Design and characterization of novel hybrid antimicrobial peptides based on cecropin A, LL-37 and magainin II

Antimicrobial peptides (AMPs) are a naturally occurring component of the innate immune response of many organisms and can have activity against both Gram-negative and Gram-positive bacterial species. In order to optimize and improve the direct antimicrobial effect of AMPs against a broad spectrum of bacterial species, novel synthetic hybrids were rationally designed from cecropin A, LL-37 and magainin II. AMPs were selected based on their α-helical secondary structure and fragments of these were analyzed and combined in silico to determine which hybrid peptides would form the best amphipathic cationic α-helices. Four hybrid peptides were synthesized (CaLL, CaMA, LLaMA and MALL) and evaluated for direct antimicrobial activity against a range of bacterial species (Bacillus anthracis, Burkholderia cepacia, Francisella tularensis LVS and Yersinia pseudotuberculosis) alongside the original 'parent' AMPs. The hybrid peptides showed greater antimicrobial effects than the parent AMPs (in one case a parent is completely ineffective while a hybrid based on it removes all traces of bacteria by 3h), although they also demonstrated higher hemolytic properties. Modifications were then carried out to the most toxic hybrid AMP (CaLL) to further improve the therapeutic index. Modifications made to the hybrid lowered hemolytic activity and also lowered antimicrobial activity by various degrees. Overall, this work highlights the potential for rational design and synthesis of improved AMPs that have the capability to be used therapeutically for treatment of bacterial infections.     

Comparative Evaluation of the Antimicrobial Activity of Different Antimicrobial Peptides against a Range of Pathogenic Bacteria

Analysis of a Selected Set of Antimicrobial Peptides

The rapid emergence of resistance to classical antibiotics has increased the interest in novel antimicrobial compounds. Antimicrobial peptides (AMPs) represent an attractive alternative to classical antibiotics and a number of different studies have reported antimicrobial activity data of various AMPs, but there is only limited comparative data available. The mode of action for many AMPs is largely unknown even though several models have suggested that the lipopolysaccharides (LPS) play a crucial role in the attraction and attachment of the AMP to the bacterial membrane in Gram-negative bacteria. We compared the potency of Cap18, Cap11, Cap11-1-18m², Cecropin P1, Cecropin B, Bac2A, Bac2A-NH₂, Sub5-NH₂, Indolicidin, Melittin, Myxinidin, Myxinidin-NH₂, Pyrrhocoricin, Apidaecin and Metalnikowin I towards Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Aeromonas salmonicida, Listeria monocytogenes, Campylobacter jejuni, Flavobacterium psychrophilum, Salmonella typhimurium and Yersinia ruckeri by minimal inhibitory concentration (MIC) determinations. Additional characteristics such as cytotoxicity, thermo and protease stability were measured and compared among the different peptides. Further, the antimicrobial activity of a selection of cationic AMPs was investigated in various E. coli LPS mutants.

Cap18 Shows a High Broad Spectrum Antimicrobial Activity

Of all the tested AMPs, Cap18 showed the most efficient antimicrobial activity, in particular against Gram-negative bacteria. In addition, Cap18 is highly thermostable and showed no cytotoxic effect in a hemolytic assay, measured at the concentration used. However, Cap18 is, as most of the tested AMPs, sensitive to proteolytic digestion in vitro. Thus, Cap18 is an excellent candidate for further development into practical use; however, modifications that should reduce the protease sensitivity would be needed. In addition, our findings from analyzing LPS mutant strains suggest that the core oligosaccharide of the LPS molecule is not essential for the antimicrobial activity of cationic AMPs, but in fact has a protective role against AMPs.     

Novel antimicrobial peptides identified from an endoparasitic wasp cDNA library

We screened an endoparasitic wasp (Pteromalus puparum) cDNA library for DNA sequences having antimicrobial activity using a vital dye exclusion assay. Two dozens of clones were isolated that inhibited the growth of host Escherichia coli cells due to expression of the cloned genes. Three peptides (PP13, PP102 and PP113) were synthesized chemically based on the amino acid sequences deduced from these clones and assayed for their antimicrobial activity. These peptides have net positive charges and are active against both Gram‐negative and ‐positive bacteria, but are not active against fungi tested. Their hemolytic activity on human red blood cells was measured, and no hemolytic activity was observed after 1‐h incubation at a concentration of 62.5 µM or below. A Blast search indicated that the three peptides have not been previously characterized as antimicrobial peptides (AMPs). Salt‐dependency studies revealed that the biocidal activity of these peptides against E. coli decreased with increasing concentration of NaCl. Transmission electron microscopic (TEM) examination of PP13‐treated E. coli cells showed extensive damage of cell membranes. The CD spectroscopy studies noted that the enhanced α‐helical characteristics of PP13 strongly contribute to its higher antimicrobial properties. These results demonstrate the feasibility to identify novel AMPs by screening the expressional cDNA library. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Production,purification, and characterization of the cecropin from <Emphasis Type="Italic">Plutella xylostella</Emphasis>, pxCECA1, using an intein-induced self-cleavable system in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Antimicrobial peptides (AMPs) are widely expressed and play an important role in innate immune defense against infectious agents such as bacteria, viruses, fungi, and parasites. Cecropins are a family of AMPs synthesized in the fat body of insects that have proven effective at killing specific pathogens. In order to fulfill their clinical potential as antimicrobial drugs, a simple, cost-effective method to express AMPs is sorely needed. In this study, we expressed and characterized the cecropin from Plutella xylostella (pxCECA1) using an intein-dependent expression system in Escherichia coli. We cloned the pxCECA1 gene from larva by RT-PCR and fused the encoding sequence of mature pxCECA1 with an intein gene and a chitin-binding domain gene (CBD) in pTWIN1 plasmid. The fusion protein CBD–intein–pxCECA1 was expressed in E. coli BL21 (DE3) and separated by flowing cell extracts through a chitin column. Subsequently, self-cleavage of the intein at its C-terminus was induced in a temperature- and pH-dependent manner, resulting in the release of mature pxCECA1. The optimal conditions for self-cleavage were determined to be pH 6.0 for 48 h at 4°C, under which 12.3 mg of recombinant pxCECA1 could be recovered from 1 l of E. coli culture. The purified pxCECA1 displayed antimicrobial activity against a broad variety of gram-positive and gram-negative bacteria. This preparation was especially effective against Staphylococcus aureus, including methicillin-resistant strains. Catalase release assays demonstrated that pxCECA1 acts as a microbicidal agent. These results show for the first time that the IMPACT-TWIN expression system is an efficient, cost-effective way to produce fully functional AMPs and that the AMP pxCECA1 is a novel microbicidal agent with promising therapeutic applications.     

Differential activity of innate defense antimicrobial peptides against <Emphasis Type="Italic">Nocardia</Emphasis> species

Background  

Members of the genus Nocardia are ubiquitous environmental saprophytes capable to cause human pulmonary, disseminated and cutaneous nocardiosis or bovine mastitis. Innate immunity appears to play an important role in early defense against Nocardia species. To elucidate the contribution of antimicrobial peptides (AMPs) in innate defense against Nocardia, the activity of human α-defensins human neutrophil peptides (HNPs) 1-3, human β-defensin (hBD)-3 and cathelicidin LL-37 as well as bovine β-defensins lingual and tracheal antimicrobial peptides (LAP, TAP) and bovine neutrophil-derived indolicidin against four important Nocardia species was investigated.     

Inhibition and destruction of Pseudomonas aeruginosa biofilms by antibiotics and antimicrobial peptides

Pseudomonas aeruginosa is one of the major nosocomial pathogen that can causes a wide variety of acute and chronic infections P. aeruginosa is a dreaded bacteria not just because of the high intrinsic and acquired antibiotic resistance rates but also the biofilm formation and production of multiple virulence factors. We investigated the in vitro activities of antibiotics (ceftazidime, tobramycin, ciprofloxacin, doripenem, piperacillin and colistin) and antimicrobial cationic peptides (AMPs; LL-37, CAMA: cecropin(1–7)-melittin A(2–9) amide, melittin, defensin and magainin-II) alone or in combination against biofilms of laboratory strain ATCC 27853 and 4 clinical strains of P. aeruginosa. The minimum inhibitory concentrations (MIC), minimum bactericidal concentration (MBC) and minimum biofilm eradication concentrations (MBEC) were determined by microbroth dilution technique. The MBEC values of antibiotics and AMPs were 80–>5120 and 640–>640 mg/L, respectively. When combined with the LL-37 or CAMA at 1/10× MBEC, the MBEC values of antibiotics that active against biofilms, were decreased up to 8-fold. All of the antibiotics, and AMPs were able to inhibit the attachment of bacteria at the 1/10× MIC and biofilm formation at 1× or 1/10× MIC concentrations. Time killing curve studies showed 3-log₁₀ killing against biofilms in 24 h with almost all studied antibiotics and AMPs. Synergism were seen in most of the studied combinations especially CAMA/LL-37 + ciprofloxacin against at least one or two strains’ biofilms. Since biofilms are not affected the antibiotics at therapeutic concentrations, using a combination of antimicrobial agents including AMPs, or inhibition of biofilm formation by blocking the attachment of bacteria to surfaces might be alternative methods to fight with biofilm associated infections.     

Broad activity against porcine bacterial pathogens displayed by two insect antimicrobial peptides moricin and cecropin B

In response to infection, insects produce a variety of antimicrobial peptides (AMPs) to kill the invading pathogens. To study their physicochemical properties and bioactivities for clinical and commercial use in the porcine industry, we chemically synthesized the mature peptides Bombyx mori moricin and Hyalophora cecropia cecropin B. In this paper, we described the antimicrobial activity of the two AMPs. Moricin exhibited antimicrobial activity on eight strains tested with minimal inhibitory concentration values (MICs) ranging between 8 and 128 μg/ml, while cecropin B mainly showed antimicrobial activity against the Gramnegative strains with MICs ranging from 0.5 to 16 μg/ml. Compared to the potent antimicrobial activity these two AMPs displayed against most of the bacterial pathogens tested, they exhibited limited hemolytic activity against porcine red blood cells. The activities of moricin and cecropin B against Haemophilus parasuis SH 0165 were studied in further detail. Transmission electron microscopy (TEM) of moricin and cecropin B treated H. parasuis SH 0165 indicated extensive damage to the membranes of the bacteria. Insights into the probable mechanism utilized by moricin and cecropin B to eliminate pathogens are also presented. The observations from this study are important for the future application of AMPs in the porcine industry.     

High level expression and purification of antimicrobial human cathelicidin LL-37 in Escherichia coli

The human antimicrobial peptide LL-37 is a cationic peptide with antimicrobial activity against both Gram-positive and Gram-negative microorganisms. This work describes the development of an expression system based on Escherichia coli capable of high production of the recombinant LL-37. The fusion protein Trx-LL-37 was expressed under control of T7 promoter. The expression of T7 polymerase in the E. coli strain constructed in this work was controlled by regulation mechanisms of the arabinose promoter. The expression plasmid was stabilized by the presence of parB locus which ensured higher homology of the culture during cultivation without antibiotic selection pressure. This system was capable of producing up to 1 g of fusion protein per 1 l of culture. The subsequent semipreparative HPLC allowed us to isolate 40 mg of pure LL-37. LL-37 showed high antimicrobial activity against both Gram-negative and Gram-positive microorganisms. Its activity against Candida albicans was practically nonexistent. Minimal Inhibition Concentration (MIC) determined for E. coli was 1.65 μM; for Staphylococcus aureus 2.31 μM, and for Enterococcus faecalis 5.54 μM. The effects of cathelicidin on E. coli included the ability to permeabilize both cell membranes, as could be observed by the increase of β-galactosidase activity in extracellular space in time. Physiological changes were studied by scanning electron microscopy; Gram-positive microorganisms did not show any visible changes in cell shapes while the changes observed on E. coli cells were evident. The results of this work show that the herein designed expression system is capable of producing adequate quantities of active human antimicrobial peptide LL-37.     

Two novel families of antimicrobial peptides from skin secretions of the Chinese torrent frog, Amolops jingdongensis

The characterization of new natural antimicrobial peptides (AMPs) can help to solve the serious problem of bacterial resistance to currently used antibiotics. In the current study, we analyzed two families of AMPs from the Chinese torrent frog Amolops jingdongensis with a range of bioactivities. The first family of peptides, named jindongenin-1a, is 24 amino acids in length; a BLAST search of jindongenin-1a revealed no sequence similarity with other AMPs. The second family consists of two peptides containing 29 amino acid residues each. These peptides have high sequence similarity with the AMPs of palustrin-2 and are therefore designated palustrin-2AJ1 and palustrin-2AJ2. The cDNA sequences encoding these AMPs have been cloned and the deduced protein sequence of each AMP has been determined by protein sequencing. Sequence and structural analysis showed that each precursor is composed of a putative signal peptide, an N-terminal spacer, a processing site and a disulfide-bridged heptapeptide segment at the C-terminus. We synthesized jindongenin-1a and palustrin-AJ1 to test their antimicrobial, hemolytic, antioxidative and cytotoxic activities. These two peptides showed broad-spectrum antimicrobial activity to standard and clinically isolated strains of bacteria. In addition, they exhibited weak hemolytic activity to human and rabbit erythrocytes under our experimental conditions. Moreover, these peptides also displayed cytotoxic activity against the K562 and HT29 mammalian cell lines and low anti-oxidant activity. These findings provide helpful insight that will be useful in the design of anti-infective peptide agents.     

Efficacy of Indolicidin,Cecropin A (1-7)-Melittin (CAMA) and Their Combination Against Biofilm-Forming Multidrug-Resistant Enteroaggregative Escherichia coli

The present study examined the anti-biofilm efficacy of two short-chain antimicrobial peptides (AMPs), namely, indolicidin and cecropin A (1-7)-melittin (CAMA) against biofilm-forming multidrug-resistant enteroaggregative Escherichia coli (MDR-EAEC) isolates. The typical EAEC isolates re-validated by PCR and confirmed using HEp-2 cell adherence assay was subjected to antibiotic susceptibility testing to confirm its MDR status. The biofilm-forming ability of MDR-EAEC isolates was assessed by Congo red binding, microtitre plate assays and hydrophobicity index; broth microdilution technique was employed to determine minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs). The obtained MIC and MBEC values for both AMPs were evaluated alone and in combination against MDR-EAEC biofilms using crystal violet (CV) staining and confocal microscopy-based live/dead cell quantification methods. All the three MDR-EAEC strains revealed weak to strong biofilm-forming ability and were found to be electron-donating and weakly electron-accepting (hydrophobicity index). Also, highly significant (P < 0.001) time-dependent hydrodynamic growth of the three MDR-EAEC strains was observed at 48 h of incubation in Dulbecco’s modified Eagle’s medium (DMEM) containing 0.45% D-glucose. AMPs and their combination were able to inhibit the initial biofilm formation at 24 h and 48 h as evidenced by CV staining and confocal quantification. Further, the application of AMPs (individually and combination) against the preformed MDR-EAEC biofilms resulted in highly significant eradication (P < 0.001) at 24 h post treatment. However, significant differences were not observed between AMP treatments (individually or in combination). The AMPs seem to be an effective candidates for further investigations such as safety, stability and appropriate biofilm-forming MDR-EAEC animal models.

     

Identification and Characterization of Novel Antibacterial Peptides from Skin Secretions of Euphlyctis cyanophlyctis

In this study, we extracted and purified antimicrobial peptides (AMPs) secreted from skin of Euphlyctis cyanophlyctis using reverse phase-high performance liquid chromatography. Three AMPs were isolated from skin secretions of this frog and sequenced using tandem mass spectrometry. The purified peptides were named buforin-EC (1875.05 ± 0.5 Da), cyanophlyctin (2347.50 ± 0.5 Da) and temporin-ECa (1013.33 ± 0.5 Da). Multiple alignments and homology search showed that buforin-EC, cyanophlyctin and temporin-ECa had a homology of 71.43, 47.1, and 69.23% to buforin II, brevinin-2EC, and temporin-1CSc, respectively. Antimicrobial tests demonstrated that our peptides have a great antimicrobial effect on both gram-positive and gram-negative bacteria. The results indicated that they have an overall minimum inhibitory concentration (MIC) below 13 μM against E. coli. No hemolysis was observed in around of their MIC values. In conclusion, skin secretions of E. cyanophlyctis contain a novel class of AMPs with the proper characteristics.     

A novel cysteine-rich antimicrobial peptide from the mucus of the snail of Achatina fulica

Antimicrobial peptides (AMPs) are important components of the innate immunity. Many antimicrobial peptides have been found from marine mollusks. Little information about AMPs of mollusks living on land is available. A novel cysteine-rich antimicrobial peptide (mytimacin-AF) belonging to the peptide family of mytimacins was purified and characterized from the mucus of the snail of Achatina fulica. Its cDNA was also cloned from the cDNA library. Mytimacin-AF is composed of 80 amino acid residues including 10 cysteines. Mytimacin-AF showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria and the fungus Candida albicans. Among tested microorganisms, it exerted strongest antimicrobial activity against Staphylococcus aureus with a minimal peptide concentration (MIC) of 1.9 μg/ml. Mytimacin-AF had little hemolytic activity against human blood red cells. The current work confirmed the presence of mytimacin-like antimicrobial peptide in land-living mollusks.     

Coupling Molecular Dynamics Simulations with Experiments for the Rational Design of Indolicidin-Analogous Antimicrobial Peptides

Antimicrobial peptides (AMPs) have attracted much interest in recent years because of their potential use as new-generation antibiotics. Indolicidin (IL) is a 13-residue cationic AMP that is effective against a broad spectrum of bacteria, fungi, and even viruses. Unfortunately, its high hemolytic activity retards its clinical applications. In this study, we adopted molecular dynamics (MD) simulations as an aid toward the rational design of IL analogues exhibiting high antimicrobial activity but low hemolysis. We employed long-timescale, multi-trajectory all-atom MD simulations to investigate the interactions of the peptide IL with model membranes. The lipid bilayer formed by the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was chosen as the model erythrocyte membrane; lipid bilayers formed from a mixture of POPC and the negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol were chosen to model bacterial membranes. MD simulations with a total simulation time of up to 4 μs revealed the mechanisms of the processes of IL adsorption onto and insertion into the membranes. The packing order of these lipid bilayers presumably correlated to the membrane stability upon IL adsorption and insertion. We used the degree of local membrane thinning and the reduction in the order parameter of the acyl chains of the lipids to characterize the membrane stability. The order of the mixed 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol/POPC lipid bilayer reduced significantly upon the adsorption of IL. On the other hand, although the order of the pure-POPC lipid bilayer was perturbed slightly during the adsorption stage, the value was reduced more dramatically upon the insertion of IL into the membrane's hydrophobic region. The results imply that enhancing IL adsorption on the microbial membrane may amplify its antimicrobial activity, while the degree of hemolysis may be reduced through inhibition of IL insertion into the hydrophobic region of the erythrocyte membrane. In addition, through simulations, we identified the amino acids that are most responsible for the adsorption onto or insertion into the two model membranes. Positive charges are critical to the peptide's adsorption, whereas the presence of hydrophobic Trp8 and Trp9 leads to its deeper insertion. Combining the hypothetical relationships between the membrane disordering and the antimicrobial and hemolytical activities with the simulated results, we designed three new IL-analogous peptides: IL-K7 (Pro7 → Lys), IL-F89 (Trp8 and Trp9 → Phe), and IL-K7F89 (Pro7 → Lys; Trp8 and Trp9 → Phe). The hemolytic activity of IL-F89 is considerably lower than that of IL, whereas the antimicrobial activity of IL-K7 is greatly enhanced. In particular, the de novo peptide IL-K7F89 exhibits higher antimicrobial activity against Escherichia coli; its hemolytic activity decreased to only 10% of that of IL. Our simulated and experimental results correlated well. This approach—coupling MD simulations with experimental design—is a useful strategy toward the rational design of AMPs for potential therapeutic use.     

Expression and Purification of an Antimicrobial Peptide, Bovine Lactoferricin Derivative LfcinB-W10 in Escherichia coli

Antimicrobial peptides (AMPs) are extremely attractive candidate for therapeutic agents due to their wide spectrum of antimicrobial activity and action mechanism different from antibiotics. In this study, a method using genetic engineering for obtaining an antimicrobial peptide, bovine lactoferricin derivative peptide LfcinB-W10, has been developed. According to the coden usage of Escherichia coli, a gene encoding the peptide was synthesized and a recombinant vector of E. coli expression pGEX-EN-LFW was constructed. The LfcinB-W10 peptide fused with glutathione S-transferase (GST) was successfully expressed and about 20 mg fusion protein with 90% purity was obtained from 1 l culture. The recombinant LfcinB-W10 (rLfcinB-W10) was released from fusion protein by the enterokinase digestion, and about the LfcinB-W10 yield reached 300 μg per 1 l culture. The purified rLfcinB-W10 was found to have growth inhibition activity against Staphylococcus aureus (S. aureus) ATCC25923.     

Antimicrobial Activity of Novel Synthetic Peptides Derived from Indolicidin and Ranalexin against Streptococcus pneumoniae

Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics in order to defeat multidrug-resistant bacteria such as Streptococcus pneumoniae. In this study, thirteen antimicrobial peptides were designed based on two natural peptides indolicidin and ranalexin. Our results revealed that four hybrid peptides RN7-IN10, RN7-IN9, RN7-IN8, and RN7-IN6 possess potent antibacterial activity against 30 pneumococcal clinical isolates (MIC 7.81-15.62µg/ml). These four hybrid peptides also showed broad spectrum antibacterial activity (7.81µg/ml) against S. aureus, methicillin resistant S. aureus (MRSA), and E. coli. Furthermore, the time killing assay results showed that the hybrid peptides were able to eliminate S. pneumoniae within less than one hour which is faster than the standard drugs erythromycin and ceftriaxone. The cytotoxic effects of peptides were tested against human erythrocytes, WRL-68 normal liver cell line, and NL-20 normal lung cell line. The results revealed that none of the thirteen peptides have cytotoxic or hemolytic effects at their MIC values. The in silico molecular docking study was carried out to investigate the binding properties of peptides with three pneumococcal virulent targets by Autodock Vina. RN7IN6 showed a strong affinity to target proteins; autolysin, pneumolysin, and pneumococcal surface protein A (PspA) based on rigid docking studies. Our results suggest that the hybrid peptides could be suitable candidates for antibacterial drug development.     

The functional interaction between abaecin and pore-forming peptides indicates a general mechanism of antibacterial potentiation

Long-chain proline-rich antimicrobial peptides such as bumblebee abaecin show minimal activity against Gram-negative bacteria despite binding efficiently to specific intracellular targets. We recently reported that bumblebee abaecin interacts with Escherichia coli DnaK but shows negligible antibacterial activity unless it is combined with sublethal doses of the pore-forming peptide hymenoptaecin. These two bumblebee peptides are co-expressed in vivo in response to a bacterial challenge. Here we investigated whether abaecin interacts similarly with pore-forming peptides from other organisms by replacing hymenoptaecin with sublethal concentrations of cecropin A (0.3 μM) or stomoxyn (0.05 μM). We found that abaecin increased the membrane permeabilization effects of both peptides, confirming that it can reduce the minimal inhibitory concentrations of pore-forming peptides from other species. We also used atomic force microscopy to show that 20 μM abaecin combined with sublethal concentrations of cecropin A or stomoxyn causes profound structural changes to the bacterial cell surface. Our data indicate that the potentiating functional interaction between abaecin and pore-forming peptides is not restricted to specific co-expressed peptides from the same species but is likely to be a general mechanism. Combination therapies based on diverse insect-derived peptides could therefore be used to tackle bacteria that are recalcitrant to current antibiotics.     

Folding profiles of antimicrobial scorpion venom-derived peptides on hydrophobic surfaces: a molecular dynamics study

Abstract

Most helical antimicrobial peptides (AMPs) are usually unfolded in aqueous solution; however they acquire their secondary structure in the presence of a hydrophobic environment such as lipid membranes. Being the biological membranes the main target of many AMPs it is necessary to understand their way of action. Pandinin 2 (Pin2) is an alpha-helical AMP isolated from the venom of the African scorpion Pandinus imperator which shows high antimicrobial activity against Gram-positive bacteria and it is less active against Gram-negative bacteria, nevertheless, it has strong hemolytic activity. Its chemically synthesized Pin2GVG analog has low hemolytic activity while keeping its antimicrobial activity. With the aim of exploring the partition and subsequent folding of these peptides, in this work we report the results of extensive molecular dynamics simulations of Pin2 and Pin2GVG peptides in the presence of 2 hydrophobic environments such as dodecyl-phosphocholine (DPC) micelle and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) membrane. Our results indicate that Pin2 folds in DPC with a 79% of alpha-helical content, which is in agreement with the experimental results, while in POPC it has 62.5% of alpha-helical content. On the other hand, Pin2GVG presents a higher percentage of alpha-helical structure in POPC and a smaller content in DPC when compared with Pin2. These results can help to better choose the starting structures in future molecular dynamics simulations of AMPs, because these peptides can adopt slightly different conformations depending on the hydrophobic environment.

Communicated by Ramaswamy H. Sarma     

Antimicrobial and antibiofilm activity of cathelicidins and short, synthetic peptides against Francisella

Francisella infects the lungs causing pneumonic tularemia. Focusing on the lung’s host defense, we have examined antimicrobial peptides as part of the innate immune response to Francisella infection. Interest in antimicrobial peptides, such as the cathelicidins, has grown due their potential therapeutic applications and the increasing problem of bacterial resistance to commonly used antibiotics. Only one human cathelicidin, LL-37, has been characterized. Helical cathelicidins have also been discovered in snakes including the Chinese King Cobra, Naja atra (NA-CATH). Four synthetic 11-residue peptides (ATRA-1, -2, -1A and -1P) containing variations of a repeated motif within NA-CATH were designed. We hypothesized that these smaller synthetic peptides could have excellent antimicrobial effectiveness with shorter length (and less cost), making them strong potential candidates for development into broad-spectrum antimicrobial compounds. We tested the susceptibility of F. novicida to four ATRA peptides, LL-37, and NA-CATH. Two of the ATRA peptides had high antimicrobial activity (μM), while the two proline-containing ATRA peptides had low activity. The ATRA peptides did not show significant hemolytic activity even at high peptide concentration, indicating low cytotoxicity against host cells. NA-CATH killed Francisella bacteria more quickly than LL-37. However, LL-37 was the most effective peptide against F. novicida (EC50 = 50 nM). LL-37 mRNA was induced in A549 cells by Francisella infection. We recently demonstrated that F. novicida forms in vitro biofilms. LL-37 inhibited F. novicida biofilm formation at sub-antimicrobial concentrations. Understanding the properties of these peptides, and their endogenous expression in the lung could lead to potential future therapeutic interventions for this lung infection.     

Diversity and exploration of bioactive marine actinomycetes in the Bay of Bengal of the Puducherry coast of India

The present study was designed to investigate the Puducherry coast of the Bay of Bengal, India for the diversity of bioactive actinomycetes. A total of 50 actinomycete strains were isolated from the marine sediments and most of the strains were belongs to Streptomyces. These strains were identified by means of morphological physiological, biochemical and cultural characteristics. The isolates were subjected to shake flask fermentation and the secondary metabolites were extracted with ethyl acetate and screened for cytotoxicity, hemolytic activity and antimicrobial activity against selected bacterial and fungal pathogens. The cytotoxic activity was evaluated using HeLa cell lines by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole (MTT) assay, hemolytic activity on mouse erythrocytes and the antifungal activity was evaluated by MTT cytotoxic assay against Aspergillus niger, Aspergillus fumigatus and Candida albicans. The antibacterial activity was studied against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Klebsiella pneumoniae. The cytotoxicity and antimicrobial activity of secondary metabolite was found to be concentration dependent and nearly 24% of isolates showed significant antimicrobial, hemolytic and cytotoxic activity. The results of our study indicate the diversity and bioactive potential of marine actinomycetes isolated in the Puducherry coast.