Expression And Hydrolytic Activity Analysis Of The TatD-like DNase Of Trypanosoma Brucei Brucei

Trypanosoma brucei is a kind of protozoan flagellate parasites that grow in the bloodstreams of various mammals causing human and animal African trypanosomiasis.Human African trypanosomiasis is also called sleeping sickness,the patients suffer from psychiatric symptoms and sleep all day in the second stage of infection.The death rate can reach 100 percent if left untreated.Animal African trypanosomiasis is also known as nagana,which causes animal anemia,wasting and death.T.brucei inflicts a terrible burden on life and agriculture.Trypanosoma brucei can escape the immune clearance of the host.At present,it is believed that evasion of the acquired immune response in trypanosomes is principally mediated by antigenic variation.Innate immune response is the first line of defense against pathogens.Upon activation,after the initial microbial contact,neutrophils export filamentous elements composed of DNA and proteases,which confine and kill the invading pathogens.The filamentous structures released from neutrophils are called neutrophil extracellular traps(NETs),and have been proven to contribute to pathogen capture as important part of the innate immune response.Correspondingly,some pathogens successfully escape the trap by secreting DNases to degrade the DNA.According the genomic sequence analysis,there are two gene sequences coding for the two variants of putative TatD-like DNases of T.brucei brucei.Whether trypanosome is also in this way to escape the innate immune response of the host is unknown,so this test will be a preliminary validation.In this experiment,the two gene sequences were retrieved from the NCBI database and resynthesized chemically,then cloned to p GEX-4T-1 and p ET-28 a expression vectors.The recombinant proteins TatD-like DNase with GST-tag and His-tag were expressed in E.coli.The rabbits and rats were immunized with the purified His-tagged recombinant proteins.The specific antibody Ig G was purified by affinity chromatography,and applied to the Western-blot and indirect immunofluorescence analysis of the TatD-like DNase expression in T.brucei brucei,these two proteins were lcated around the trypanosomal membrane and flagellum.We further demonstrated the DNA hydrolase activity of TbTatD by incubating r TbTatD-1155-GST and r TbTatD-1005-GST with mouse DNA,and these two proteins were able to hydrolyze doublestranded DNA in vitro.The neutrophils were stimulated with PMA to export neutrophil extracellular traps(NETs)and r TbTatD-1155-GST and r TbTatD-1005-GST were added to incubate together.It was found that both recombinant proteins could degrade DNA of NETs and destroy the structure of NETs.This experiment has initially verified the distribution and hydrolysis of TatD-like DNase in T.b.brucei,lays a foundation for futher study of its correlation with patasite pathogenicity,as well as for the mechanism of trypanosomal innate immune evasion.