treating high-density infections in a murine cutaneous abscess model. Formulated peptide 73 reduced abscess size by 36% and bacterial loads by 2.2-fold compared to the parent peptide aurein 2.2Δ3. Micelle encapsulated peptides 73c and D-73 exhibited superior activity, further reducing abscess sizes by 85% and 63% and lowering bacterial loads by 510- and 9-fold compared to peptide 73.

Probing the interaction between U24 and the SH3 domain of Fyn tyrosine kinase.

The putative membrane protein U24 from HHV-6A shares a seven-residue sequence identity (which includes a PxxP motif) with myelin basic protein (MBP), a protein responsible for the compaction of the myelin sheath in the central nervous system. U24 from HHV-6A also shares a PPxY motif with U24 from the related virus HHV-7, allowing them both to block early endosomal recycling. Recently, MBP has been shown to have protein-protein interactions with a range of proteins, including proteins containing SH3 domains. Given that this interaction is mediated by the proline-rich segment in MBP, and that similar proline-rich segments are found in U24, we investigate here whether U24 also interacts with SH3 domain-containing proteins and what the nature of that interaction might be. The implications of a U24-Fyn tyrosine kinase SH3 domain interaction are discussed in terms of the hypothesis that U24 may function like MBP through molecular mimicry, potentially contributing to the disease state of multiple sclerosis or other demyelinating disorders.

Broad-spectrum anti-biofilm peptide that targets a cellular stress response.

Bacteria form multicellular communities known as biofilms that cause two thirds of all infections and demonstrate a 10 to 1000 fold increase in adaptive resistance to conventional antibiotics. Currently, there are no approved drugs that specifically target bacterial biofilms. Here we identified a potent anti-biofilm peptide 1018 that worked by blocking (p)ppGpp, an important signal in biofilm development. At concentrations that did not affect planktonic growth, peptide treatment completely prevented biofilm formation and led to the eradication of mature biofilms in representative strains of both Gram-negative and Gram-positive bacterial pathogens including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus, Salmonella Typhimurium and Burkholderia cenocepacia. Low levels of the peptide led to biofilm dispersal, while higher doses triggered biofilm cell death. We hypothesized that the peptide acted to inhibit a common stress response in target species, and that the stringent response, mediating (p)ppGpp synthesis through the enzymes RelA and SpoT, was targeted. Consistent with this, increasing (p)ppGpp synthesis by addition of serine hydroxamate or over-expression of relA led to reduced susceptibility to the peptide. Furthermore, relA and spoT mutations blocking production of (p)ppGpp replicated the effects of the peptide, leading to a reduction of biofilm formation in the four tested target species. Also, eliminating (p)ppGpp expression after two days of biofilm growth by removal of arabinose from a strain expressing relA behind an arabinose-inducible promoter, reciprocated the effect of peptide added at the same time, leading to loss of biofilm. NMR and chromatography studies showed that the peptide acted on cells to cause degradation of (p)ppGpp within 30 minutes, and in vitro directly interacted with ppGpp. We thus propose that 1018 targets (p)ppGpp and marks it for degradation in cells. Targeting (p)ppGpp represents a new approach against biofilm-related drug resistance.

Biomembrane interactions reveal the mechanism of action of surface-immobilized host defense IDR-1010 peptide.

Dissecting the mechanism of action of surface-tethered antimicrobial and immunomodulatory peptides is critical to the design of optimized anti-infection coatings on biomedical devices. To address this, we compared the biomembrane interactions of host defense peptide IDR-1010cys (1) in free form, (2) as a soluble polymer conjugate, and (3) with one end tethered to a solid support with model bacterial and mammalian lipid membranes. Our results show that IDR-1010cys in all three distinct forms interacted with bacterial and mammalian lipid vesicles, but the extent of the interactions as monitored by the induction of secondary structure varied. The enhanced interaction of surface-tethered peptides is well correlated with their very good antimicrobial activities. Our results demonstrate that there may be a difference in the mechanism of action of surface-tethered versus free IDR-1010cys.

Overexpression and purification of U24 from human herpesvirus type-6 in E. coli: unconventional use of oxidizing environments with a maltose binding protein-hexahistine dual tag to enhance membrane protein yield.

BACKGROUND: Obtaining membrane proteins in sufficient quantity for biophysical study and biotechnological applications has been a difficult task. Use of the maltose binding protein/hexahistidine dual tag system with E.coli as an expression host is emerging as a high throughput method to enhance membrane protein yield, solubility, and purity, but fails to be effective for certain proteins. Optimizing the variables in this system to fine-tune for efficiency can ultimately be a daunting task. To identify factors critical to success in this expression system, we have selected to study U24, a novel membrane protein from Human Herpesvirus type-6 with potent immunosuppressive ability and a possible role in the pathogenesis of the disease multiple sclerosis. RESULTS: We expressed full-length U24 as a C-terminal fusion to a maltose binding protein/hexahistidine tag and examined the effects of temperature, growth medium type, cell strain type, oxidizing vs. reducing conditions and periplasmic vs. cytoplasmic expression location. Temperature appeared to have the greatest effect on yield; at 37°C full-length protein was either poorly expressed (periplasm) or degraded (cytoplasm) whereas at 18°C, expression was improved especially in the periplasm of C41(DE3) cells and in the cytoplasm of oxidizing Δtrx/Δgor mutant strains, Origami 2 and SHuffle. Expression of the fusion protein in these strains were estimated to be 3.2, 5.3 and 4.3 times greater, respectively, compared to commonly-used BL21(DE3) cells. We found that U24 is isolated with an intramolecular disulfide bond under these conditions, and we probed whether this disulfide bond was critical to high yield expression of full-length protein. Expression analysis of a C21SC37S cysteine-free mutant U24 demonstrated that this disulfide was not critical for full-length protein expression, but it is more likely that strained metabolic conditions favour factors which promote protein expression. This hypothesis is supported by the fact that use of minimal media could enhance protein production compared to nutrient-rich LB media. CONCLUSIONS: We have found optimal conditions for heterologous expression of U24 from Human Herpesvirus type-6 in E.coli and have demonstrated that milligram quantities of pure protein can be obtained. Strained metabolic conditions such as low temperature, minimal media and an oxidizing environment appeared essential for high-level, full-length protein production and this information may be useful for expressing other membrane proteins of interest.

The biocompatibility and biofilm resistance of implant coatings based on hydrophilic polymer brushes conjugated with antimicrobial peptides.

Bacterial colonization on implant surfaces and subsequent infections are one of the most common reasons for the failure of many indwelling devices. Several approaches including antimicrobial and antibiotic-eluting coatings on implants have been attempted; however, none of these approaches succeed in vivo. Here we report a polymer brush based implant coating that is non-toxic, antimicrobial and biofilm resistant. These coating consists of covalently grafted hydrophilic polymer chains conjugated with an optimized series of antimicrobial peptides (AMPs). These tethered AMPs maintained excellent broad spectrum antimicrobial activity in vitro and in vivo. We found that this specially structured robust coating was extremely effective in resisting biofilm formation, and that the biofilm resistance depended on the nature of conjugated peptides. The coating had no toxicity to osteoblast-like cells and showed insignificant platelet activation and adhesion, and complement activation in human blood. Since such coatings can be applied to most currently used implant surfaces, our approach has significant potential for the development of infection-resistant implants.

Structural studies of a peptide with immune modulating and direct antimicrobial activity.

The structure and function of the synthetic innate defense regulator peptide 1018 was investigated. This 12 residue synthetic peptide derived by substantial modification of the bovine cathelicidin bactenecin has enhanced innate immune regulatory and moderate direct antibacterial activities. The solution state NMR structure of 1018 in zwitterionic dodecyl phosphocholine (DPC) micelles indicated an α-helical conformation, while secondary structures, based on circular dichroism measurements, in anionic sodium dodecyl sulfate (SDS) and phospholipid vesicles (POPC/PG in a 1:1 molar ratio) and simulations revealed that 1018 can adopt a variety of folds, tailored to its different functions. The structural data are discussed in light of the ability of 1018 to potently induce chemokine responses, suppress the LPS-induced TNF-α response, and directly kill both Gram-positive and Gram-negative bacteria.

Synthetic fusion peptides of tick-borne encephalitis virus as models for membrane fusion.

The fusion peptide of TBEV is a short segment of the envelope protein that mediates viral and host cell membrane fusion at acidic pH. Previous studies on the E protein have shown that mutations at L107 have an effect on fusogenic activity. Structural studies have also suggested that during the fusion process the E protein rearranges to form a trimer. In the present study, a number of short peptides were synthesized, and their structure/activity was examined: (1) monomers consisting of residues 93-113 of the wild-type E protein with Leu at position 107 (WT) and two mutants, namely, L107F and L107T; (2) a monomer consisting of residues 93-113 of the E protein with a C105A mutation (TFPmn); (3) a trimer consisting of three monomers described in (2), linked at the C-terminus via 1 Lys (TFPtr); (4) a monomer consisting of residues 93-113 of the E protein plus six additional Lys at the C-terminus; and (5) a trimer consisting of three monomers described in (3), linked via the side chain of the sixth lysine. The secondary structure content of all peptides was investigated using circular dichroism (CD). Approximately seven of the residues were in beta-strand conformation, in the presence of POPC/POPE/cholesterol. The structures did not depend on pH significantly. The fusogenicity of the peptides was measured by FRET and photon correlation spectroscopy. The data suggest that TFPtr is the most fusogenic at acidic pH and that the mutation from L107 to T reduces activity. Molecular dynamics simulations of WT, L107T, and L107F suggest that this reduction in activity may be related to the fact that the mutations disrupt trimer stability. Finally, tryptophan fluorescence experiments were used to localize the peptides in the membrane. It was found that WT, L107F, TFPmn, and TFPtr could penetrate better into the acyl chain region of the lipids than the other peptides tested. The implications of these results on the fusion mechanism of TBEV E protein will be presented.

Effect of divalent cations on the structure of the antibiotic daptomycin.

Daptomycin, a cyclic anionic lipopeptide antibiotic, whose three-dimensional structure was recently solved using solution state NMR (Ball et al. 2004; Jung et al. 2004; Rotondi and Gierasch 2005), requires calcium for function. To date, the exact nature of the interaction between divalent cations, such as Ca(2+) or Mg(2+), has not been fully characterized. It has, however, been suggested that addition of Ca(2+) to daptomycin in a 1:1 molar ratio induces aggregation. Moreover, it has been suggested that certain residues, e.g. Asp3 and Asp7, which are essential for activity (Grunewald et al. 2004; Kopp et al. 2006), may also be important for Ca(2+) binding (Jung et al. 2004). In this work, we have tried: (1) to further pinpoint how Ca(2+) affects daptomycin structure/oligomerization using analytical ultracentrifugation; and (2) to determine whether a specific calcium binding site exists, based on one-dimensional (13)C NMR spectra and molecular dynamics (MD) simulations. The centrifugation results indicated that daptomycin formed micelles of between 14 and 16 monomers in the presence of a 1:1 molar ratio of Ca(2+) and daptomycin. The (13)C NMR data indicated that addition of calcium had a significant effect on the Trp1 and Kyn13 residues, indicating that either calcium binds in this region or that these residues may be important for oligomerization. Finally, the molecular dynamics simulation results indicated that the conformational change of daptomycin upon calcium binding might not be as significant as originally proposed. Similar studies on the divalent cation Mg(2+) are also presented. The implication of these results for the biological function of daptomycin is discussed.

Characterization of de novo four-helix bundles by molecular dynamics simulations.

We have investigated the structure and dynamics of three cavitand-based four-helix bundles (caviteins) by computer simulation. In these systems, designed de novo, each of the four helices contain the identical basis sequence EELLKKLEELLKKG (N1). Each cavitein consists of a rigid macrocycle (cavitand) with four aryl linkages, to each of which is connected an N1 peptide by means of a linker peptide. The three caviteins studied here differ only in the linker peptide, which consist of one, two, or three glycine residues. Previous experimental work has shown that these systems exhibit very different behavior in terms of stability and oligomerization states despite the small differences in the linker peptide. Given that to date no three-dimensional structure is available for these caviteins, we have undertaken a series of molecular dynamics (MD) simulations in explicit water to try to rationalize the large differences in the experimentally observed behavior of these systems. Our results provide insight, for the first time, into why and how the cavitein with a single glycine linker forms dimers. In addition, our results indicate why although the two- and three-glycine-linked caviteins have similar stabilities, they have different native-like characteristics: the cavitein with three glycines can form a supercoiled helix, whereas the one with two glycines cannot. These findings may provide a useful guide in the rational de novo design of novel proteins with finely tunable structures and functions in the future.

Mode of action of the new antibiotic for Gram-positive pathogens daptomycin: comparison with cationic antimicrobial peptides and lipopeptides.

With the steady rise in the number of antibiotic-resistant Gram-positive pathogens, it has become increasingly important to find new antibacterial agents which are highly active and have novel and diversified mechanisms of action. Two classes will be discussed here: the cationic antimicrobial peptides, which are amphiphilic in nature, targeting membranes and increasing their permeability; and lipopeptides, which consist of linear or cyclic peptides with an N-terminus that is acylated with a fatty acid side chain. One member of the cyclic lipopeptide family, the anionic molecule daptomycin, has been extensively studied and is the major focus of this review. Models will be presented on its mode of action and comparisons will be made to the known modes of action of cationic antimicrobial peptides and other lipopeptides.

Interaction of alamethicin with ether-linked phospholipid bilayers: oriented circular dichroism, 31P solid-state NMR, and differential scanning calorimetry studies.

The arrangement of the antimicrobial peptide alamethicin was studied by oriented circular dichroism, 31P solid-state NMR, and differential scanning calorimetry in ether-linked phospholipid bilayers composed of 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DHPC). The measurements were performed as a function of alamethicin concentration relative to the lipid concentration, and results were compared to those reported in the literature for ester-linked phospholipid bilayers. At ambient temperature, alamethicin incorporates into the hydrophobic core of DHPC bilayers but results in more lipid disorder than observed for ester-linked 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayers. This orientational disorder appears to depend on lipid properties such as bilayer thickness. Moreover, the results suggest that alamethicin inserts into the hydrophobic core of the bilayers (at high peptide concentration) for both ether- and ester-linked lipids but using a different mechanism, namely toroidal for DHPC and barrel-stave for POPC.

Plasticity of influenza haemagglutinin fusion peptides and their interaction with lipid bilayers.

A detailed molecular dynamics study of the haemagglutinin fusion peptide (N-terminal 20 residues of the HA2 subunits) in a model bilayer has yielded useful information about the molecular interactions leading to insertion into the lipids. Simulations were performed on the native sequence, as well as a number of mutant sequences, which are either fusogenic or nonfusogenic. For the native sequence and fusogenic mutants, the N-terminal 11 residues of the fusion peptides are helical and insert with a tilt angle of approximately 30 degrees with respect to the membrane normal, in very good agreement with experimental data. The tilted insertion of the native sequence peptide leads to membrane bilayer thinning and the calculated order parameters show larger disorder of the alkyl chains. These results indicate that the lipid packing is perturbed by the fusion peptide and could be used to explain membrane fusion. For the nonfusogenic sequences investigated, it was found that most of them equilibrate parallel to the interface plane and do not adopt a tilted conformation. The presence of a charged residue at the beginning of the sequence (G1E mutant) resulted in a more difficult case, and the outcomes do not fall straightforwardly into the general picture. Sequence searches have revealed similarities of the fusion peptide of influenza haemagglutinin with peptide sequences such as segments of porin, amyloid alpha eta peptide, and a peptide from the prion sequence. These results confirm that the sequence can adopt different folds in different environments. The plasticity and the conformational dependence on the local environment could be used to better understand the function of fusion peptides.

Recent developments in solid-state magic-angle spinning, nuclear magnetic resonance of fully and significantly isotopically labelled peptides and proteins.

In recent years, a large number of solid-state nuclear magnetic resonance (NMR) techniques have been developed and applied to the study of fully or significantly isotopically labelled ((13)C, (15)N or (13)C/(15)N) biomolecules. In the past few years, the first structures of (13)C/(15)N-labelled peptides, Gly-Ile and Met-Leu-Phe, and a protein, Src-homology 3 domain, were solved using magic-angle spinning NMR, without recourse to any structural information obtained from other methods. This progress has been made possible by the development of NMR experiments to assign solid-state spectra and experiments to extract distance and orientational information. Another key aspect to the success of solid-state NMR is the advances made in sample preparation. These improvements will be reviewed in this contribution. Future prospects for the application of solid-state NMR to interesting biological questions will also briefly be discussed.

Membrane protein structure determination using solid-state NMR.

Solid-state NMR is emerging as a method for resolving structural information for large biomolecular complexes, such as membrane-embedded proteins. In principle, there is no molecular weight limit to the use of the approach, although the complexity and volume of data is still outside complete assignment and structural determinations for any large (Mr > approx 30,000) complex unless specific methods to reduce the information content to a manageable amount are employed. Such methods include specific residue-type labeling, labeling of putative segments of a protein, or examination of complexes made up of smaller, manageable units, such as oligomeric ion channels. Labeling possibilities are usually limited to recombinant or synthesized proteins, and labeling strategies often follow models from a bioinformatics approach. In all cases, and in common with most membrane studies, sample preparation is vital, and this activity alone can take considerable effort before NMR can be applied--peptide or protein production (synthesis or expression) followed by reconstitution into bilayers and resolution of suitable sample geometry is still technically challenging. As experience is gained in the field, this development time should decrease. Here, the practical aspects of the use of solid-state NMR for membrane protein structural determinations are presented, as well as how the methodology can be applied. Some successes to date are discussed, with an indication of how the area might develop.

Expression, purification, and activities of full-length and truncated versions of the integral membrane protein Vpu from HIV-1.

Vpu is an 81-residue accessory protein of HIV-1. Because it is a membrane protein, it presents substantial technical challenges for the characterization of its structure and function, which are of considerable interest because the protein enhances the release of new virus particles from cells infected with HIV-1 and induces the intracellular degradation of the CD4 receptor protein. The Vpu-mediated enhancement of the virus release rate from HIV-1-infected cells is correlated with the expression of an ion channel activity associated with the transmembrane hydrophobic helical domain. Vpu-induced CD4 degradation and, to a lesser extent, enhancement of particle release are both dependent on the phosphorylation of two highly conserved serine residues in the cytoplasmic domain of Vpu. To define the minimal folding units of Vpu and to identify their activities, we prepared three truncated forms of Vpu and compared their structural and functional properties to those of full-length Vpu (residues 2-81). Vpu(2-37) encompasses the N-terminal transmembrane alpha-helix; Vpu(2-51) spans the N-terminal transmembrane helix and the first cytoplasmic alpha-helix; Vpu(28-81) includes the entire cytoplasmic domain containing the two C-terminal amphipathic alpha-helices without the transmembrane helix. Uniformly isotopically labeled samples of the polypeptides derived from Vpu were prepared by expression of fusion proteins in E. coli and were studied in the model membrane environments of lipid micelles by solution NMR spectroscopy and oriented lipid bilayers by solid-state NMR spectroscopy. The assignment of backbone resonances enabled the secondary structure of the constructs corresponding to the transmembrane and the cytoplasmic domains of Vpu to be defined in micelle samples by solution NMR spectroscopy. Solid-state NMR spectra of the polypeptides in oriented lipid bilayers demonstrated that the topology of the domains is retained in the truncated polypeptides. The biological activities of the constructs of Vpu were evaluated. The ion channel activity is confined to the transmembrane alpha-helix. The C-terminal alpha-helices modulate or promote the oligomerization of Vpu in the membrane and stabilize the conductive state of the channel, in addition to their involvement in CD4 degradation.