Cationic Amphipathic Peptides Research Articles

Amphipathicity Determines Different Cytotoxic Mechanisms of Lysine- or Arginine-Rich Cationic Hydrophobic Peptides in Cancer Cells.

Cationic amphipathic peptides (CAPs) are known to be able to cause membrane destabilization and induce cell death, yet how the hydrophobicity, amphipathicity, and lysine(K)/arginine(R) composition synergistically affect the peptide activity remains incompletely understood. Here, we designed a panel of peptides based on the well-known anticancer peptide KLA. Increasing hydrophobicity enhanced the cytotoxicities of both the K- and R-rich peptides. Peptides with an intact amphipathic helical interface can cause instant cell death through a membrane lysis mechanism. Interestingly, rearranging the residue positions to minimize amphipathicity caused a great decrease of cytotoxicity to the K-rich peptides but not to the R-rich peptides. The amphipathicity-minimized R-rich peptide 6 (RL2) (RLLRLLRLRRLLRL-NH2) penetrated the cell membrane and induced caspase-3-dependent apoptotic cell death. We found that the modulation of hydrophobicity, amphipathicity, and K/R residues leads to distinct mechanisms of action of cationic hydrophobic peptides. Amphipathicity-reduced, arginine-rich cationic hydrophobic peptides (CHPs) may represent a new class of peptide therapeutics.

536. The Viral Transduction Enhancer Vectofusin-1 Is a Nanofibrillar Peptide Capable of Increasing the Contact between Viral Vectors and Target Cells

Gene transfer into hCD34+ hematopoietic stem/progenitor cells (HSPCs) using HIV-1-based lentiviral vectors (LVs) has several therapeutic applications ranging from monogenic diseases, infectious diseases and cancer. In such therapeutic context, the gene therapy could be improved by enhancing transduction levels of target cells and by reducing the amount of LVs used on the cells for greater safety and reduced costs. We recently identified a new cationic amphipathic peptide, Vectofusin-1, with viral transduction enhancing capacity, enabling higher transduction levels with low amounts of LV. Vectofusin-1 promotes the entry of several retroviral pseudotypes into target cells when added to the culture medium and is not toxic to HSPCs. Here, we present the first insights into the mechanism of action of this new transduction enhancer. First, a viral pull down assay showed that viral particles were easily pelleted by low speed centrifugation in presence of Vectofusin-1, suggesting that this latter may form unsoluble nanofibrils, trapping lentiviral particles. Atomic force (AFM) and electron microscopy (EM) of Vectofusin-1 confirmed that this peptide is rapidly forming annular aggregates and nanofibres in culture medium. Furthermore, these fibres were shown to be auto-fluorescent in medium with high-protein content (X-Vivo20), allowing the observation of a nanofibrillar network of Vectofusin-1 using confocal microscopy. Next, Vectofusin-1 was shown to strongly interact with Congo Red, especially in presence of lentiviral particles, but labeling with Thioflavin T was inefficient, suggesting that Vectofusin-1 fibres are not amyloid-type fibrils. Structural studies by circular dichroism confirmed this result. The capacity to form nanofibrils appears to be essential for the mechanism of action of Vectofusin-1 since a defective mutant called LAH2-A4, unable to promote lentiviral transduction (Majdoul S. et al (2016) J. Biol. Chem.), was also unable to form nanofibrils. In conclusion, biophysical, nanoscopic and microscopic observations have helped us to define Vectofusin-1 as a new nanofibrillar peptide capable of enhancing lentiviral transduction of target cells in conditions well-adapted to cGMP and scalable gene therapy protocols.

The immunology of host defence peptides: beyond antimicrobial activity.

Host defence peptides (HDPs) are short, cationic amphipathic peptides with diverse sequences that are produced by various cells and tissues in all complex life forms. HDPs have important roles in the body's response to infection and inflammation. This Review focuses on human HDPs and explores the diverse immunomodulatory effects of HDPs from a systems biology perspective, which highlights the interconnected nature of the effect (or effects) of HDPs on the host. Studies have demonstrated that HDPs are expressed throughout the body and mediate a broad range of activities, which explains their association with various inflammatory diseases and autoimmune disorders. The diverse actions of HDPs, such as their roles in wound healing and in the maintenance of the microbiota, are also explored, in addition to potential therapeutic applications.

Potentiating the Anticancer Properties of Bisphosphonates by Nanocomplexation with the Cationic Amphipathic Peptide, RALA.

Bisphosphonates (BPs) are a class of bone resorptive drug with a high affinity for the hydroxyapatite structure of bone matrices that are used for the treatment of osteoporosis. However, clinical application is limited by a common toxicity, BP-related osteonecrosis of the jaw. There is emerging evidence that BPs possess anticancer potential, but exploitation of these antiproliferative properties is limited by their toxicities. We previously reported the utility of a cationic amphipathic fusogenic peptide, RALA, to traffic anionic nucleic acids into various cell types in the form of cationic nanoparticles. We hypothesized that complexation with RALA could similarly be used to conceal a BP's hydroxyapatite affinity, and to enhance bioavailability, thereby improving anticancer efficacy. Incubation of RALA with alendronate, etidronate, risedronate, or zoledronate provoked spontaneous electrostatic formation of cationic nanoparticles that did not exceed 100 nm in diameter and that were stable over a range of temperatures and for up to 6 h. The nanoparticles demonstrated a pH responsiveness, possibly indicative of a conformational change, that could facilitate release of the BP cargo in the endosomal environment. RALA/BP nanoparticles were more potent anticancer agents than their free BP counterparts in assays investigating the viability of PC3 prostate cancer and MDA-MB-231 breast cancer cells. Moreover, RALA complexation potentiated the tumor growth delay activity of alendronate in a PC3 xenograft model of prostate cancer. Taken together, these findings further validate the use of BPs as repurposed anticancer agents.

Mismatch Dependent Tilt of Amphipathic α-Helical Antimicrobial Peptides Inserted in Positive Spontaneous Curvature Lipid Membranes

We have used solid state NMR to study the orientation of membrane-active peptides in model membranes with different properties. We found that the peptide behavior can to a large extent be explained by the simple biophysical concepts of hydrophobic matching and lipid spontaneous curvature. Hydrophobic peptides insert in the membrane in a transmembrane orientation, and it is well established that peptides which have a longer hydrophobic stretch than the bilayer thickness start to tilt to compensate the hydrophobic mismatch. Here we show that the hydrophobic matching principle also applies to cationic amphipathic α-helical antimicrobial peptides. Using a series of peptides called KIAn, based on the repetitive amino acid sequence KIAGKIA, we found that only peptides long enough to span the hydrophobic thickness of the membrane induce leakage in lipid vesicles. Interestingly, a certain minimum length was also needed to induce hemolysis and to kill bacteria, and this minimum length was different for different bacterial strains, so that the series of peptides can be used as a “molecular ruler” to determine the thickness of erythrocyte or bacterial membranes in vivo. Using solid-state NMR we found that the orientation depends strongly on the lipid spontaneous curvature. The KIA peptides are always staying flat on the membrane surface in lipids with a negative spontaneous curvature. On the other hand, in lipids with a positive spontaneous curvature, like lyso-lipids, they can go deeper into the membrane and have a tilted or even inserted orientation. In the inserted state, the tilt of the peptides is found to be mismatch dependent. This is the first time a mismatch effect has been found for amphipathic peptides, and shows that the effect is more generally valid than has been appreciated until now.

Solid State NMR Structural and Topolocical Studies of Antimicrobial Peptides LPcin Analogs

The wide use of antibiotics has led to pathogens' increased drug resistance. Most of the drugs in development are analogs of earlier antibiotics which work on a select number of bacterial targets. There will be an increasing demand for novel drugs to combat bacterial infections. One approach to this problem of resistance to current antibiotics is to look to evolutionary ancient weapons like antimicrobial peptides(AMP). Lactophoricin (LPcin), a cationic amphipathic peptide consists of 23-mer peptide, corresponds to the carboxy terminal 113-135 region of component-3 of proteose-peptone found in bovine milk. LPcin is a good candidate as a peptide antibiotic because it has an antibacterial activity but no hemolytic activity. Three different analogs of LPcin, LPcin-yk2 which has mutant amino acids, LPcin-yk1 and LPcin-yk3 that has shorter mutant amino acids are recently developed by using peptide engineering in our laboratory. These three LPcin analogs show better antibioitic activities than wild-type LPcin and no toxicity at all.In order to understand the structural correlation between LPcin analogs structure and antimicrobial activity under the membrane environments, we tried to express and purify as large as amounts of LPcin and three different LPcin analogs. We finally optimized and succeed to overexpress in the form of fusion protein in Escherichia coli and purified with biophysical techniques like Ni-affinity chromatography, dialysis, centrifuge, chemical cleavage, and reversed-phase semiprep HPLC. In here, we will present the optimizing processes for high-yield expression and purification and structural topological studies using solution NMR and solid state NMR spectroscopy to figure out antimicrobial killing mechanisms.

NMR Structural Studies of Antimicrobial Peptides: LPcin Analogs

Lactophoricin (LPcin), a component of proteose peptone (113–135) isolated from bovine milk, is a cationic amphipathic antimicrobial peptide consisting of 23 amino acids. We designed a series of N- or C-terminal truncated variants, mutated analogs, and truncated mutated analogs using peptide-engineering techniques. Then, we selected three LPcin analogs of LPcin-C8 (LPcin-YK1), LPcin-T2WT6W (LPcin-YK2), and LPcin-T2WT6W-C8 (LPcin-YK3), which may have better antimicrobial activities than LPcin, and successfully expressed them in E. coli with high yield. We elucidated the 3D structures and topologies of the three LPcin analogs in membrane environments by conducting NMR structural studies. We investigated the purity of the LPcin analogs and the α-helical secondary structures by performing 1H-15N 2D HSQC and HMQC-NOESY liquid-state NMR spectroscopy using protein-containing micelle samples. We measured the 3D structures and tilt angles in membranes by conducting 15N 1D and 2D 1H-15N SAMMY type solid-state NMR spectroscopy with an 800 MHz in-house-built 1H-15N double-resonance solid-state NMR probe with a strip-shield coil, using protein-containing large bicelle samples aligned and confirmed by molecular-dynamics simulations. The three LPcin analogs were found to be curved α-helical structures, with tilt angles of 55–75° for normal membrane bilayers, and their enhanced activities may be correlated with these topologies.

540. Vectofusin-1 Significantly Enhances Transduction of Ex-Vivo Activated T Cells With RD114-TR-Pseudotyped Lentiviral Vectors Derived from the RD3-MolPack Stable Packaging Cells

Cell therapy with T cells genetically modified with lentiviral vectors (LVs) carrying therapeutic genes is growing as a promising tool for tumor treatment. Noticeably, the clinical production of these cells relies on the availability of either LVs, whose production is cumbersome from a safety, cost and reproducibility standpoint, or transduction enhancers to get optimal transduction rates. Development of LV packaging cells represents therefore an obliged goal in LV-based gene therapy, allowing reduction of the manufacturing cost and increasing the overall quality of the vectors. Similarly, the identification of the optimal transduction conditions is crucial. We investigated the efficiency of the recently described transduction enhancer Vectofusin-1® in promoting transduction of human ex-vivo activated T cells with a RD114-TR-pseudotyped SIN-GFP LV derived from MolMed's proprietary RD3-MolPack-SIN-GFP producer cells. Vectofusin-1® is an histidine-rich cationic amphipathic peptide that has been recently shown to increase LV gene transfer in hematopoietic stem cells without inducing toxicity through improvement of adhesion and fusion of LVs with the cellular membrane. Vectofusin-1® is easily synthetized, purified, and can be added to the viral supernatant without a pre-coating step, thus requiring less overall manipulation. RD3-MolPack-SIN-GFP was generated exploiting our RD-MolPack technology which is based on the RD114-TR envelope glycoprotein that, in contrast to the most utilized VSV-G envelope, permits generation of constitutive packaging cells for the production of SIN-LV. We selected a producer clone growing continuously in disposable two-compartment bioreactor for longer than 3 months and releasing LV with a titer, on average, of 1-5 × 106 TU/ml. Here, we show that the addition of Vectofusin-1® significantly improved RD3-MolPack-SIN-GFP LV transduction rate, without spinoculation, of either CD3/CD28 or OKT-3 activated T cells (70% GFP+) compared to the absence of adjuvant (6% GFP+) at MOI 1. The analysis of the vector copy number (VCN) integration confirmed these results (VCN=4 in the presence of Vectofusin-1® vs VCN=0.09 in the absence of Vectofusin-1®, respectively). When Vectofusin-1® was compared with Retronectin® and polybrene a similar effect was registered. No major differences in the immune phenotype and expansion rate were observed between cells transduced with Vectofusin-1® and cells transduced without any adjuvant. In summary, we conclude that combination of SIN-LV produced by RD3-MolPack cells and Vectofusin-1® might be considered for clinical manufacturing of LV-transduced T cells.

Optimized Expression and Characterization of Antimicrobial Peptides, LPcin Analogs

Lactophoricin (LPcin or LPcin‐I), a cationic amphipathic antimicrobial peptide consisting of 23 amino acids, is a part of proteose peptone (113–135) isolated from bovine milk. The 23 amino acids of LPcin are relatively long for industrial uses as antimicrobial peptides. Therefore, a series of N‐ or C‐terminal truncated variant mutated analogs and truncated mutated analogs were designed using peptide engineering techniques to investigate the structure–activity relationships. Then, three analogs of LPcin‐C8 [(LPcin‐YK1), LPcin‐T2WT6W (LPcin‐YK2), and LPcin‐T2WT6W‐C8 (LPcin‐YK3)] were optimized to express with high yield and characterized for their structural studies. Successful expression of these three LPcin analogs in Escherichia coli system was accomplished through the formation of a fusion protein with higher yield. The peptides were purified using several biophysical techniques and finally isolated using reversed‐phase preparative HPLC. The purity, molecular mass, and the secondary structure of the purified and isolated LPcin analogs were characterized by polyacrylamide gel electrophoresis, mass spectrometry, and circular dichroism spectrometry. The secondary structure of LPcin and its three analog peptides clearly showed α‐helical characteristics in membrane environments and random‐coil conformation in water. Consequently, our results so far have shown that the three LPcin analogs could become potent antimicrobial peptides like LPcin as an alternative of new antibiotics in the near future.

Cationic amphipathic peptides KT2 and RT2 are taken up into bacterial cells and kill planktonic and biofilm bacteria

We investigated the mechanisms of two tryptophan-rich antibacterial peptides (KT2 and RT2) obtained in a previous optimization screen for increased killing of both Gram-negative and Gram-positive bacteria pathogens. At their minimal inhibitory concentrations (MICs), these peptides completely killed cells of multidrug-resistant, enterohemorrhagic pathogen Escherichia coli O157:H7 within 1–5min. In addition, both peptides exhibited anti-biofilm activity at sub-MIC levels. Indeed, these peptides prevented biofilm formation and triggered killing of cells in mature E. coli O157:H7 biofilms at 1μM. Both peptides bound to bacterial surface LPS as assessed using the dansyl-polymyxin displacement assay, and were able to interact with the lipids of liposomes as determined by observing a tryptophan blue shift. Interestingly, even though these peptides were highly antimicrobial, they did not induce pore formation or aggregates in bacterial cell membranes. Instead these peptides readily penetrated into bacterial cells as determined by confocal microscopy of labeled peptides. DNA binding assays indicated that both peptides bound to DNA with higher affinity than the positive control peptide buforin II. We propose that cationic peptides KT2 and RT2 bind to negatively-charged LPS to enable self-promoted uptake and, subsequently interact with cytoplasmic membrane phospholipids through their hydrophobic domains enabling translocation across the bacterial membrane and entry into cells within minutes and binding to DNA and other cytoplasmic membrane. Due to their dual antimicrobial and anti-biofilm activities, these peptides may find use as an alternative to (or in conjunction with) conventional antibiotics to treat acute infections caused by planktonic bacteria and chronic, biofilm-related infections.

Translocation of Cationic Amphipathic Peptides Across Phospholipid Bilayers

We have developed a method to measure translocation of amphipathic peptides across lipid membranes that uses giant unilamellar vesicles (GUVs) containing inner vesicles. When the GUVs are added to a peptide solution containing a water-soluble fluorescent dye, permeabilization of the membrane of the outer vesicle by the peptide results in the appearance of fluorescence in its lumen. The inner vesicles remain dark if the peptide does not translocate. However, the appearance of dye in the inner vesicles indicates peptide translocation across the outer membrane of the GUVs. This is because to cause dye flux into the inner vesicles, the peptide must have crossed the membrane of the outer vesicle. The method does not require that the peptide itself be fluorescently labeled; but if it is, this approach can be used to measure the kinetics of translocation. Initially we tested the method with three peptides derived from each class of antimicrobial, cytolytic, and cell-penetrating peptides. Those three peptides also had very different Gibbs energies of insertion into the bilayer, approximated by the difference between the Gibbs energy of binding to the membrane and the Gibbs energy of transfer from water to octanol. The initial measurements appeared to indicate that the probability of translocation was inversely correlated with the Gibbs energy of insertion. Now we extended this study to several variants of the original peptides. Two hypotheses were tested: (1) The probability of translocation simply decreases as the Gibbs energy of peptide insertion increases; (2) The probability of translocation increases if the distribution of positively charged residues along the peptide sequence is such that the inserted peptide has similar probabilities of returning the outside and crossing the membrane, to the inside of the vesicle. The results of these new tests are discussed.

Solid State NMR Structural Studies of Antimicrobial Peptides LPcin Analogs with Enhanced Activities

The availability of antibiotics has allowed for the successful treatment of many bacterial infections as well as the ability to perform invasive medical procedures including surgery and chemotherapy. However, their wide use has led to pathogens’ increased drug resistance and the need to find novel classes of antimicrobial peptides as alternatives to antibiotics. Lactophoricin (LPcin), a cationic amphipathic peptide consists of 23-mer peptide, corresponds to the carboxy terminal 113-135 region of component-3 of proteose-peptone. LPcin is a good candidate as a peptide antibiotic because it has an antibacterial activity but no hemolytic activity. Three different analogs of LPcin, LPcin-yk2 which has mutant amino acids, LPcin-yk1 and LPcin-yk3 that has shorter mutant amino acids are recently developed by using peptide engineering in our laboratory. These three LPcin analogs show better antibioitic activities than wild-type LPcin and no toxicity at all.In order to understand the structural correlation between LPcin analogs structure and antimicrobial activity under the membrane environments, we tried to express and purify as large as amounts of LPcin and three different LPcin analogs. We finally optimized and succeed to overexpress in the form of fusion protein in Escherichia coli and purified with biophysical techniques like Ni-affinity chromatography, dialysis, centrifuge, chemical cleavage, and reversed-phase semiprep HPLC. In here, we will present the optimizing processes with high-yield expression and purification of three LPcin analogs and solid- state NMR structural studies to figure out antibacterial killing mechanisms.

Antimicrobial and immunomodulatory properties of PGLa-AM1, CPF-AM1, and magainin-AM1: Potent activity against oral pathogens

Cationic amphipathic α-helical peptides are intensively studied classes of host defence peptides (HDPs). Three peptides, peptide glycine–leucine–amide (PGLa-AM1), caerulein-precursor fragment (CPF-AM1) and magainin-AM1, originally isolated from norepinephrine-stimulated skin secretions of the African volcano frog Xenopus amieti (Pipidae), were studied for their antimicrobial and immunomodulatory activities against oral and respiratory pathogens. Minimal effective concentrations (MECs), determined by radial diffusion assay, were generally lower than minimal inhibitory concentrations (MICs) determined by microbroth dilution. PGLa-AM1 and CPF-AM1 were particularly active against Streptococcus mutans and all three peptides were effective against Fusobacterium nucleatum, whereas Enterococcus faecalis and Candida albicans proved to be relatively resistant micro-organisms. A type strain of Pseudomonas aeruginosa was shown to be more susceptible than the clinical isolate studied. PGLa-AM1 displayed the greatest propensity to bind lipopolysaccharide (LPS) from Escherichia coli, P. aeruginosa and Porphyromonas gingivalis. All three peptides showed less binding to P. gingivalis LPS than to LPS from the other species studied. Oral fibroblast viability was unaffected by 50μM peptide treatments. Production of the pro-inflammatory cytokine IL-8 by oral fibroblasts was significantly increased following treatment with 1 or 10μM magainin-AM1 but not following treatment with PGLa-AM1 or CPF-AM1. In conclusion, as well as possessing potent antimicrobial actions, the X. amieti peptides bound to LPS from three human pathogens and had no effect on oral fibroblast viability. CPF-AM1 and PGLa-AM1 show promise as templates for the design of novel analogues for the treatment of oral and dental diseases associated with bacteria or fungi.

Lipid interactions of LAH4, a peptide with antimicrobial and nucleic acid transfection activities.

The cationic amphipathic designer peptide LAH4 exhibits potent antimicrobial, nucleic acid transfection and cell penetration activities. Closely related derivatives have been developed to enhance viral transduction for gene therapeutic assays. LAH4 contains four histidines and, consequently, its overall charge and membrane topology in lipid bilayers are strongly pH dependent. In order to better understand the differential interactions of this amphipathic peptide with negatively-charged membranes its interactions, topologies, and penetration depth were investigated in the presence of lipid bilayers as a function of pH, buffer, phospholipid head group, and fatty acyl chain composition using a combination of oriented synchrotron radiation circular dichroism spectroscopy as well as oriented and non-oriented solid-state NMR spectroscopy. This combination of methods indicates that in the presence of lipids with phosphatidylglycerol head groups, the topological equilibria of LAH4 is shifted towards more in-plane configurations even at neutral pH. In contrast, a transmembrane alignment is promoted when LAH4 interacts with membranes made of dimyristoyl phospholipids rather than palmitoyl-oleoyl-phospholipids. Finally, the addition of citrate buffer favours LAH4 transmembrane alignments, even at low pH, probably by complex formation with the cationic charges of the peptide. In summary, this study has revealed that the membrane topology of this peptide is readily modulated by the environmental conditions.

Peptide-siRNA nanocomplexes targeting NF-κB subunit p65 suppress nascent experimental arthritis.

The NF-κB signaling pathway is implicated in various inflammatory diseases, including rheumatoid arthritis (RA); therefore, inhibition of this pathway has the potential to ameliorate an array of inflammatory diseases. Given that NF-κB signaling is critical for many immune cell functions, systemic blockade of this pathway may lead to detrimental side effects. siRNAs coupled with a safe and effective delivery nanoplatform may afford the specificity lacking in systemic administration of small-molecule inhibitors. Here we demonstrated that a melittin-derived cationic amphipathic peptide combined with siRNA targeting the p65 subunit of NF-κB (p5RHH-p65) noncovalently self-assemble into stable nanocomplexes that home to the inflamed joints in a murine model of RA. Specifically, administration of p5RHH-p65 siRNA nanocomplexes abrogated inflammatory cytokine expression and cellular influx into the joints, protected against bone erosions, and preserved cartilage integrity. The p5RHH-p65 siRNA nanocomplexes potently suppressed early inflammatory arthritis without affecting p65 expression in off-target organs or eliciting a humoral response after serial injections. These data suggest that this self-assembling, largely nontoxic platform may have broad utility for the specific delivery of siRNA to target and limit inflammatory processes for the treatment of a variety of diseases.

Interaction of a novel antimicrobial peptide isolated from the venom of solitary bee Colletes daviesanus with phospholipid vesicles and Escherichia coli cells.

The peptide named codesane (COD), consisting of 18 amino acid residues and isolated from the venom of wild bee Colletes daviesanus (Hymenoptera : Colletidae), falls into the category of cationic α-helical amphipathic antimicrobial peptides. In our investigations, synthetic COD exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria and Candida albicans but also noticeable hemolytic activity. COD and its analogs (collectively referred to as CODs) were studied for the mechanism of their action. The interaction of CODs with liposomes led to significant leakage of calcein entrapped in bacterial membrane-mimicking large unilamellar vesicles made preferentially from anionic phospholipids while no calcein leakage was observed from zwitterionic liposomes mimicking membranes of erythrocytes. The preference of CODs for anionic phospholipids was also established by the blue shift in the tryptophan emission spectra maxima when the interactions of tryptophan-containing COD analogs with liposomes were examined. Those results were in agreement with the antimicrobial and hemolytic activities of CODs. Moreover, we found that the studied peptides permeated both the outer and inner cytoplasmic membranes of Escherichia coli. This was determined by measuring changes in the fluorescence of probe N-phenyl-1-naphthylamine and detecting cytoplasmic β-galactosidase released during the interaction of peptides with E. coli cells. Transmission electron microscopy revealed that treatment of E. coli with one of the COD analogs caused leakage of bacterial content mainly from the septal areas of the cells.

Cationic amphipathic D-enantiomeric antimicrobial peptides with in vitro and ex vivo activity against drug-resistant Mycobacterium tuberculosis

Tuberculosis (TB) is the leading cause of bacterial death worldwide. Due to the emergence of multi-drug resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB), and the persistence of latent infections, a safe and effective TB therapy is highly sought after. Antimicrobial peptides (AMPs) have therapeutic potential against infectious diseases and have the ability to target microbial pathogens within eukaryotic cells. In the present study, we investigated the activity of a family of six AMPs containing all-D amino acids (D-LAK peptides) against MDR and XDR clinical strains of Mycobacterium tuberculosis (Mtb) both in vitro and, using THP-1 cells as a macrophage model, cultured ex vivo. All the D-LAK peptides successfully inhibited the growth of Mtb in vitro and were similarly effective against MDR and XDR strains. D-LAK peptides effectively broke down the heavy clumping of mycobacteria in broth culture, consistent with a 'detergent-like effect' that could reduce the hydrophobic interactions between the highly lipidic cell walls of the mycobacteria, preventing bacteria cell aggregation. Furthermore, though not able to eradicate the intracellular mycobacteria, D-LAK peptides substantially inhibited the intracellular growth of drug-resistant Mtb clinical isolates at concentrations that were well tolerated by THP-1 cells. Finally, combining D-LAK peptide with isoniazid could enhance the anti-TB efficacy. D-LAK peptide, particularly D-LAK120-A, was effective as an adjunct agent at non-toxic concentration to potentiate the efficacy of isoniazid against drug-resistant Mtb in vitro, possibly by facilitating the access of isoniazid into the mycobacteria by increasing the surface permeability of the pathogen.

Antimicrobial activity of cationic peptides in endodontic procedures.

Objectives:The present study aimed to investigate the antimicrobial and biofilm inhibition activity of synthetic antimicrobial peptides (AMPs) against microbes such as Enterococcus faecalis, Staphylococcus aureus, and Candida albicans which are involved in endodontic infections.Materials and Methods:Agar diffusion test was done to determine the activity of peptides. The morphological changes in E. faecalis and reduction in biofilm formation after treatment with peptides were observed using scanning electron microscope. The efficacy of peptides using an ex vivo dentinal model was determined by polymerase chain reaction and confocal laser scanning microscopy. Platelet aggregation was done to determine the biocompatibility of peptides.Results:Among 11 peptides, two of the amphipathic cationic peptides were found to be highly active against E. faecalis, S. aureus, C. albicans. Efficacy results using dentinal tubule model showed significant reduction in microbial load at 400 μm depth. The peptides were also biocompatible.Conclusion:These results suggest that synthetic AMPs have the potential to be developed as antibacterial agents against microorganisms involved in dental infections and thus could prevent the spread and persistence of endodontic infections improving treatment outcomes and teeth preservation.

Length-Dependent Activity of Membrane-Bound Cationic Amphipathic Alpha-Helical Peptides

MSI-103 [sequence (KIAGKIA)3-NH2] is a designer-made antimicrobial peptide based on the sequence of PGLa, a host defense peptide from the African frog Xenopus laevis, with high activity against bacteria [1, 2]. It forms an amphipathic α-helix upon binding to a lipid bilayer, and has been proposed to kill bacteria by forming membrane pores. If this were the case, shorter analogs that are not long enough to span the membrane should not be able to form pores and should be inactive. To test this hypothesis we have synthesized a series of analogs of MSI-103, called KIA peptides, with a length of 14 to 28 amino acids, all of which were shown to be α-helical by circular dichroism spectroscopy. We tested their antimicrobial and hemolytic activities and the ability to induce vesicle leakage, and found that there is a threshold length needed for activity, supporting the pore formation hypothesis. Using solid-state 15N-NMR on isotopically labeled peptides, we also investigated the orientation of the different KIA peptides in membranes of different lipid composition, and also here observed a systematic dependence on peptide length.

NMR Structural Studies of Antimicrobial Peptides as In-Plane Helix of Membrane Proteins

Antibiotics from organic compounds are a long established part of our defense against bacterial pathogens. Their wide use has led to pathogens' increased drug resistance and the need to find novel classes of antimicrobial peptides as alternatives to antibiotics. Lactophoricin (LPcin), a cationic amphipathic peptide consists of 23-mer peptide, corresponds to the carboxy terminal 113-135 region of component-3 of proteose-peptone. LPcin is a good candidate as a peptide antibiotic because it has an antibacterial activity but no hemolytic activity. Three different analogs of LPcin, LPcin-yk2 which has mutant amino acids, LPcin-yk1 and LPcin-yk3 that has shorter mutant amino acids are recently developed by using peptide engineering in our laboratory. These three LPcin analogs show better antibioitic activities than wild-type LPcin and no toxicity at all. In order to understand the structural correlation between LPcin analogs structure and antimicrobial activity under the membrane environments, we tried to express and purify as large as amounts of LPcin and three different LPcin analogs. We finally optimized and succeed to overexpress in the form of fusion protein in Escherichia coli and purified with biophysical techniques like Ni-affinity chromatography, dialysis, entrifuge, chemical cleavage, and reversed-phase semiprep HPLC. In here, we will present the optimizing processes for high-yield expression and purification and solution NMR spectra and solid state NMR spectra for antimicrobial mechanisms.