The Cloning, Expression And Function Of Mycobacteriophage SWU1Polysaccharide Depolymerase-A321gp39

Tuberculosis （TB） is a kind of harm to people’s major infectious diseases of public health. The pathogenic bacterial of tuberculosis is mycobacterium tuberculosis（MTB） In recent years, due to the global AIDS epidemic, the increase of population mobility and the emergence of multi-drug resistant strains, leading to global tuberculosis epidemic situation more serious. It is estimated that about one-third of the world’s population is infected with mycobacterium tuberculosis and about two million people died of TB in each year. Tuberculosis （TB） has closed to the HIV/AIDS and become the second most deadly infectious diseases. Currently, the main method for the treatment of tuberculosis （TB） is one of the traditional chemical treatment methods, such as isoniazid and rifampicin e.tc. But with the increase of the resistance, leading to the treatment effect of these drugs is more and more not ideal. Therefore, urgently needs to develop new effective method for rapid diagnosis and treatment.Bacteriophages （or’phages’） are viruses that parasitize bacteria, and can specifically infect the host bacterium, including some pathogens. Polysaccharide depolymerase, a polysaccharide hydrolase encoded by bacteriophages （or’phages’）, can specifically degrade the macromolecule carbohydrates of the host bacterial envelope. Most of the known phage was tailed phage or Caudovirales. In the process of tailed phage infected some host bacteria that was packaged with capsule, can be form a circular bull’s eye outside the plaque, termed the "halo’. The formation of the halo may be due to in the process of infection, the bacteriophage will produce some free depolymerases, these depolymerases not only spread on the agar plate, and degrade host bacteria around the plaque, thereby form halo. At present, it has been found in many phage existence polysaccharide depolymerase, such as Pseudomonas aeruginosa phage, pneumonia klebsiella phage, streptococcus pyogenes phage, E. coli phage,e.tc. These enzymes mainly includes lysin or endolysin, degradation of bacterial cell wall peptidoglycan layer framework, endo-Rhamnosidase, destroyed bacteria lipopolysaccharide O antigen and degradation bacteria capsular polysaccharide of alginate lyase and endosialidase, etc. But there is no report finds lyase with depolymerase activity in mycobacteriophage.SWU1is a new separation of mycobacteriophage, and with unique structural features of the plaque. SWU1also can be form a circular bull’s eye outside the plaque and generate a turbid area in the center of the plaque. Therefore, The plaque characteristics of SWU1and depolymerase producing bacteriophage （DEP） are equal. Therefore, SWU1is regarded as a DEP. This article uses the MEGA4software to build volutionary tree With SWUl homologous bacteriophage. Results show that SWU1and a temperate mycobacteriophages L5have closest relationship. Although their genome similarity is as high as80%, but L5does not form’halo’. In order to looking for reasons of two bacteriophage form different plaque, we use mavue software to compare genome of SWU1and L5. Results show that there is a special gene A321gp39in SWU1genome. A321gp39was annotated for leishmanolysin-like peptidase in NCBI. The A321gp39of amino acid sequence has weak sequence similarity to putative head-binding domain of phage tailspike protein of Escherichia sp.4₁₄0B （41%identity）. HHpred analysis results showed that the A321gp39of N terminal has a glycoside hydro lase catalytic activity center. Therefore, we speculated that it may be a polysaccharide hydrolase and involved in the formation of SWU1bacteriophage of external’halo’. In here, We successfully cloned and expressed A321gp39genes, and purify the A321gp39protein. A321gp39can destroy M. smegmatis mc2155lawn and can remarkably disrupt mycobacterial bio films. In addition, we successfully built recombination M. smegmatis mc2155M.s-pALACE-A321gp39and tested the expression of A321gp39in M. smegmatis mc2155by SDS-PAGE and Western-Blot. Compared with the no-load M. smegmatis mc2155Ms-pALACE, the express of A321gp39protein in the recombination M. smegmatis mc2155not only inhibited its sliding ability, and showed sensitivity to different antibiotics.