Antibacterial And Antiviral Activities Of Antimicrobal Peptide From Plutella Xylostella,pxCECA1

Antimicrobial peptides (AMPs) are widely expressed in nature with the characteristic of small molecular mass, positive charge and amphiphilicity. So far, more than1600AMPs have been reported. Most AMPs from different organisms can be broadly divided into four groups based on common secondary structure motifs:amphipathic α-helices, β-sheet structures with two to four disulphide bonds, loop structures and extended structures. As significant components of the innate immune system, AMPs have a variety of interesting biological functions including antibacterial, antifungal, antiparasitic, antitumoural, and antiviral activities. Recent study shows that mammalian AMPs also involve in the modulation of host inflammatory response as a bridge connecting the innate immunity and adaptive immunity. Diseases keep threatening people’s health, but traditional antibacterial, antiviral and antitumoural medicines don’t work well. Especially the antibiotic-resistant bacteria raised by the abuse of antibiotic make it urgent to develop new source of antibiotic. AMPs are effective at killing both gram-positive and gram-negative bacteria by disrupting bacterial membranes or translocating across both the cellular and nuclear membranes to act on the inside targets with the mechanism which rarely induces resistance. These characteristics make pxCECAl an attractive novel therapeutic that warrants further development.A growing body of research has focused on the antimicrobial activity of AMPs, but the lack of a cost-effective means of mass production remains one major obstacle to their further development as therapeutics. Extracting AMPs from the natural resource turns out to be inefficient leading to low yield. Chemical synthesis is too expensive and the activity of protein can not be guaranteed. Numerous biological expression systems have been established to produce AMPs in hosts such as prokaryotic cells and yeast cells. However, each of these systems presents their own set of problems. Yeast-based expression systems avoid issues associated with prokaryotic hosts, they have the disadvantages of a long production cycle, relatively low yield of the target protein, and high investment on batch cultivation. In the E.coli expression system AMPs are often expressed as fusion proteins to decrease their toxicity to prokaryotic hosts. Considering the tight relationship between structure and function of AMPs, the fusion partners, usually an affinity tag, must be cut off after purification to obtain AMPs with biological activity. To date, purification and cleavage methods have proven to be expensive and time consuming procedures.In present study larvae of Plutella xylostella was stimulated by bacteria and the total RNA was extracted. pxCECAl was amplified by RT-PCR and the amino acids sequence was analyzed. pxCECAl contained a signal peptide consisted of22amino acids and another two should be deleted resulting the mature peptide. According to the structure analysis, pxCECA1was amphiphilic carrying5positive charges, and the whole molecule formed an a-helix. There were only a few different amino acids between pxCECA1and other cecropin from Plutella xylostella. pxCECA1also contained highly conserved amino acid sequence when compared to cecropin family members from other species. To address the issue of efficient production of pxCECA1in E.coli, we employed the pET and IMPACT-TWIN expression system in the study. We insert pxCECA1into plasmids pET-32a(+) and pTWINl respectively and the transformed bacteria were induced by IPTG to express fusion protein. Using the pET system, pxCECAl was expressed as a fusion protein with a His-tag at the N-terminus in E. coli for isolation and purification. The purified fusion protein was cleaved by enterokinase and released native pxCECAl. Using the IMPACT-TWIN system, the fusion protein CBD-intein-pxCECA1was purified by the chitin beads. After exploration of the impact on the cleavage efficiency and protein yields of pH and temperature, the best condition for production was pH6.0for48hours at4℃. Compared with pET system, the IMPACT-TWIN system is a much easier and cheaper platform for AMPs’production, also resulting the relative high yields as12.3mg from1L culture.pxCECAl exerted good antibacterial activity against Escherichia coli, Staphyloccocus aureus, Pseudomonas aeruginosa, Bacillus pumilus Meyer and Bacillus subtilis. The MICs of pxCECA1against Escherichia coli was4.1μg/ml. The MICs of pxCECAl against Staphyloccocus aureus was8.2μg/ml which was lower than the other reported cecropin. We also observed that pxCECA1displayed equal activity against Staphyloccocus aureus as well as the antibiotic-resistant strains MRS A P1381and P1386. Growth inhibition tests and microbicidal tests showed that the antimicrobial ability was dependent on the peptide concentration in a microbicidal way. Catalase release assay showed pxCECA1killded bacteria in a membrane-disruptive way.pxCECAl didn’t exert hemolytic activity and MTT assay showed that pxCECAl didn’t exert cytotoxicity towards MDCK cells under the concentration of128μ g/ml. After treatment with pxCECAl, influenza A virus H3N2was not inhibited according to the MTT and PFU assay which demonstrated pxCECAl may not involve in the process of virus adsorption. Hemagglutination inhibition tests showed that, pxCECAl didn’t inhibit the hemagglutination of virus. When we added pxCECAl to the cells post infection immediately, pxCECAl could protect MDCK cells from death and decrease the relative quantity released from the cells. Both the protection and inhibition effects were concentration dependent. To explore the possible target pxCECAl might affect, the neuraminidase activity of virus treated by pxCECA1was identified. The result showed that pxCECAl didn’t inhibit influenza virus by blocking the budding process.In summary, we have expressed a previously uncharacterized microbicide, the AMP pxCECAl, using the novel IMPACT-TWIN system. The pH and temperature controlled self-cleavage mechanism utilized by this platform proved to be a simple, efficient method to express and purify a fully functional AMP. Furthermore, this study is the first to our knowledge to examine the biological activity of pxCECAl. Purified pxCECA1displayed broad spectrum antibacterial activity and antiviral activity against influenza A virus H3N2in vitro. The results of this study validate the efficiency and feasibility of the IMPACT-TWIN platform for producing functional AMPs, and demonstrate that the cecropin pxCECAl is a novel antimicrobial agent with broad spectrum activity and therapeutic promise.