Preliminary Study On The Biological Functions Of Brucella Type ? Secretion System Effector Proteins BPE123 And BPE043

Brucellosis is a zoonotic disease mainly characterized by abortion and fever caused by Brucella,which poses a serious threat to human and animal health.Brucella is a facultative intracellular parasitic Gram-negative bacterium,and its virulence is mainly manifested by chronic persistent infection.Brucella can escape the degradation of host lysosome by forming brucella-containing vacuole(BCV),and achieve intracellular survival and replication.At the same time,Brucella infection doesn't cause apoptosis,the infected cells can avoid recognition and elimination of host immune system,by which Brucella can achieve its long-term survival in the host cells and cause persistent infection.The type IV secretion system(T4SS)of Brucella is a complex composed of proteins encoded by 12 genes that penetrates the cell wall and outer membrane,and is the main virulence factor of Brucella.T4SS interferes with the normal function of host cells mainly by secreting effector proteins.Effector proteins of Brucella have been continuously identified in recent years,but the function and mechanism of most effector proteins are unknown.Among them,BPE123 and BPE043 are validated Brucella effector proteins,their function and mechanism in the intracellular survival of Brucella are still unclear.Therefore,the interacting proteins and biological functions of Brucella BPE123 and BPE043 were investigated.The results were listed as follows:(1)Identification of BPE123 interacting protein IFT20 and its effect on intracellular survival of Brucella.Our previous studies have demonstrated that BPE123 is an important virulence factor for intracellular survival of Brucella.In this study,we analyzed the localization of BPE123 in HeLa cells by laser confocal experiment,and found that BPE123 was distributed around the nucleus in the form of vesicles,suggesting that BPE123 may be involved in vesicle transport of host cells.Then we screened the cDNA library of human spleen by yeast two-hybrid and identified IFT20 as its host target,the same result was verified by co-immunoprecipitation.After that,we constructed cell lines with IFT20 knockdown,IFT20 stable low-expression and IFT20 heterozygote respectively and established brucella infection models.The results showed that the intracellular viability of Brucella was enhanced in these IFT20 low-expressing cell lines.(2)The effect of interaction between BPE123 and IFT20 on autophagy.Western Blot was used to detect the change in the expression of autophagy-related proteins when BPE123 and IFT20 were overexpressed.It was found that overexpression of IFT20 could promote autophagy,overexpression of BPE123 could inhibit autophagy,and BPE123 could antagonize the autophagy induced by IFT20 when they were co-expressed.Furthermore,the knockdown of IFT20 could reduce the inhibition of autophagy induced by BPE123.(3)Identification of BPE043 as a virulence factor of Brucella.The bpe043 gene deletion strain of Brucella ?bpe043 was constructed by homologous recombination.The Brucella wild-type strain 104 and ? bpe043 were cultured under the same initial concentration,and their growth characteristics were observed.It was found that the growth trends of the two strains were basically the same in vitro.Then the mice infection model were established by infection with 104 and ? bpe043 strain respectively,and the spleen weight of mice was measured and the bacteria load in the spleen was calculated.The results showed that splenomegaly was observed in both 104 and ?bpe043 strain infected groups on day 7 post-infection(pi),while the spleen weight of mice infected with the ? bpe043 was lower than that of mice infected with wild strain.In addition,the splenomegaly of both infected groups reduced after day 14 pi.The bacteria load in the spleen of mice infected with ? bpe043 was lower than that of mice infected with wild strain at day 7 pi,and reduced sharply after day 14 pi.Further,using co-immunoprecipitation,we identified Rab4 and Rab11(small GTPases)as host targets of BPE043.The preliminary study on the biological functions of Brucella type IV secretory system effector proteins BPE123 and BPE043 was conducted to pave the way for further studies on how Brucella disturbs the signaling pathway of host cells and promote intracellular survival of Brucella by regulating autophagy by secreting effector proteins.It has laid a foundation for the prevention and treatment of Brucella and the development of vaccine.