Establishment Of VirB4 Knockout Strain Of Bartonella Tribocorum And Effects On Infectivity

Bartonella is a gram negative bacteria that mainly infects host erythrocytes and vascular endothelial cells,leading to the occurrence of bacterial angiomatosis.Bartonella belongs to α-Proteobacteria.Total 40 Bartonella species or subspecies have been confirmed so far.Bartonella tribocorum was selected to construct the knockout strain of VirB4 gene in IV secretion system,and to investigate the function of VirB4 in Bartonella tribocorum infection process.A.The establishment of VirB4 gene knockout strain in Bartonella tribocorumUpstream and downstream homologous fragment of VirB4 gene were amplified,and were introduced into PJM05 plasmid(PJM05-?VirB4).Then PJM05-?VirB4 was transformed into Escherichia coli S17-1 for double parents mating assay.Transconjugants were screened after growth on sucrose plate based on SacB selection by colony PCR and sequencing.The results showed 6 VirB4 gene inframe deletion strains were identified from 22 double exchange strains.Sequencing results showed that 2136 bp was in-frame deleted.No point mutations were observed in rest sequence.B.Study on the function of VirB4 geneIn this experiment,a comparative study of wild type strain and VirB4 deletion strain was conducted to investigate VirB4 gene function involving intracellular survival in macrophage,invasion of lymph circulation and Hela cell infection.Wild type strain and VirB4 deletion strain were used to infect rat peritoneal macrophages for 30 min.Extracellular bacteria was killed by gentamicin protection assay.The intracellular bacteria were released from the cells after freezing and thawing.Colony forming unit(CFU)of intracellular bacteria was counted on chocolate plates.The results showed that both the wild type strain and VirB4 deletion strain were able to survive in macrophages in 12 h.VirB4 deletion strain was more efficient to be phagocytized by macrophages in the early stage of infection,but there was no significant difference in the survival rate.It indicated that VirB4 gene was not a key factor for Bartonella tribocorum to surive in macrophage.Based on the rat thoracic duct drainage model,wild type strain and VirB4 deletion strain were used to infect rats,respectively.The bacterial load in lymph fluid was detected by Taqman probe.The results showed that both the wild type strain and VirB4 deletion strain were able to invade lymphatic circulation at different time points.However,the bacterial load of VirB4 deletion strain in the lymphatic circulation significantly increased in comparsion with wild type strain 8 hours post infection,which indicated VirB4 mutant strain was more efficient to invade host lymphatic circulation.Hela cells were infected with the wild type strain and VirB4 mutant strain for 12 hours.Gentamicin was added to kill the extracellular bacteria.Thereafter,infected Hela cells were immunostained.The results showed that wild type strain infected Hela cells more efficient than mutant strain,but there was no significant statistical difference.It indicated that VirB4 gene does not directly effect endocytosis of Bartonella druing infection epithelial cells.