A Comparison of Antibacterial Properties of Tachyplesin, Thanatin, and Enterocin P on Enterococcus faecalis.

OBJECTIVE: Enterococcus faecalis (E. faecalis) is one of the persistent microorganisms responsible for the failure of root canal treatments. This study investigated the antibacterial property, the time-killing of 3 peptides, namely Tachyplesin, Thanatin, and Enterocin P, on E. faecalis. METHODS: In this study, recombinant peptides were synthesized via secretory synthesis. The peptides were then purified and isolated using affinity chromatography after which their purification was evaluated through SDS-PAGE. The antimicrobial activity of peptides against E. faecalis was tested using the minimum inhibitory concentration test (MIC), the minimum bactericidal concentration test (MBC), and the time-killing assay. RESULTS: Based on antimicrobial tests, a similar value was observed for the MIC and MBC in the recombinant peptide of Enterocin P. The concentration of MBC was twice as much as that of MIC for Tanatin and Tachyplesin. The time-killing-assay antimicrobial test showed that Enterocin P has a better pattern and antimicrobial activity than the other two peptides; all three peptides have weaker antimicrobial activities than sodium hypochlorite. CONCLUSION: Considering the equivalence of MIC and MBC in the recombinant peptide of Enterocin P, it can be a viable replacement for traditional disinfectants and medicaments used in root canal treatment procedures.

The Application of a Recombinant Antimicrobial Peptide of Thrombocidin-1 Expressed in Pichia pastoris as a Novel Approach Against Some Oral Pathogenic Bacteria: An In Vitro Study.

OBJECTIVE: Oral infections and dental caries are considered serious health problems. Therefore, searching for new agents with antimicrobial properties seems to be crucial. This study aimed to evaluate the antimicrobial activity of the recombinant Thrombocidin-1 [TC-1] peptide on some oral pathogens. Also, the cytotoxicity of this peptide on human gingival fibroblast cells was investigated. METHODS: In this study, Pichia pastoris was used for the expression of recombinant TC-1. The microbroth dilution method was used to determine the minimum inhibitory concentration [MIC] and minimum bacterial concentration [MBC]. It tested against four main oral pathogens; Streptococcus mutans, Streptococcus salivarius, Streptococcus oralis, and Enterococcus faecalis. Moreover, the cytotoxicity analysis was done on gingival fibroblast cells by the MTT method. The data were analyzed using a two-way analysis of variance [ANOVA] and Tukey's HSD tests. RESULTS: The most bactericidal effect of TC-1 was against S. salivarius, the highest bacteriostatic effect was against S. salivarius, and S. oralis had the lowest MIC value of 1.512 µg/ml. The Thrombocidin- 1 peptide showed lower antibacterial properties against E. faecalis compared with CHX, unlike the stronger antimicrobial effect on examined streptococci. According to cytotoxicity examination, no concentration of TC-1 presented over 50% growth inhibition [IC50] of the fibroblasts cells. CONCLUSION: Based on antimicrobial tests and cytotoxicity results, the Thrombocidin-1 peptide may be useful as a safe antibacterial agent against some oral pathogens in dental materials.

A New Approach Against Some Oral Pathogenic Bacteria Using a Chimeric Antimicrobial Peptide Derived from the Camel Milk; Lactoferrampin - Lactoferricin Chimer.

OBJECTIVES: The present study was conducted to evaluate the antimicrobial effects of the recombinant chimer present in the lactoferrampin-lactoferricin [LFA-LFC] derived from the camel milk on some oral bacteria responsible for dental caries and endodontic failures. METHODS AND MATERIAL: The antimicrobial activity was assessed on the Streptococcus mutans [ATCC 35668], Streptococcus salivarius [ATCC 9222], Streptococcus oralis [ATCC 35037], and Enterococcus faecalis [ATCC 29212], using the microbroth dilution method. The cytotoxicity analysis was done through the MTT method on the human gingival fibroblasts. The data were reported using the descriptive methods and were analyzed by the one-way analysis of variance (ANOVA) and Tukey HSD test. RESULTS: Results showed that the chimeric peptide had the highest bacteriostatic effect on S. salivarius with the lowest minimum inhibitory concentration value of 1.22 µg/Ml. Also, LFA-LFC chimer was more effective against S. mutans and S. salivarius compared to using 0.2% chlorhexidine mouthwash. The minimum bactericidal concentration analysis showed the most bactericidal effect against S. mutans [1.256 µg/mL]. In spite of the greater antibacterial effect on the evaluated streptococci, this peptide showed lower bacteriostatic and bactericidal properties against E. faecalis compared to the chlorhexidine. Based on cytotoxicity assay, over 50% of the cells were viable in all the evaluation times, demonstrating the biocompatibility of the peptide. CONCLUSION: The LFA-LFC chimer revealed comparable or even more effective antibacterial properties compared to the chlorhexidine mouthwash against the caries-inducing bacteria with no toxicity on the human gingival fibroblast cells. So, this peptide can be used as a safe alternative to chlorhexidine and other chemicals in dental applications for the prevention and management of dental caries.

Comparative study of antimicrobial activity between some medicine plants and recombinant Lactoferrin peptide against some pathogens of cultivated button mushroom.

The adverse effects of chemical pesticides on human health and environment cannot be ignored, hence it seems that novel alternative compounds should be applied to control plant pathogens. Among various alternative sources, natural compounds such as plant essential oils, plant extracts and recombinant antimicrobial peptides are of significance. The aim of the present study was to investigate antimicrobial activity of plants essential oils and plant extracts of six medicinal plants (Lippia citriodora, Ferula gummosa, Bunium persicum, Mentha piperita, Plantago major and Salvadora persica) along with a chimera peptide of camel lactoferrin, which is the most important antimicrobial component of camel milk, against Pseudomonas tolaasii and Trichoderma harzianum as pathogens of white button mushroom. The antibacterial activity test was conducted under in vitro conditions through disc diffusion method. The results showed that chimera camel lactoferrin peptide, with the highest amount of inhibitory zone (14.63 mm in 20 µg/mL concentration), has a significant difference in antibacterial activity compared to other treatments. Ferula gummosa conferred no antibacterial activity. Also, the results of antifungal effects indicated that plant essential oils and extracts have more antifungal activity than recombinant peptide. Generally, L. citriodora, B. persicum, M. piperita treatments could completely prevent growth of fungal in in vitro conditions. Therefore, using the mentioned plants can be a good replacement for reducing the chemical pesticides against pathogenic agents of button mushroom, without any adverse effects on environment and human health.

Generation of an engineered food-grade Lactococcus lactis strain for production of an antimicrobial peptide: in vitro and in silico evaluation.

BACKGROUND: Foodborne pathogens and their biofilms are considered as one of the most serious problems in human health and food industry. Moreover, safety of foods is a main global concern because of the increasing use of chemical food additives. Ensuring food safety enhances interest in discovery of new alternative compounds such as antimicrobial peptides (AMPs), which can be used as bio-preservatives in the food industry. In this study, the most important antimicrobial peptides of camel milk lactoferrin (lactoferrampin and lactoferricin) were recombinantly expressed in the form of chimeric peptide (cLFchimera) in a food-grade L. lactis strain. P170 expression system was used to express secreted cLFchimera using pAMJ1653 expression vector which harbors a safe (non-antibiotic) selectable marker. RESULTS: Peptide purification was carried out using Ni-NTA agarose column from culture medium with concentration of 0.13 mg/mL. The results of disk diffusion test revealed that cLFchimera had considerable antimicrobial activity against a number of major foodborne bacteria. Furthermore, this chimeric peptide showed strong and weak inhibitory effect on biofilm formation against P. aeruginosa, S. aureus E. faecalis, and E. coli, respectively. Antioxidant activity and thermal stability of the chimeric peptide was determined. The results showed that cLFchimera had antioxidant activity (IC50: 310 µ/mL) and its activity was not affected after 40 min of boiling. Finally, we evaluated the interaction of the peptide with LPS and DNA in bacteria using molecular dynamic simulation as two main intra and extra cellular targets for AMPs, respectively. Our in silico analysis showed that cLFchimera had strong affinity to both of these targets by positive charged residues after 50 ns molecular dynamic simulation. CONCLUSIONS: Overall, the engineered food-grade L. lactis generated in the present study successfully expressed a secreted chimeric peptide with antimicrobial properties and could be considered as a promising bio-preservative in the food industry.

Expression of Thanatin in HEK293 Cells and Investigation of its Antibacterial Effects on Some Human Pathogens.

BACKGROUND: Thanatin is the smallest member of Beta-hairpin class of cationic peptide derived from insects with vast activities against various pathogens. OBJECTIVE: In this study, the antimicrobial activity of this peptide against some species of human bacterial pathogens as well as its toxicity on NIH cells were evaluated. METHODS: Thanatin DNA sequence was cloned into pcDNA3.1+ vector and transformed into a DH5α bacterial strain. Then the recombinant plasmids were transfected into HEK-293 cells by calcium phosphate co-precipitation. After applying antibiotic treatment, the supernatant medium containing thanatin was collected. The peptide quantity was estimated by SDS-PAGE and GelQuant software. The antimicrobial activity of this peptide was performed with Minimum Inhibitory Concentration (MIC) method. In addition, its toxicity on NIH cells were evaluated by MTT assay. RESULTS: The peptide quantity was estimated approximately 164.21 µmolL-1. The antibacterial activity of thanatin was estimated between 0.99 and 31.58 µmolL-1 using MIC method. The result of cytotoxicity test on NIH cell line showed that the peptide toxicity up to the concentration of 394.10 µmolL-1 and for 48 hours, was not statistically significant from negative control cells (P>0.05). The antimicrobial assay demonstrated that thanatin had an antibacterial effect on some tested microorganisms. The results obtained in this study also showed that thanatin had no toxicity on mammalian cell lines including HEK293 and NIH. CONCLUSION: Antimicrobial peptides such as thanatin are considered to be appropriate alternatives to conventional antibiotics in treating various human pathological diseases bacteria.

Assessment of a Novel Antimicrobial Peptide Against Clinically Isolated Animal Pathogens and Prediction of Its Thermal-Stability.

Multidrug resistance of pathogenic microorganisms is a common problem in the treatment of infections. Therefore, searching for new agents with antimicrobial activities appears to be essential. Thrombocidin-1 (TC-1) is an antimicrobial peptide (AMP) derived from platelets. The present study aimed to produce recombinant TC-1 (rTC-1) with His tag to evaluate its antimicrobial activity and also predict its thermal-stability through molecular dynamic (MD) simulations. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration of rTC-1 against bacterial isolates were determined. Considering the importance of thermal-stability of proteins in their therapeutic applications, thermal-stability of rTC-1 predicted through MD simulation during 25ns at two important temperatures including avian normal body temperature and water boiling temperature at sea level. MIC results revealed that rTC1 had the most and least potency against Salmonella enteritidis and Escherichia coli, respectively. The root-mean-square deviation of rTC-1 during 25ns MD simulations revealed that this protein is stable at avian normal body temperature (40°C) but might lose stability at water boiling temperature at sea level. rTC-1 as an AMP has a good potency against some bacterial pathogens especially Salmonella spp. and E. coli 0157:H7. rTC-1 can be used as an alternative for common antimicrobial agents used in clinical settings.

Biosynthesis and antibiotic activity of silver nanoparticles using different sources: Glass industrial sewage-adapted Bacillus sp. and herbaceous Amaranthus sp.

Synergistic effects of metallic nanoparticles (NPs) with commonly used antibiotics have encouraged the exploration of novel biological entities, including bacteria and weed plants. The present study for the first time reports the capability of an extracellular fraction of Bacillus sp. isolated from effluents of a glass-manufacturing unit to biosynthesis silver nanoparticles (AgNPs) without hazardous materials. Besides, the biosynthesis of AgNPs using an aqueous extract of herbaceous weed plant (Amaranthus sp.), as a low-cost natural source, has been addressed in this study. Our findings confirmed the fabrication of microbial and plant-sourced AgNPs, being thoroughly characterized by UV-vis, transmission electron microscopy, X-ray diffraction, dynamic light scattering, energy dispersive X-ray spectroscopy, and zeta potential measurements. Further, biological activities of the plant- and bacterium-derived AgNPs were investigated against several pathogenic bacteria, in combination with streptomycin. The antibacterial effectiveness of the antibiotic coated with 400 µg/disk of AgNPs increased over 50% toward all the pathogenic bacteria. The data presented here demonstrate that both industrial wastewater-adapted Bacillus sp. and wild-growing Amaranthus sp. are efficient natural sources with excellent capabilities for creating biologically active AgNPs, which would be of considerable interest for circumventing bacterial resistance to current antibiotics.

Study on Antiviral Activity of Two Recombinant Antimicrobial Peptides Against Tobacco Mosaic Virus.

Antimicrobial peptides (AMPs) are generally small peptides with less than 50 amino acid residues, which have been considered as the first line of defense system in plants and animals. These small cationic peptides belong to a family of antimicrobials that are multifunctional effectors of innate immunity. The direct antimicrobial activity of AMPs against different bacteria, viruses, fungi, and parasites has been confirmed in different studies. In this study, the antiviral activity of two recombinant AMPs named thanatin and lactoferricin+lactoferrampin was evaluated against Tobacco mosaic virus (TMV) using half-leaf and leaf disk methods under in vivo and in vitro condition, respectively. The obtained result indicated that both recombinant AMPs have shown an antiviral activity against TMV. Compared to the chimeric lactoferricin+lactoferrampin, recombinant thanatin showed a higher rate of antiviral activity against TMV. Three types of effects, including protective, curative, and inactivation, were evaluated during an antiviral activity test. In the present study, the antiviral activity of two recombinant AMPs is represented for the first time: thanatin and chimeric lactoferricin+lactoferrampin against TMV as a viral plant pathogen.

Expression in eukaryotic cells and purification of synthetic gene encoding enterocin P: a bacteriocin with broad antimicrobial spectrum.

Due to the emergence of multidrug-resistant bacteria, treatment options for infectious diseases are decreasing. Bacteriocins are small antimicrobial peptides produced by numerous bacteria that offer alternative therapeutic strategies to combat multidrug-resistant bacterial infections. We evaluated the cloning, functional expression, and antimicrobial activities of enterocin P (EntP), a class II bacteriocin member, in Chinese hamster ovary (CHO) cells. A synthetic gene matching CHO cell codon usage was designed from the known mature amino acid sequence of EntP and cloned into the protein expression vector pcDNA™3.1(+). CHO cells were transformed with the recombinant plasmid and cultured, and the recombinant protein was purified by affinity chromatography. Antimicrobial activities of the recombinant EntP were evaluated on Gram-positive, Gram-negative, and multidrug-resistant pathogens. Recombinant EntP inhibited growth of a variety of bacteria, including pathogenic species known to cause nosocomial infections, often with multidrug-resistant strains. In addition, recombinant EntP demonstrated broad antimicrobial activities in both high salt medium and human plasma and was stable at high temperatures. The broad antimicrobial activity and stability of EntP make it an attractive therapeutic candidate, particularly for treatment of multidrug-resistant bacterial infections.

Heterologous expression of Thrombocidin-1 in Pichia pastoris: Evaluation of its antibacterial and antioxidant activity.

The antimicrobial peptide Thrombocidin-1 (TC-1) isolated from human blood that derived from NAP-2 by deleting of two amino acids from C-terminal region. In this study, a C-terminal 6 _ His tagged recombinant TC-1 was expressed as a secreted peptide in Pichia pastoris, for the first time. The recombinant P. pastoris was inoculated in to BMMY culture medium, incubation with 5 µl/ml absolute methanol for 72 h at 30 °C. The TC-1 peptide was concentrated with nickel affinity chromatography and electrophoresis on 16% acrylamide gels. The molecular weight of recombinant TC-1 is approximately 8 kDa and under these conditions, the concentration of TC-1 considered 190 µg/ml that determined by the Bradford method. The antimicrobial activity test (Minimum Inhibitory Concentration and Minimum Bactericidal Concentration) was done against: Listeria monocytogenes, Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa. The growth of these pathogenic bacteria was limited when we used peptide at a concentration of as low as 19.56 µg/ml. Based on DPPH radical scavenging (DPPH-RS) activity and reducing power assays, this peptide showed relatively good antioxidant potential in comparison with standard antioxidant used in this study (BHT). Due to the existence of TC-1 in blood, which makes it safe for human consumption, and the good results of its antimicrobial and antioxidant activity, it can be introduced as a good alternative and a novel effective peptide to food industry for bio-preservation.

Secretory Expression of a Chimeric Peptide in Lactococcus lactis: Assessment of its Cytotoxic Activity and a Deep View on Its Interaction with Cell-Surface Glycosaminoglycans by Molecular Modeling.

Nowadays, cancer remains a major cause of death affecting millions of people. Currently, the antimicrobial peptides (AMPs) as potent anticancer therapeutic agents offer specificity and low levels of side effects in cancer therapy. In the present study, a cationic chimeric peptide (cLFchimera), derived from camel lactoferrin, was expressed as a secretory peptide using P170 expression system in L. lactis. Peptide purification was carried out using Ni-NTA agarose column from culture medium with 21 µ/mL concentration. The recombinant peptide was investigated for its activity against four tumor and one normal cell line. The cLFchimera was more active against two tumor cell lines (chondrosarcoma and colorectal cancer cells), but the activity against two other tumor cell lines (hepatoma and breast cancer cell line) and normal cells was low. Finally, to have better insight into the mode of action of the peptide on cytotoxic activity, we examined the interaction of cationic peptide with two glycosaminoglycans (GAGs), heparan sulfate (HS) and chondroitin sulfate (CS), as the two most anionic molecules on the cell surface by molecular dynamic simulation. The results of in silico analysis showed that the cLFchimera interacted with HS and CS with a totally different amino acid profile. Hydrogen bonding screening in GAGs-peptide complexes revealed K21, V23 and I3, R16 are the dominant amino acids involved in peptide-HS and CS interaction, respectively. Overall, the results of this investigation showed the P170 expression system successfully expressed a cationic peptide with potent anticancer activity. Moreover, molecular docking analysis revealed the pattern of peptide interaction with negatively charged membrane molecules.

Antifungal activity of recombinant thanatin in comparison with two plant extracts and a chemical mixture to control fungal plant pathogens.

The most common method for controlling plant diseases is the application of chemical pesticides and sometimes use of resistant cultivars. Due to the effects of chemical pesticides on human and environmental health, mutation in pathogens and resistance to various toxins besides the challenges with resistant cultivar production, the constant use of these methods are not recommended any longer. Thus, use of biological control agents along with the natural ingredient extracted from plants and application of peptide with antimicrobial activity, have been the focus of many researchers. In the present study, the antifungal activity of two plant extracts named Turmeric and Persian lilac in comparison with a chemical mixture and recombinant thanatin were evaluated against five following fungal plant pathogens; Geotrichum candidum, Botrytis cinerea, Rhizoctonia solani, Alternaria tenuissima and Gibberella fujikuroi. The results showed that, all treatments have antifungal activity against tested fungi. Both plant extracts were shown an acceptable antifungal activity against tested fungi but their inhibition effects was not comparable with chemical mixture. Turmeric showed a higher rate of mycelial inhibition than Persian lilac. Amongst all treatment, thanatin showed a great antifungal activity by its application at µg level under both in vitro and in vivo condition. Considering to the compatibility of thanatin with human health and environmental safety we could imagine a clear perspective for the application of this recombinant peptide in sustainable agriculture.

Expression and Purification of the Main Component Contained in Camel Milk and Its Antimicrobial Activities Against Bacterial Plant Pathogens.

Lactoferrin is the most dominant protein in milk after casein. This protein plays a crucial role in many biological processes including the regulation of iron metabolism, induction and modulation of the immune system, the primary defense against microorganisms, inhibiting lipid peroxidation and presenting antimicrobial activity against various pathogens such as parasites, fungi, bacteria, and viruses. The major antimicrobial effect of lactoferrin is related to its N-terminal tail where different peptides for instance lactoferricin and lactoferrampin which are important for their antimicrobial abilities are present. The growth rate of bacterial cells in camel milk is lower than that of the cow milk due to having more antimicrobial compounds. In this study, we have fused a codon-optimized partial camel lactoferrcin and lactoferrampin DNA sequences in order to construct a fused peptide via a lysine. This chimeric 42-mer peptide consists of complete and partial amino acid sequence of camel lactoferrampin and lactoferricin, respectively. Human embryonic kidney 293 (HEK-293) cells were used for synthesizing this recombinant peptide. Finally, the antibacterial activities of this constructed peptide were investigated under in vitro condition. The result showed that, all construction, cloning and expression processes were successfully performed in HEK-293. One His-tag tail was added to the chimera in order to optimize the isolation and purification processes and also reduce the cost of production. Additionally, His-tag retained the antimicrobial activity of the chimera. The antimicrobial tests showed that the growth rate in the majority of bacterial plant pathogens, including gram negative and positive bacteria, was inhibited by recombinant chimera as the level of MIC values were evaluated between 0.39 and 25.07 µg/ml for different bacterial isolates.