Characteristics Of A Broad Lytic Spectrum Endolysin From Phage VPp1 Of Vibrio Parahaemolyticus

Vibrio parahaemolyticus, a Gram-negative bacterium, widely exists in ocean environment and various seafood, including fish and shellfish, which can arose zoonosis. It not only be consisted as opportunistic pathogen of mariculture animals, but also can cause large-scale human food poisoning. To reduce the harm of Vibrio parahaemolyticus, heavy use of antibiotics contributes to the problem of antimicrobial resistance and it is quiet urgent to develop more effective and environment friendly inhibitors. Phages and endolysin are the natural enemies of bacteria and well known for their extreme host specificity, making them ideal candidates for applications designed to prevent target pathogenic bacteria. Phage encoded endolysin was considered to be one of the most promising enzyme antibiotics in 21st century.In this study, phage VPp1 was selected among Vibrio parahaemolyticus phages stored in our lab after the initial screening of their physiological-biochemical characteristics. According to the genetic analysis of phage genome, the study tried to explain how the phage VPp1 lytic bacteria in the angle of bioinformatics. The gene gp31, which was predicted to encode endolysin, cloned and expression in E.coil by genetic engineering technology and its lytic activity was verified by physicochemical method.114 strain of Vibrio parahaemolyticus phages were digested by the restriction enzyme Hind Illand solid proliferation and lysis liguid were performed. As a result, phage VPp1 had high titer about 1010pfu/ml and could lyse three kind of V. parahaemolyticus with clarify lysate. Besides, previous study has shown that the phage had good performance in depuration to reduce V. parahaemolyticus in oysters and detecting V. parahaemolyticus. So phage VPp1 was selected for the futher research. The sequence of the phage VPp1 genome has been deposited in GenBank and accession number was KJ936628.2 According to the genetic analysis of phage VPp1 genome, it was proposed primarily that ORF31 was predicted to encode phage endolysin by BLAST and functional analysis. ORF31 was predicted to be a kind of 28.2kDa glucoside hydrolase with non-signal peptide, two hydrophobic regions and no transmembrane domain, whose isoelectric point was 4.58. Secondary and tertiary structure showed its similarity with other phage endolysin. Furthermore, no genes encoded holin were detected, which might due to the diversity of holin sequences. It is speculated that phage VPp1 endolysin might lyse bacteria with the help of holin.3 Phage VPp1 genomic DNA was extracted and used as a template to amplify the entire endolysin gene. PCR products were purified and inserted into the BamU1/Xho1 site of pET-28a. which contains a His-tag for protein purification. The recombinant plasmids were transformed into Rosetta (DE3) and induced by the addition of 0.1 mM IPTG at 37℃ for 4 h. The recombinant endolysins were purified on a Ni2+-NTA columns with a protein concentrations about 1mg/ml. SDS-PAGE analysis reveale its molecular weight is about 40kDa.4 Diffusion and turbidimetric assays were performed to vetify the LysVPpl enymtic activity and it could lyse bacteria peptidoglycan, dead and living bacteria. The antimicrobial activity of LysVPp1 was stable at pH 5.0 to 10and temperature 30℃ to 55 ℃. After six months storage at -20 ℃, the acivity of LysVPp1 could keep 80%. Low iron concentration (Zn2+, Ca2+) helps to increase the lytic activity of LysVPp1. EDTA was used to increase outer membrane permeability, which could enhance the activity of LysVPpl. The host range test of LysVPp1 revealed that it has a more broad inhibition range than phage VPpl. The characteristic of LysVPpl display its potential value in control V. parahaemolyticus as an effective and environment friendly inhibitor.In conclusion, the phage VPp1 endolysin was predicted to be a kind of glucoside hydrolase by sequence and structure analysis. And then the gp31 was amplified, expression and LysVPpl was obtained as a fusion protein. Diffusion and turbidimetric assays showed the LysVPp1 enymtic activity in lysing bacteria peptidoglycan, dead and living bacteria with a stable environmental characteristic. The result has enriched the phage genome annotation data and filled the gap of V. parahaemolyticus phage endolysin researh. which could provide theoretical basis and instructions as biological agents to control V. parahaemolyticus.