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

Abstract

Background: Thanatin is the smallest member of Beta-hairpin class of cationic peptide derived from insects with vast activities against various pathogens. Objective: n 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. Method: 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.

Antibacterial effects assessment on some livestock pathogens, thermal stability and proposing a probable reason for different levels of activity of thanatin

“…This peptide was utilized from our previous study 12 .…”

“…The SDS-PAGE used for monitoring of peptide that secreted in culture media. 30 µL of the culture media supernatant (transfected cells by recombinant and self-ligated pcNDA3.1+) were run on SDS-PAGE in Tris/Glycine/SDS buffer using a 16% polyacrylamide gel and was stained with Coomassie Brilliant Blue 12 . This peptide was not purified, thus the concentration of the peptide was estimated with the GelQuant software v 1.8.2 ( www.biochemlabsolutions.com ) from the SDS-PAGE image.…”

“…The plate was incubated at 37 °C for 24 h without shaking. In this study, the lowest concentration of thanatin that could inhibit the bacterial growth was used as MIC for different bacterial isolates 12 . The McFarland standard 0.5 was used as standard to measure the turbidity.…”

“…Unfortunately, some reasons exist for preventing the use of some antimicrobial peptides such as hemolytic toxicity 10 , as well as the emerging of drug resistance 11 . Several studies demonstrated that thanatin was not toxic on some human cell lines even at very high concentrations which is considered an appropriate feature in utilizing this peptide in the treatment section 10 , 12 . Unlike pore-forming peptides, the primary mechanism of thanatin was through agglutination and clumping of cells that culminate reducing of releasing toxic substances by bacteria 13 , 14 .…”

“…In this study, we used the recombinant thanatin peptide from our previous study 12 to assess the minimum inhibitory concentration and the minimum bactericidal concentration on some farm animal and poultry bacterial pathogens. Furthermore, the thermal-stability of thanatin was investigated by both in silico and a culture-based method.…”