The Correlation Between T4SS Effector Bepe Of Bartonella And Cell Migration

Bartonella spp.are gram-negative,facultative and intracellular pathogen.Bartonella are transmitted by blood-sucking arthropods.Bartonella infection causes long-lasting intra-erythrocytic bacteraemia,cat-scratch disease,and bacillary angiomatosis or bacillary peliosis in immunodeficient patients.Bartonella harbors a Vir B4 type 4 secretion system totranslocate Bartonella effector protein(Bep)into host cells,modulating host cell apoptosis,cytoskeleton rearrangement and inflammation.Among these,BepE was reported to promote host dendrtic cell(DCs)migration,so that Bartonella can hijack DCs as “trojan horse” to disseminate Bartonella infection in vivo.In viewing that endothelial cells are targeted by Bartonella to induce angiomatosis formation,the present study aims to explore the correlation between BepE and endothelial cell migrantion.SacB-mediated in frame deletion strategy was used in our study to establish the Bartonella henselae(Bhe)BepE knock out strain(Bhe-?BepE).Bhe-BepE complemented in trans in Bhe-?BepE strain was also constructed.Firstly,upstream and downstream homologous fragment of BepE gene were amplified,and were introduced into pJM05 plasmid to build pJM05-?BepE.Then pJM05-?BepE was transformed into Bhe by conjugation with Escherichia coli S17-1.Bhe-?BepE strains were screened based on SacB selection and PCR.Then,whole BepE gene was amplified and introduced into p ANT4 plasmid,and p ANT4-BepE was transformed into Bhe-?BepE by conjugative transfer assay between Escherichia coli S17-1 and Bhe-?BepE to construct Bhe-pBepE.Subsequently,human vascular endothelial cells(HUVECs)were infected by wild type Bhe?Bhe-?BepE and Bhe-pBepE for 24 hours.And then,wound was produced by needle scratch.Wound area were photographed at the time of 0 h,6 h,12 h and 18 h post infection.Migration ability of HUVECs was compared by calculating the cell migration rates.The results showed that there was no significant difference of the migration ability of HUVECs after the infection of three strains,indicating BepE of Bhe failed to promote the migration of HUVECs.Subsequently,we infected HUVECs with Bhe-BepE expression lentivirus.Cell invasion and migration was evaluated by using transwell strategy.The results showed that there was no significant difference of the migration ability of HUVECs after the infection of Bhe-BepE-coated lentivirus and control virus.However,we confirmed that BepE significantly promoted disassembly of focal adhesions by immuostainning with Paxillin and Vinculin.BepE induced the formation of cell membrane ruffles where focal adhesions were totally disappeared.As conclusion,our study demonstrated that BepE of Bhe fails to promote the migration of HUVECs.However,it was found that BepE of Bhe promote the disassembly of focal adhesions of cells.In viewing that coordinated effect on cell migration of focal adhesion dynamics and actin contractile formation,we speculated that BepE requires another Bartonella T4 SS effector to reach a cooperative effect on cell migration,by using its ability to promote focal adhesion disassembly and potential ability of another effect on anctin-myosin stress fiber formation.