Effect of proline position on the antimicrobial mechanism of buforin II

Buforin II (BF2) is a histone-derived antimicrobial peptide that causes cell death by translocating across membranes and interacting with nucleic acids. It contains one proline residue critical for its function. Previous research found that mutations replacing proline lead to decreased membrane translocation and antimicrobial activity as well as increased membrane permeabilization. This study further investigates the role of proline in BF2's antimicrobial mechanism by considering the effect of changing proline position on membrane translocation, membrane permeabilization, and antimicrobial activity. For this purpose, four mutants were made with proline substitution (P11A) or relocation (P11A/G7P, P11A/V12P, P11A/V15P). These mutations altered the amount of helical content. Although antimicrobial activity correlated with the α-helical content for the peptides containing proline, membrane translocation did not. This observation suggests that factors in BF2's bactericidal mechanism other than translocation must be altered by these mutations. To better explain these trends we also measured the nucleic acid binding and membrane permeabilization of the mutant peptides. A comparison of mutant and wild type BF2 activity revealed that BF2 relies principally on membrane translocation and nucleic acid binding for antimicrobial activity, although membrane permeabilization may play a secondary role for some BF2 variants. A better understanding of the role of proline in the BF2 antimicrobial mechanism will contribute to the further design and development of BF2 analogs. Moreover, since proline residues are prevalent among other antimicrobial peptides, this systematic characterization of BF2 provides general insights that can promote our understanding of other systems.     

Structure–activity relationships of a snake cathelicidin-related peptide,BF-15

Cathelicidin-BF15 (BF-15) is a 15-mer peptide derived from Cathelicidin-BF (BF-30), which is found in the venom of the snake Bungarus fasciatus and exhibits broad antimicrobial activity. Since BF-15 retains most part of the antimicrobial activity of BF-30 but has significantly reduced haemolytic activity and a much shorter sequence length (and less cost), it is a particularly attractive template around which to design novel antimicrobial peptides. However, the structure–activity relationship of it is still unknown. We designed and synthesized a series of C-terminal amidated analogs of BF-15 based on its amphipathic α-helix structure. And we characterized their antimicrobial potency and haemolytic activity. We identified the amidated BF-15 (analog B1) with potent antimicrobial activity against several antibiotic-resistant bacteria (MICs between 1 and 64 μg/mL, 2–16-folds higher than BF-30) and much lower haemolytic activity. The subsequent circular dichroism study results showed a typical α-helix pattern of analog B1 and the content of the α-helix structure of it increased significantly comparing with BF-30, which indicates the peptide sequence of BF-15 may provide a major contribution to the α-helix content of the whole BF-30 sequence. The peptide induced chaotic membrane morphology and cell debris as determined by electron microscopy. This suggests that the antimicrobial activity of B1 is based on cytoplasmic membrane permeability. Taken together, our results suggested that peptide B1 should be considered as an excellent candidate for developing therapeutic drugs.     

Structure-activity relationships of a snake cathelicidin-related peptide, BF-15

Cathelicidin-BF15 (BF-15) is a 15-mer peptide derived from Cathelicidin-BF (BF-30), which is found in the venom of the snake Bungarus fasciatus and exhibits broad antimicrobial activity. Since BF-15 retains most part of the antimicrobial activity of BF-30 but has significantly reduced haemolytic activity and a much shorter sequence length (and less cost), it is a particularly attractive template around which to design novel antimicrobial peptides. However, the structure–activity relationship of it is still unknown. We designed and synthesized a series of C-terminal amidated analogs of BF-15 based on its amphipathic α-helix structure. And we characterized their antimicrobial potency and haemolytic activity. We identified the amidated BF-15 (analog B1) with potent antimicrobial activity against several antibiotic-resistant bacteria (MICs between 1 and 64 μg/mL, 2–16-folds higher than BF-30) and much lower haemolytic activity. The subsequent circular dichroism study results showed a typical α-helix pattern of analog B1 and the content of the α-helix structure of it increased significantly comparing with BF-30, which indicates the peptide sequence of BF-15 may provide a major contribution to the α-helix content of the whole BF-30 sequence. The peptide induced chaotic membrane morphology and cell debris as determined by electron microscopy. This suggests that the antimicrobial activity of B1 is based on cytoplasmic membrane permeability. Taken together, our results suggested that peptide B1 should be considered as an excellent candidate for developing therapeutic drugs.     

Membrane active antitumor activity of NK-18, a mammalian NK-lysin-derived cationic antimicrobial peptide

As the increasing emergence of multi-drug resistant tumor cells, there is an urgent need for developing new chemotherapeutic agents. NK-lysin was a novel effector of cytotoxic T cells and natural killer (NK) cells and had broad antimicrobial activity. In this study, we developed a core region of NK-lysin termed NK-18, and studied its antitumor activity and possible action mode. Our results showed that NK-18 (with 18 amino acids) possesses potent antitumor activity against bladder and prostate cancer cells by disrupting the integrity of cell membrane, but has negligible hemolysis activity against mouse erythrocytes. In addition, CD spectra was employed to study its conformation in membrane mimicking environment. NK-18 takes a standard α-helical conformation in membrane mimicking environment, which could be accounted for its more potent antitumor activity compared with its low α-helical content homologous derivatives. These findings together with its shorter amino acid sequence and lower synthesis cost suggest that NK-18 could present an alternative therapeutic strategy to cancer chemotherapy and play a promising role in fighting the multi-drug resistant tumors.     

Mechanism of action and specificity of antimicrobial peptides designed based on buforin IIb

Buforin IIb-a synthetic analog of buforin II that contains a proline hinge between the two α-helices and a model α-helical sequence at the C-terminus (3× RLLR)-is a potent cell-penetrating antimicrobial peptide. To develop novel antimicrobial peptides with enhanced activities and specificity/therapeutic index, we designed several analogs (Buf III analogs) by substitutions of amino acids in the proline hinge region and two α-helices of buforin IIb, and examined their antimicrobial activity and mechanism of action. The substitution of hydrophobic residues ([F(6)] and [V(8)]) in the proline hinge region with other hydrophobic residues ([W(6)] and [I(8)]) did not affect antimicrobial activity, while the substitution of the first four amino acids RAGL with a model α-helical sequence increased the antimicrobial activity up to 2-fold. Like buforin IIb, Buf III analogs penetrated the bacterial cell membranes without significantly permeabilizing them and were accumulated inside Escherichia coli. Buf III analogs were shown to bind DNA in vitro and the DNA binding affinity of the peptides correlated linearly with their antimicrobial potency. Among the Buf III analogs, the therapeutic index of Buf IIIb and IIIc (RVVRQWPIG[RVVR](3) and KLLKQWPIG[KLLK](3), respectively) were improved 7-fold compared to that of buforin IIb. These results indicate that Buf III analogs appear to be promising candidates for future development as novel antimicrobial agents.     

Antitumor effects, cell selectivity and structure-activity relationship of a novel antimicrobial peptide polybia-MPI

A novel antimicrobial peptide, polybia-MPI, was purified from the venom of the social wasp Polybia paulista. It has potent antimicrobial activity against both Gram-positive and Gram-negative bacteria, but causing no hemolysis to rat erythrocytes. To date, there is no report about its antitumor effects on any tumor cell lines. In this study we synthesized polybia-MPI and studied its antitumor efficacy and cell selectivity. Our results revealed that polybia-MPI exerts cytotoxic and antiproliferative efficacy by pore formation. It can selectively inhibit the proliferation of prostate and bladder cancer cells, but has lower cytotoxicity to normal murine fibroblasts. In addition, to investigate the structure–activity relationship of polybia-MPI, three analogs in which Leu⁷, Ala⁸ or Asp⁹ replaced by l-Pro were designed and synthesized. l-Pro substitution of Leu⁷ or Asp⁹ significantly reduces the content of -helix conformation, and l-Pro substitution of Ala⁸ can disrupt the -helix conformation thoroughly. The l-Pro substitution induces a significant reduction of antitumor activity, indicating that the -helix conformation of polybia-MPI is important for its antitumor activity. In summary, polybia-MPI may offer a novel therapeutic strategy in the treatment of prostate cancer and bladder cancer, considering its relatively lower cytoxicity to normal cells.     

Comparison of the effects of hydrophobicity,amphiphilicity, and α-helicity on the activities of antimicrobial peptides

Multiple linear regression was used to quantify the dependence of the antimicrobial activity of 13 peptides upon three calculated or experimentally determined parameters: mean hydrophobicity, mean hydrophobic moment, and α-helix content. Mean hydrophobic moment is a measure of the amphiphilicity of peptides in an α-helical conformation. Antimicrobial activity was quantified as the reciprocal of the measured minimal inhibitory concentration (MIC) against Escherichia coli. One of the peptides was magainin 2, and the remainder were novel peptides designed for this study. The multiple linear regression results revealed that the amphiphilicity of the peptides was the most important factor governing anti-microbial activity compared to mean hydrophobicity orα-helix content. A better regression cf the data was obtained using In(1/MIC + constant) as the dependent variable than with either 1/MIC or In(1/MIC). These results should be useful in designing peptides with higher antimicrobial activity. © 1995 Wiley-Liss, Inc.     

Solution structure of P01, a natural scorpion peptide structurally analogous to scorpion toxins specific for apamin-sensitive potassium channel

The venom of the North African scorpion Androctonus mauretanicus mauretanicus possesses numerous highly active neurotoxins that specifically bind to various ion channels. One of these, P05, has been found to bind specifically to calcium-activated potassium channels and also to compete with apamin, a toxin extracted from bee venom. Besides the highly potent ones, several of these peptides (including that of P01) have been purified and been found to possess only a very weak, although significant, activity in competition with apamin. The amino acid sequence of P01 shows that it is shorter than P05 by two residues. This deletion occurs within an α-helix stretch (residues 5–12). This α-helix has been shown to be involved in the interaction of P05 with its receptor via two arginine residues. These two arginines are absent in the P01 sequence. Furthermore, a proline residue in position 7 of the P01 sequence may act as an α-helix breaker. We have determined the solution structure of P01 by conventional two-dimensional ¹H nuclear magnetic resonance and show that 1) the proline residue does not disturb the α-helix running from residues 5 to 12; 2) the two arginines are topologically replaced by two acidic residues, which explains the drop in activity; 3) the residual binding activity may be due to the histidine residue in position 9; and 4) the overall secondary structure is conserved, i.e., an α-helix running from residues 5 to 12, two antiparallel stretches of β-sheet (residues 15–20 and 23–27) connected by a type I′ β-turn, and three disulfide bridges connecting the α-helix to the β-sheet.     

Antimicrobial lipopeptaibol trichogin GA IV: role of the three Aib residues on conformation and bioactivity

The lipopeptaibol trichogin GA IV is a natural, non-ribosomally synthesized, antimicrobial peptide remarkably resistant to the action of hydrolytic enzymes. This feature may be connected to the multiple presence in its sequence of the non-coded residue α-aminoisobutyric acid (Aib), which is known to be responsible for the adoption of particularly stable helical structures already at the level of short peptides. To investigate the role of Aib residues on the 3D-structure and bioactivity of trichogin GA IV, we synthesized and fully characterized four analogs where one or two Aib residues are replaced by L-Leu. Our extensive conformational studies (including an X-ray diffraction analysis) and biological assays performed on these analogs showed that the Aib to L-Leu replacements do not affect the resistance to proteolysis, but modulate the bioactivity of trichogin GA IV in a 3D-structure related manner.     

Effect of hydrocarbon stapling on the properties of α-helical antimicrobial peptides isolated from the venom of hymenoptera

The impact of inserting hydrocarbon staples into short α-helical antimicrobial peptides lasioglossin III and melectin (antimicrobial peptides of wild bee venom) on their biological and biophysical properties has been examined. The stapling was achieved by ring-closing olefin metathesis, either between two S-2-(4′-pentenyl) alanine residues (S ₅) incorporated at i and i + 4 positions or between R-2-(7′-octenyl) alanine (R ₈) and S ₅ incorporated at the i and i + 7 positions, respectively. We prepared several lasioglossin III and melectin analogs with a single staple inserted into different positions within the peptide chains as well as analogs with double staples. The stapled peptides exhibited a remarkable increase in hemolytic activity, while their antimicrobial activities decreased. Some single stapled peptides showed a higher resistance against proteolytic degradation than native ones, while the double stapled analogs were substantially more resistant. The CD spectra of the singly stapled peptides measured in water showed only a slightly better propensity to form α-helical structure when compared to native peptides, whereas the doubly stapled analogs exhibited dramatically enhanced α-helicity.     

Synthesis, activity on NK-3 tachykinin receptor and conformational solution studies of scyliorhinin II analogs modified at position 16.

Two analogs of a tachykinin family peptides - scyliorhinin II (ScyII): [Aib(16)]ScyII and [Sar(16)]ScyII were synthesized by the solid-phase method using Fmoc chemistry. Conformational studies in water and DMSO-d(6) on these peptides were performed using a combination of two-dimensional NMR and theoretical conformational analysis. The solution structure of the peptides studied is interpreted as an equilibrium of several conformers with different statistical weights. The structure of [Sar(16)]ScyII in water appeared to be more flexible, especially in the C-terminal fragment. A better defined structure for this analog was obtained in DMSO-d(6), in which the analysis resulted in a family of conformers with similar shapes. Some of these conformers were characterized by the presence of a 3(10)-helix in the N-terminal fragment and middle part of the molecule. The introduction of the Aib residue in position 16 significantly rigidifies the structure. For [Aib(16)]ScyII in both solvent systems very similar populations of conformations were obtained which are characterized by the presence of a 3(10)-helix in the 13-18 fragment. A common structural motif was found in conformationally constrained Cys(7)-Cys(13) fragment, which resembles the Greek letter 'omega'. The differences in the solution structure of the C-terminal fragment of the peptides studied are responsible for their specificity. [Aib(16)]ScyII showed 25% the agonistic activity of selective NK-3 agonist - senktide, but it also showed antagonist effect vs. this peptide, whereas [Sar(16)]ScyII appeared to be a full agonist of NK-3 tachykinin receptor.     

Enhanced cell selectivity of hybrid peptides with potential antimicrobial activity and immunomodulatory effect

BackgroundHybridization is a useful strategy to bond the advantages of different peptides into novel constructions. We designed a series of AMPs based on the structures of a synthetic AMP KFA3 and a naturally-occurred host defense peptide substance P (SP) to obtain peptides retaining the high antibacterial activity of KFA3 and the immunomodulatory activity and low cytotoxicity of SP.MethodsTwo repeats of KFA and different C terminal fragments of SP were hybridized, generating a series of novel AMPs (KFSP1–8). The antibacterial activities, host cell toxicity and immunomodulation were measured. The antibacterial mechanisms were investigated.ResultsHybrid peptides KFSP1–4 exerted substantial antibacterial activities against Gram-negative bacteria of standard strains and clinical drug-resistant isolates including E.coli, A.baumannii and P.aeruginosa, while showing little toxicity towards host cells. Compared with KFA3, moderate reduction in α-helix content and the interruption in α-helix continuality were indicated in CD spectra analysis and secondary-structure simulation in these peptides. Membrane permeabilization combined with time-kill studies and FITC-labeled imaging, indicated a selective membrane interaction of KFSP1 with bacteria cell membranes. By specially activating NK1 receptor, the hybrid peptides kept the ability of SP to induce intracellular calcium release and ERK1/2 phosphorylation, but unable to stimulate NF-κB phosphorylation. KFSP1 facilitated the survival of mouse macrophage RAW264.7, directly interacting with LPS and inhibiting the LPS-induced NF-κB phosphorylation and TNF-α expression.ConclusionHybridization is a useful strategy to bond the advantages of different peptides. KFSP1 and its analogs are worth of advanced efforts to explore their potential applications as novel antimicrobial agents.     

Competing interactions for antimicrobial selectivity based on charge complementarity

Antimicrobial peptides (AMPs) are an evolutionary conserved component of the innate immune system and possible templates for the development of new antibiotics. An important property of antimicrobial peptides is their ability to discriminate bacterial from eucaryotic cells which is attributed to the difference in lipid composition of the outer leaflet of the plasma membrane between the two types of cells. Whereas eucaryotic cells usually expose zwitterionic lipids, procaryotic cells expose also anionic lipids which bind the cationic antimicrobial peptides electrostatically. An example is the antimicrobial peptide NK-2 which is highly cationic and favors binding to anionic membranes. In the present study, the difference in binding affinity of NK-2 for palmitoyl-oleoyl-phosphatidyl-glycerol (POPG) and palmitoyl-oleoyl-phosphatidyl-choline (POPC) is studied using molecular dynamics simulations in conjunction with a coarse grained model and thermodynamic integration, by computing the change in free energy and its components upon the transfer of NK-2 from POPC to POPG. The transfer is indeed found to be highly favorable. Interestingly, the favorable contribution from the electrostatic interaction between the peptide and the anionic lipids is overcompensated by an unfavorable contribution from the change in lipid-cation interactions due to the release of counterions from the lipids. The increase in entropy due to the release of the cations is compensated by other entropic components. The largest favorable contribution arises from the solvation of the counterions. Overall the interaction between NK-2 and POPG is not determined by a single driving force but a subtle balance of competing interactions.     

Antimicrobial and anti-inflammatory activities of chemokine CXCL14-derived antimicrobial peptide and its analogs

CXCL14 is a CXC chemokine family that exhibits antimicrobial activity and contains an amphipathic cationic α-helical region in the C-terminus, a characteristic structure of antimicrobial peptides (AMPs). In this study, we designed three analogs of CXCL14_59–75 (named CXCL14-C17) corresponding to the C-terminal α-helix of CXCL14, which displayed potential antimicrobial activity against a wide variety of gram-negative and gram-positive bacteria with minimum inhibitory concentrations of 4?16?μM without mammalian cell toxicity. Furthermore, two CXCL14-C17 analogs (CXCL14-C17-a1 and CXCL14-C17-a3) with improved cell selectivity were engineered by introducing Lys, Arg, or Trp in CXCL14-C17. Additionally, CXCL14-C17 analogs showed much greater synergistic effect (FICI: 0.3125–0.375) with chloramphenicol and ciprofloxacin against multidrug-resistant Pseudomonas aeruginosa (MDRPA) than LL-37 did (FICI: 0.75–1.125). CXCL14-C17 analogs were more active against antibiotic-resistant bacteria including methicillin-resistant Staphylococcus aureus (MRSA), MDRPA, and vancomycin-resistant Enterococcus faecium (VREF) than LL-37 and melittin. In particular, CXCL14-C17-a2 and CXCL14-C17-a3 completely inhibited the biofilm formation at sub-MIC and all of the peptides were able to eliminate pre-formed biofilm as well. Membrane depolarization, flow cytometry, sytox green uptake, ONPG hydrolysis and confocal microscopy revealed the possible target of the native peptide (CXCL14-C17) to likely be intracellular, and the amphipathic designed analogs targeted the bacterial membrane. CXCL14-C17 also showed DNA binding characteristic activity similar to buforin-2. Interestingly, CXCL14-C17-a2 and CXCL14-C17-a3 effectively inhibited the production and expression of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 from lipopolysaccharide (LPS)-stimulated RAW264.7 cells, suggesting that these peptides could be promising anti-inflammatory and antimicrobial agents.     

Conformational Flexibility Determines Selectivity and Antibacterial, Antiplasmodial, and Anticancer Potency of Cationic ��-Helical Peptides

We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.     

Molecular basis for membrane selectivity of NK-2, a potent peptide antibiotic derived from NK-lysin

Increasing resistance of pathogenic bacteria against antibiotics is a severe problem in health care. Natural antimicrobial peptides and derivatives thereof have emerged as promising candidates for “new antibiotics”. In contrast to classical antibiotics, these peptides act by direct physical destabilization of the target cell membrane. Nevertheless, they exhibit a high specificity for bacteria over mammalian cells. However, the precise mechanism of action and the molecular basis for membrane selectivity are still a matter of debate. We have designed a new peptide antibiotic (NK-2) with enhanced antimicrobial activity based on an effector protein of mammalian immune cells (NK-lysin). Here we describe the interaction of this α-helical synthetic peptide with membrane mimetic systems, designed to mimic the lipid compositions of mammalian and bacterial cytoplasmic membranes. Utilizing fluorescence and biosensor assays, we could show that on one hand, NK-2 strongly interacts with negatively charged membranes; on the other hand, NK-2 is able to discriminate, without the necessity of negative charges, between the zwitterionic phospholipids phosphatidylethanolamine (PE) and phosphatidylcholine (PC), the major constituents of the outer leaflet of the cytoplasmic membranes of bacteria and mammalian cells, respectively.     

Panurgines, novel antimicrobial peptides from the venom of communal bee Panurgus calcaratus (Hymenoptera: Andrenidae)

Three novel antimicrobial peptides (AMPs), named panurgines (PNGs), were isolated from the venom of the wild bee Panurgus calcaratus. The dodecapeptide of the sequence LNWGAILKHIIK-NH₂ (PNG-1) belongs to the category of α-helical amphipathic AMPs. The other two cyclic peptides containing 25 amino acid residues and two intramolecular disulfide bridges of the pattern Cys8–Cys23 and Cys11–Cys19 have almost identical sequence established as LDVKKIICVACKIXPNPACKKICPK-OH (X=K, PNG-K and X=R, PNG-R). All three peptides exhibited antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria, antifungal activity, and low hemolytic activity against human erythrocytes. We prepared a series of PNG-1 analogs to study the effects of cationicity, amphipathicity, and hydrophobicity on the biological activity. Several of them exhibited improved antimicrobial potency, particularly those with increased net positive charge. The linear analogs of PNG-K and PNG-R having all Cys residues substituted by α-amino butyric acid were inactive, thus indicating the importance of disulfide bridges for the antimicrobial activity. However, the linear PNG-K with all four cysteine residues unpaired, exhibited antimicrobial activity. PNG-1 and its analogs induced a significant leakage of fluorescent dye entrapped in bacterial membrane-mimicking large unilamellar vesicles as well as in vesicles mimicking eukaryotic cell membrane. On the other hand, PNG-K and PNG-R exhibited dye-leakage activity only from vesicles mimicking bacterial cell membrane.     

Membrane-active antimicrobial peptides and human placental lysosomal extracts are highly active against mycobacteria

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, manifests discreet strategies to subvert host immune responses, which enable the pathogen to survive and multiply inside the macrophages. This problem is further worsened by the emergence of multidrug resistant mycobacterial strains, which make most of the anti-tuberculous drugs ineffective. It is thus imperative to search for and design better therapeutic strategies, including employment of new antibiotics. Recently, naturally produced antimicrobial molecules such as enzymes, peptides and their synthetic analogs have emerged as compounds with potentially significant therapeutical applications. Although, many antimicrobial peptides have been identified only very few of them have been tested against mycobacteria. A major limitation in using peptides as therapeutics is their sensitivity to enzymatic degradation or inactivity under certain physiological conditions such as relatively high salt concentration. Here, we show that NK-2, a peptide representing the cationic core region of the lymphocytic effector protein NK-lysin, and Ci-MAM-A24, a synthetic salt-tolerant peptide derived from immune cells of Ciona intestinalis, efficiently kill Mycobacterium smegmatis and Mycobacterium bovis-BCG. In addition, NK-2 and Ci-MAM-A24 showed a synergistic killing effect against M. smegmatis, no cytotoxic effect on mouse macrophages at bactericidal concentrations, and were even found to kill mycobacteria residing inside the macrophages. We also show that human placental lysosomal contents exert potent killing effect against mycobacteria under acidic and reducing growth conditions. Electron microscopic studies demonstrate that the lysosomal extract disintegrate bacterial cell membrane resulting in killing of mycobacteria.     

Role of α-helical conformation of histatin-5 in candidacidal activity examined by proline variants

Human salivary histatin-5 (Hsn-5) is a potent in vitro anticandidal agent. The aim of this study was to investigate the importance of α-helical structure of Hsn-5 for its candidacidal activity. The following three Hsn-5 variants, where one or more functionally nonessential residues were replaced with proline (potent α-helix breaker), were produced by Escherichia coli expression system: H21P (1P), H19P/H21P (2P), and E16P/H19P/H21P (3P). The activities of purified proteins were determined by candidacidal assays, and the secondary structures by circular dichroism (CD) spectroscopy in trifluoroethanol (TFE) that is considered the helix-promoting solvent, and lysophosphatidyl-glycerol (LPG) micelles, the environment that more closely resembles the biological membranes. Our results indicated that 3P variant displayed a candidacidal activity which was similar to that of unaltered Hsn-5 (0P), while 1P and 2P variants showed lower cidal activity. The CD spectra in TFE indicated that 3P variant has less helical characteristics than the 0P, 1P and 2P. These results suggested that the α-helical content of Hsn-5 proline variants does not correlate with the candidacidal activity. Further, the CD spectral analysis of peptides in LPG micelles indicated the formation of β-turn structures in 0P and 3P variants. In conclusion, 3P variant which exhibited comparable candidacidal activity to 0P contains lower percentage of α-helical structure than 1P and 2P variants, which exhibited lower candidacidal activity. This suggests α-helix may not be important for anticandidal activity of Hsn-5.     

Relative spatial positions of tryptophan and cationic residues in helical membrane-active peptides determine their cytotoxicity

The cytotoxic activity of 10 analogs of the idealized amphipathic helical 21-mer peptide (KAAKKAA)3, where three of the Ala residues at different positions have been replaced with Trp residues, has been investigated. The peptide's cytotoxic activity was found to be markedly dependent upon the position of the Trp residues within the hydrophobic sector of an idealized α-helix. The peptides with Trp residues located opposite the cationic sector displayed no antitumor activity, whereas those peptides with two or three Trp residues located adjacent to the cationic sector exhibited high cytotoxic activity when tested against three different cancer cell lines. Dye release experiments revealed that in contrast to the peptides with Trp residues located opposite the cationic sector, the peptides with Trp residues located adjacent to the cationic sector induced a strong permeabilizing activity from liposomes composed of a mixture of zwitterionic phosphatidylcholine and negatively charged phosphatidylserine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS)) (2:1) but not from liposomes composed of zwitterionic phosphatidylcholine, POPC. Fluorescence blue shift and quenching experiments revealed that Trp residues inserted deeper into the hydrophobic environment of POPC/POPS liposomes for peptides with high cytotoxic activity. Through circular dichroism studies, a correlation between the cytotoxic activity and the α-helical propensity was established. Structural studies of one inactive and two active peptides in the presence of micelles using NMR spectroscopy showed that only the active peptides adopted highly coiled to helical structures when bound to a membrane surface.