Identification and characterization of a hexapeptide with activity against phytopathogenic fungi that cause postharvest decay in fruits

A hexapeptide of amino acid sequence Ac-Arg-Lys-Thr-Trp-Phe-Trp-NH2 was demonstrated to have antimicrobial activity against selected phytopathogenic fungi that cause postharvest decay in fruits. The peptide synthesized with either all D- or all L-amino acids inhibited the in vitro growth of strains of Penicilium italicum, P. digitatum, and Botrytis cinerea, with MICs of 60 to 80 microM and 50% inhibitory concentration (IC50) of 30 to 40 microM. The inhibitory activity of the peptide was both sequence- and fungus-specific since (i) sequence-related peptides lacked activity (including one with five residues identical to the active sequence), (ii) other filamentous fungi (including some that belong to the genus Penicllium) were insensitive to the peptide's antifungal action, and (iii) the peptide did not inhibit the growth of several yeast and bacterial strains assayed. Experiments on P. digitatum identified conidial germination as particularly sensitive to inhibition although mycelial growth was also affected. Our findings suggest that the inhibitory effect is initially driven by the electrostatic interaction of the peptide with fungal components. The antifungal peptide retarded the blue and green mold diseases of citrus fruits and the gray mold of tomato fruits under controlled inoculation conditions, thus providing evidence for the feasibility of using very short peptides in plant protection. This and previous studies with related peptides indicate some degree of peptide amino acid sequence and structure conservation associated with the antimicrobial activity, and suggest a general sequence layout for short antifungal peptides, consisting of one or two positively charged residues combined with aromatic amino acid residues.     

Parallel evaluation of antimicrobial peptides derived from the synthetic PAF26 and the human LL37

The antimicrobial hexapeptide PAF26 was de novo designed towards phytopathogenic fungi of agricultural importance. To analyze its clinical potential, the activity of PAF26 has been determined against several microorganisms of clinical relevance including Staphylococcus, Candida, and several dermatophytes. For comparison purposes, the peptides KR20 and KI26 derived from the human cathelicidin LL37 were selected and fungal pathogens of agronomic relevance were included. PAF26 has similar antimicrobial activity in vitro compared to KR20 despite their different lengths and amino acid compositions. Moreover, neither peptide is lytic to human erythrocytes or keratinocytes. The hybrid peptide PAF26:KR20 showed better antimicrobial properties than the original peptides against most of the pathogens tested. The structural properties of PAF26:KR20 compared to related 26-amino acid peptides support the idea that the increment in toxicity correlates with positive charge and hydrophobicity. However, the degree of peptide helicity was not a predictor of antimicrobial activity.     

Characterization of antimicrobial peptides against a US strain of the rice pathogen Rhizoctonia solani

AIM: To identify antimicrobial peptides with high lytic activity against Rhizoctonia solani strain LR172, causal agent of rice sheath blight and aerial blight of soyabeans in the US. METHODS AND RESULTS: Among 12 natural and synthetic antimicrobial peptides tested in vitro, the wheat-seed peptide, purothionin, showed the strongest inhibitory activity that was similar to the antifungal antibiotics, nystatin and nikkomycin Z. Cecropin B, a natural peptide from cecropia moth, and synthetic peptide D4E1 produced the highest inhibitory activity against R. solani among linear peptides. Membrane permeabilization levels strongly correlated with antifungal activity of the peptides. Noticeable changes in membrane integrity were observed at concentrations of >/=0.5 micromol l(-1) for purothionin, 2 micromol l(-1) for cecropin B, D4E1, D2A21, melittin, and phor21, and 8 micromol l(-1) for magainin II and phor14. An increase of nuclear membrane permeabilization was observed in fungal cells treated with cecropin B, but not with purothionin. Diffusion of nuclear content was observed by fluorescent microscopy 10 min after adding a lethal concentration of cecropin B. Evaluation by electron microscopy confirmed severe cytoplasmic degradation and plasma membrane vesiculation. Purothionin and cecropin B were the most stable against proteolytic degradation when added to liquid cultures of R. solani. CONCLUSIONS: Purothionin, cecropin B, D4E1 and phor21 were shown to exhibit high in vitro lytic activity against R. solani strain LR172 for rice and soyabean. These peptides are greater than 16 amino acids long and rapidly increase fungal membrane permeabilization. Resistance to proteolysis is important for sufficient antifungal activity of antimicrobial peptides. SIGNIFICANCE AND IMPACT OF THE STUDY: Selected antimicrobial peptides offer an attractive alternative to traditional chemicals that could be utilized in molecular breeding to develop crops resistant to rice sheath blight and aerial blight of soyabean.     

Cationic amphipathic peptides, derived from bovine and human lactoferrins, with antimicrobial activity against oral pathogens

Peptides derived from the N-terminal domain that comprises an amphipathic alpha-helix in human lactoferrin (LFh 18-31 and LFh 20-38) and bovine lactoferrin (LFb 17-30 and LFb 19-37) were chemically synthesised. Since many positively charged amphipathic alpha-helices contain antimicrobial activity, the peptides were tested for their antimicrobial activity against various oral pathogens. Both peptides from bovine lactoferrin had more potent antimicrobial activities than the human equivalents. Peptide LFb 17-30, containing the largest number of positively charged amino acids, showed the highest antimicrobial activity to both Gram-positive and Gram-negative bacteria. Since native lactoferrin molecules had no killing activity, release of these peptides from the native protein should be investigated to explore the use in oral care products.     

Effect of water activity and temperature on germination and growth of Penicillium digitatum,P. italicum and Geotrichum candidum

AIMS: This study compares the effect of temperature (4-37 degrees C) and water activity (aw: 0.99-0.87) and their interactions on the germination rates, lag times prior to germination and mycelial growth 'in vitro' of Penicillium digitatum, P. italicum and Geotrichum candidum, the main postharvest pathogens affecting citrus fruits. METHODS AND RESULTS: Germination and growth were markedly influenced by temperature and aw. Generally, lag times were longer and germination and growth rates were slower when conditions of temperature and aw were far from optimum. All the studied species were able to germinate over a range of 4-30 degrees C at 0.995 aw, although in non-optimal conditions P. digitatum only reached 40-60% of germinated conidia. At low temperatures, P. italicum germinated and grew faster than P. digitatum and G. candidum, particularly at 0.95 aw. Penicillium italicum was also able to germinate and grow in the driest studied conditions (0.87 aw), while G. candidum did not germinate under 0.95 aw. CONCLUSIONS: Knowledge of the ecological requirements of these fungi is important in order to understand their behaviour in natural situations and to predict fungal spoilage on citrus fruits.     

Comparative antimicrobial activity and mechanism of action of bovine lactoferricin-derived synthetic peptides

Lactoferricin B (LfcinB), a 25 residue peptide derived from the N-terminal of bovine lactoferrin (bLF), causes depolarization of the cytoplasmic membrane in susceptible bacteria. Its mechanism of action, however, still needs to be elucidated. In the present study, synthetic LfcinB (without a disulfide bridge) and LfcinB (C–C; with a disulfide bridge) as well as three derivatives with 15-, 11- and 9-residue peptides were prepared to investigate their antimicrobial nature and mechanisms. The antimicrobial properties were measured via minimum inhibitory concentration (MIC) determinations, killing kinetics assays and synergy testing, and hemolytic activities were assessed by hemoglobin release. Finally, the morphology of peptide-treated bacteria was determined by atomic force microscopy (AFM). We found that there was no difference in MICs between LfcinB and LfcinB (C–C). Among the derivatives, only LfcinB15 maintained nearly the same level as LfcinB, in the MIC range of 16–128 μg/ml, and the MICs of LfcinB11 (64–256 μg/ml) were 4 times more than LfcinB, while LfcinB9 exhibited the lowest antimicrobial activity. When treated at MIC for 1 h, many blebs were formed and holes of various sizes appeared on the cell surface, but the cell still maintained its integrity. This suggested that LfcinB had a major permeability effect on the cytoplasmic membrane of both Gram-positive and Gram-negative bacteria, which also indicated it may be a possible intracellular target. Among the tested antibiotics, aureomycin increased the bactericidal activity of LfcinB against E. coli, S. aureus and P. aeruginosa, but neomycin did not have such an effect. We also found that the combination of cecropin A and LfcinB had synergistic effects against E. coli.     

Sensitivity of bacterial and fungal plant pathogens to the lytic peptides,MSI-99, magainin II,and cecropin B

In vitro and leaf disk assays of bacterial and fungal plant pathogens were conducted using three cationic lytic peptides, MSI-99, magainin II (MII), and cecropin B (CB). Growth of bacterial organisms was retarded or completely inhibited by low concentrations of these lytic peptides. The peptides also significantly reduced germination of fungal spores and growth of mycelia; however, higher concentrations of peptides were needed to inhibit fungal growth compared with those needed to inhibit bacteria. The relative efficacy of the peptides depended on the microorganism tested, but CB was the most inhibitory to the majority of the bacteria and fungi assayed. MSI-99, a synthetic derivative of MII with increased positive charge, showed equal or two- to fivefold higher antibacterial activity compared to MII in the in vitro assays. MSI-99 was also superior to MII against the oomycete, Phytophthora infestans but was slightly inferior to MII in assays with the true fungi, Penicillium digitatum and Alternaria solani. In the leaf disk assays, pretreating spores of Alternaria solani and Phytophthora infestans with the peptides at concentrations as low as 10 microg per ml led to significant reductions in the size of early blight lesions and prevented development of any late blight lesions on tomato leaf disks. Our results from in vitro and leaf disk assays suggest that MSI-99 can be used as a transgene to generate tomato lines with enhanced resistance to bacterial and fungal diseases of this crop.     

Design of novel analogue peptides with potent antibiotic activity based on the antimicrobial peptide,HP (2-20), derived from N-terminus of Helicobacter pylori ribosomal protein L1

HP (2-20) (AKKVFKRLEKLFSKIQNDK) is the antimicrobial sequence derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RPL1). In order to develop novel antibiotic peptides useful as therapeutic agents, potent antibiotic activities against bacteria, fungi and cancer cells without a cytotoxic effect are essential. To this end, several analogues with amino acid substitutions were designed to increase or decrease only the net hydrophobicity. In particular, the substitution of Trp for the hydrophobic amino acids, Gln and Asp at positions 17 and 19 of HP (2-20) (Anal 3), caused a dramatic increase in antibiotic activity without a hemolytic effect.In contrast, the decrease of hydrophobicity brought about by substituting Ser for Leu and Phe at positions 12 and 19 of HP (2-20), respectively (Anal 4, Anal 5), did not have a significant effect on the antibiotic activity. The antibiotic effects of these synthetic peptides were further investigated by treating prepared protoplasts of Candida albicans and conducting an artificial liposomal vesicle (PC/PS; 3:1, w/w) disrupting activity test. The results demonstrated that the Anal 3 prevented the regeneration of fungal cell walls and induced an enhanced release of fluorescent dye (carboxyfluorescein) trapped in the artificial membrane vesicles to a greater degree than HP (2-20).The potassium-release test conducted on C. albicans indicated that Anal 3 induced greater amounts of potassium ion to be released than the parent peptide, HP (2-20) did. These results indicated that the hydrophobic region of peptides is prerequisite for its effective antibiotic activity and may facilitate easy penetration of the lipid bilayers of the cell membrane.     

Antimicrobial properties of derivatives of the cationic tryptophan-rich hexapeptide PAF26

Short antimicrobial peptides represent an alternative to fight pathogen infections. PAF26 is a hexapeptide identified previously by a combinatorial approach against the fungus Penicillium digitatum and shows antimicrobial properties towards certain phytopathogenic fungi. In this work, PAF26 was used as lead compound and its properties were compared with two series of derivatives, obtained by either systematic alanine substitution or N-terminal amino acid addition. The alanine scan approach underlined the optimized sequence of PAF26 in terms of potency and permeation capability, and also the higher contribution of the cationic residues to these properties. The N-terminal addition of amino acids resulted in new heptapeptides with variations in their antimicrobial characteristics, and very low cytolysis to human red blood cells. Positive (Arg or Lys) and aromatic (Phe or Trp) residue addition increased broad spectrum activity of PAF26. Noteworthy, addition of selected residues had specific effects on the properties of derivatives of PAF26.     

Structural studies and model membrane interactions of two peptides derived from bovine lactoferricin.

The powerful antimicrobial properties of bovine lactoferricin (LfcinB) make it attractive for the development of new antimicrobial agents. An 11-residue linear peptide portion of LfcinB has been reported to have similar antimicrobial activity to lactoferricin itself, but with lower hemolytic activity. The membrane-binding and membrane-perturbing properties of this peptide were studied together with an amidated synthetic version with an added disulfide bond, which was designed to confer increased stability and possibly activity. The antimicrobial and cytotoxic properties of the peptides were measured against Staphylococcus aureus and Escherichia coli and by hemolysis assays. The peptides were also tested in an anti-cancer assay against neuroblastoma cell lines. Vesicle disruption caused by these LfcinB derivatives was studied using the fluorescent reporter molecule calcein. The extent of burial of the two Trp residues in membrane mimetic environments were quantitated by fluorescence. Finally, the solution NMR structures of the peptides bound to SDS micelles were determined to provide insight into their membrane bound state. The cyclic peptide was found to have greater antimicrobial potency than its linear counterpart. Consistent with this property, the two Trp residues of the modified peptide were suggested to be embedded deeper into the membrane. Although both peptides adopt an amphipathic structure without any regular alpha-helical or beta-sheet conformation, the 3D-structures revealed a clearer partitioning of the cationic and hydrophobic faces for the cyclic peptide.     

Purification and primary structure of two isoforms of arenicin, a novel antimicrobial peptide from marine polychaeta Arenicola marina

Two novel 21-residue antimicrobial peptides, arenicin-1 and arenicin-2, exhibiting activity against Gram-positive and Gram-negative bacteria and fungi, were purified from coelomocytes of marine polychaeta Arenicola marina (lugworm) by preparative gel electrophoresis and RP-HPLC. Molecular masses (2758.3 and 2772.3 Da) and complete amino acid sequences (RWCVYAYVRVRGVLVRYRRCW and RWCVYAYVRIRGVLVRYRRCW) were determined for each isoform. Each arenicin has one disulfide bond (Cys3-Cys20). The total RNA was isolated from the lugworm coelomocytes, RT-PCR and cloning were performed, and cDNA was sequenced. A 202-residue preproarenicin contains a putative signal peptide (25 amino acids) and a long prodomain. Arenicins have no structure similarity to any previously identified antimicrobial peptides.     

Design and characterization of short antimicrobial peptides using leucine zipper templates with selectivity towards microorganisms

Design of antimicrobial peptides with selective activity towards microorganisms is an important step towards the development of new antimicrobial agents. Leucine zipper sequence has been implicated in cytotoxic activity of naturally occurring antimicrobial peptides; moreover, this motif has been utilized for the design of novel antimicrobial peptides with modulated cytotoxicity. To understand further the impact of substitution of amino acids at ‘a’ and/or ‘d’ position of a leucine zipper sequence of an antimicrobial peptides on its antimicrobial and cytotoxic properties four short peptides (14-residue) were designed on the basis of a leucine zipper sequence without or with replacement of leucine residues in its ‘a’ and ‘d’ positions with d-leucine or alanine or proline residue. The original short leucine zipper peptide (SLZP) and its d-leucine substituted analog, DLSA showed comparable activity against the tested Gram-positive and negative bacteria and the fungal strains. The alanine substituted analog (ASA) though showed appreciable activity against the tested bacteria, it showed to some extent lower activity against the tested fungi. However, the proline substituted analog (PSA) showed lower activity against the tested bacterial or fungal strains. Interestingly, DLSA, ASA and PSA showed significantly lower cytotoxicity than SLZP against both human red blood cells (hRBCs) and murine 3T3 cells. Cytotoxic and bactericidal properties of these peptides matched with peptide-induced damage/permeabilization of mammalian cells and bacteria or their mimetic lipid vesicles suggesting cell membrane could be the target of these peptides. As evidenced by tryptophan fluorescence and acrylamide quenching studies the peptides showed similarities either in interaction or in their localization within the bacterial membrane mimetic negatively charged lipid vesicles. Only SLZP showed localization inside the mammalian membrane mimetic zwitterionic lipid vesicles. The results show significant scope for designing antimicrobial agents with selectivity towards microorganisms by substituting leucine residues at ‘a’ and/or ‘d’ positions of a leucine zipper sequence of an antimicrobial peptide with different amino acids.     

In vitro effects of water activity, temperature and solutes on the growth rate of P. italicum Wehmer and P. digitatum Sacc

AIMS: To evaluate the effect of water activity (a(w) 0.98-0.89, adjusted with glycerol, sorbitol, glucose, or NaCl) and temperature (5-25 degrees C) on the lag phase and radial growth rate (mm day(-1)) of the important citrus spoilage fungi, such as Penicillium italicum and Penicillium digitatum grown in potato dextrose agar (PDA) medium. To select, among models based on the use of different solutes, a model fitting accurately the growth of these species in relation to a(w) and temperature. METHODS AND RESULTS: Extensive data analyses showed for both Penicillium species a highly significant effect of a(w), temperature, solutes and their interactions on radial growth rate (P < 0.0001). Radial growth rate was inhibited and the lag phase (i.e. the time required for growth) lengthened as the a(w) of the medium decreased. NaCl appeared to causes the greatest stress on growth when compared with other nonionic solutes. Penicillium italicum stopped growing at 0.96 a(w) and P. digitatum at 0.93 a(w). Under the dry conditions where growth was observed, P. italicum grew faster than P. digitatum at low temperature and P. digitatum remained more active at ambient temperature. Multiple regression analysis applied to the square roots of the growth rates observed in the presence of each solute showed that both the 'glycerol model' and the 'sorbitol model' yielded a good prediction of P. italicum growth and the 'sorbitol model' gave an accurate fit for P. digitatum growth, offering high-quality prediction within the experimental limits described. CONCLUSIONS: Mathematical models describing and predicting, as a function of a(w) and temperature, the square root of the radial growth rate of the agents responsible for blue and green decays are important tools for understanding the behaviour of these fungi under natural conditions and for predicting citrus fruit spoilage. SIGNIFICANCE AND IMPACT OF THE STUDY: Implementation of these results should contribute towards a more rational control strategy against citrus spoilage fungi.     

Interactions of bovine lactoferricin with acidic phospholipid bilayers and its antimicrobial activity as studied by solid-state NMR

Bovine lactoferricin (LfcinB) is an antimicrobial peptide released by pepsin cleavage of lactoferrin. In this work, the interaction between LfcinB and acidic phospholipid bilayers with the weight percentage of 65% dimyristoylphosphatidylglycerol (DMPG), 10% cardiolipin (CL) and 25% dimyristoylphosphatidylcholine (DMPC) was investigated as a mimic of cell membrane of Staphylococcus aureus by means of quartz crystal microbalance (QCM) and solid-state (31)P and (1)H NMR spectroscopy. Moreover, we elucidated a molecular mechanism of the antimicrobial activity of LfcinB by means of potassium ion selective electrode (ISE). It turned out that affinity of LfcinB for acidic phospholipid bilayers was higher than that for neutral phospholipid bilayers. It was also revealed that the association constant of LfcinB was larger than that of lactoferrin as a result of QCM measurements. (31)P DD-static NMR spectra indicated that LfcinB interacted with acidic phospholipid bilayers and bilayer defects were observed in the bilayer systems because isotropic peaks were clearly appeared. Gel-to-liquid crystalline phase transition temperatures (Tc) in the mixed bilayer systems were determined by measuring the temperature variation of relative intensities of acyl chains in (1)H MAS NMR spectra. Tc values of the acidic phospholipid and LfcinB-acidic phospholipid bilayer systems were 21.5 degrees C and 24.0 degrees C, respectively. To characterize the bilayer defects, potassium ion permeation across the membrane was observed by ISE measurements. The experimental results suggest that LfcinB caused pores in the acidic phospholipid bilayers. Because these pores lead the permeability across the membrane, the molecular mechanism of the antimicrobial activity could be attributed to the pore formation in the bacterial membrane induced by LfcinB.     

Structure-activity relationship of buffalo antibacterial hepcidin analogs

Hepcidin is an anti-microbial peptide expressed predominantly in the liver of many species. Based on the amino acid sequence deduced from buffalo (Bubalus bubalis) hepcidin cDNA (Accession no. EU399814), six peptides Hepc(1-25), Hepc(6-25), Hepc(7-25), Hepc(9-25), Hepc(11-25) and Hepc(15-25) were synthesized using solid-phase fluorenylmethoxycarbonyl (Fmoc) chemistry. CD spectroscopy revealed different spectra of the peptides in different solvents and in all the cases beta-structure was found to be dominant with less alpha-helix as predicted. Quantitation of secondary structure indicated the highest beta-structure for all the six peptides in SDS solution, when used as mimetic for membrane-like environment. The CD spectra of all the peptides taken in water showed that degree of randomness decreased with increase in chain length of the peptide. Out of the six peptides, only Hepc(1-25), Hepc(6-25) and Hepc(7-25) showed antibacterial activity against Staphylococcus aureus (Gram-positive bacteria). The peptides did not show any sensitivity toward E. coli (Gram-negative bacteria). Minimum inhibitory concentration (MIC) showed the lowest value for Hepc(7-25) as an antibacterial agent, followed by Hepc(6-25) and Hepc(1-25). The peptides Hepc(9-25), Hepc(11-25) and Hepc(15-25) with more random structure did not show any antimicrobial activity The study demonstrated that 5 amino acids at N-terminal in buffalo hepcidin can be truncated without loss of antimicrobial activity and further reduction of length of the analog from 20 to 19 amino acids resulted increase in the activity because of increase in beta-structure of the peptide shown by CD spectroscopy.     

Antibiotic activity of reversed peptides of alpha-helical antimicrobial peptide,P18

P18 (KWKLFKKIPKFLHLAKKF-NH(2)), an a-helical antimicrobial peptide designed from cecropin Amagainin 2 hybrid, was known to have potent antimicrobial activity against bacteria as well as fungi without hemolytic activity. To find the peptides comparable or superior to the antimicrobial activity of P18, the two reversed peptides (Rev-1 and Rev-2) of P18 were designed and synthesized. These peptides were found to have similar antimicrobial activity against bacterial and fungal cells without hemolytic activity as compared with P18. Furthermore, a reversed peptide, Rev-2 was shown to have a two-fold higher activity in killing some bacterial cells than P18. Therefore, these results suggested that Rev-2 peptide seems to be an excellent candidate for developing novel peptide antibiotics.     

Design of novel analogue peptides with potent antibiotic activity based on the antimicrobial peptide,HP (2–20), derived from N-terminus of Helicobacter pylori ribosomal protein L1

HP (2–20) (AKKVFKRLEKLFSKIQNDK) is the antimicrobial sequence derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RPL1). In order to develop novel antibiotic peptides useful as therapeutic agents, potent antibiotic activities against bacteria, fungi and cancer cells without a cytotoxic effect are essential. To this end, several analogues with amino acid substitutions were designed to increase or decrease only the net hydrophobicity. In particular, the substitution of Trp for the hydrophobic amino acids, Gln and Asp at positions 17 and 19 of HP (2–20) (Anal 3), caused a dramatic increase in antibiotic activity without a hemolytic effect.In contrast, the decrease of hydrophobicity brought about by substituting Ser for Leu and Phe at positions 12 and 19 of HP (2–20), respectively (Anal 4, Anal 5), did not have a significant effect on the antibiotic activity. The antibiotic effects of these synthetic peptides were further investigated by treating prepared protoplasts of Candida albicans and conducting an artificial liposomal vesicle (PC/PS; 3:1, w/w) disrupting activity test. The results demonstrated that the Anal 3 prevented the regeneration of fungal cell walls and induced an enhanced release of fluorescent dye (carboxyfluorescein) trapped in the artificial membrane vesicles to a greater degree than HP (2–20).The potassium-release test conducted on C. albicans indicated that Anal 3 induced greater amounts of potassium ion to be released than the parent peptide, HP (2–20) did. These results indicated that the hydrophobic region of peptides is prerequisite for its effective antibiotic activity and may facilitate easy penetration of the lipid bilayers of the cell membrane.     

Structure-activity relationships of cecropin-like peptides and their interactions with phospholipid membrane

Cecropin A and papiliocin are novel 37-residue cecropin-like antimicrobial peptides isolated from insect. We have confirmed that papiliocin possess high bacterial cell selectivity and has an α-helical structure from Lys³ to Lys²¹ and from Ala²⁵ to Val³⁵, linked by a hinge region. In this study, we demonstrated that both peptides showed high antimicrobial activities against multi-drug resistant Gram negative bacteria as well as fungi. Interactions between these cecropin-like peptides and phospholipid membrane were studied using CD, dye leakage experiments, and NMR experiments, showing that both peptides have strong permeabilizing activities against bacterial cell membranes and fungal membranes as well as Trp² and Phe⁵ at the N-terminal helix play an important role in attracting cecropin-like peptides to the negatively charged bacterial cell membrane. Cecropin-like peptides can be potent peptide antibiotics against multi-drug resistant Gram negative bacteria and fungi. [BMB Reports 2013; 46(5): 282-287]     

Synthesis, spectral analysis and in vitro microbiological evaluation of novel ethyl 4-(naphthalen-2-yl)-2-oxo-6-arylcyclohex-3-enecarboxylates and 4,5-dihydro-6-(napthalen-2-yl)-4-aryl-2H-indazol-3-ols

A series of ethyl 4-(naphthalen-2-yl)-2-oxo-6-arylcyclohex-3-enecarboxylates 8-14 and 4,5-dihydro-6-(naphthalen-2-yl)-4-aryl-2H-indazol-3-ols 15-21 were synthesised and characterised by their spectroscopic data. In vitro microbiological evaluations were carried out for all the newly synthesised compounds 8-21 against clinically isolated bacterial and fungal strains. Compounds 9, 12 and 20 against Staphylococcus aureus, 10, 12, 20 against β-haemolytic streptococcus, 11, 17 against Bacillus subtilis, 12, 16 and 20 against Vibreo cholerae, 13, 16 against Escherichia coli, 13, 16, 18, 19 against Salmonella typhii, 12, 18 against Shigella flexneri, 10 against Salmonella typhii, 10, 13, 17, 18 against Aspergillus flavus, 12, 17, 21 against Aspergillus niger, 12, 15, 17, 18, 20 against Mucor, Rhizopus and Microsporeum gypsuem exhibit potent antimicrobial activity.     

Interactions of bovine lactoferricin with acidic phospholipid bilayers and its antimicrobial activity as studied by solid-state NMR

Bovine lactoferricin (LfcinB) is an antimicrobial peptide released by pepsin cleavage of lactoferrin. In this work, the interaction between LfcinB and acidic phospholipid bilayers with the weight percentage of 65% dimyristoylphosphatidylglycerol (DMPG), 10% cardiolipin (CL) and 25% dimyristoylphosphatidylcholine (DMPC) was investigated as a mimic of cell membrane of Staphylococcus aureus by means of quartz crystal microbalance (QCM) and solid-state ³¹P and ¹H NMR spectroscopy. Moreover, we elucidated a molecular mechanism of the antimicrobial activity of LfcinB by means of potassium ion selective electrode (ISE). It turned out that affinity of LfcinB for acidic phospholipid bilayers was higher than that for neutral phospholipid bilayers. It was also revealed that the association constant of LfcinB was larger than that of lactoferrin as a result of QCM measurements. ³¹P DD-static NMR spectra indicated that LfcinB interacted with acidic phospholipid bilayers and bilayer defects were observed in the bilayer systems because isotropic peaks were clearly appeared. Gel-to-liquid crystalline phase transition temperatures (Tc) in the mixed bilayer systems were determined by measuring the temperature variation of relative intensities of acyl chains in ¹H MAS NMR spectra. Tc values of the acidic phospholipid and LfcinB-acidic phospholipid bilayer systems were 21.5 °C and 24.0 °C, respectively. To characterize the bilayer defects, potassium ion permeation across the membrane was observed by ISE measurements. The experimental results suggest that LfcinB caused pores in the acidic phospholipid bilayers. Because these pores lead the permeability across the membrane, the molecular mechanism of the antimicrobial activity could be attributed to the pore formation in the bacterial membrane induced by LfcinB.