Study On The Effect Of Small Ubiquitin-related Modifier Protein SUMO During Brucella Infected Macrophage

Brucella was a kind of virulent intracellular bacteria for human beings and livestock, the infection could lead to miscarriage, orchitis, and other symptoms of rheumatoid arthritis。In China, Xinjiang and Inner Mongolia was a province of animal husbandry. In recent years, the incidence of the disease was on the rise. Therefore, the study about small ubiquitin-related modifier is significant during brucella infection.Objective:The study based on genes of mice macrophage SUMO-1/2/3 and Ubc-9,to interfere and overexpressed the genes,and then brucella melitensis 16 M infected the cells,to explore their influence to intracellular survival of brucella and host cell apoptosis caused by brucella. Preliminary screening the virulence factors of Brucella which affected small ubiquitin-related modification.Method:(1) Brucella 16 M and M5-90 infected mouse macrophage RAW264.7, and then using q RT-PCR method and Western blotting to detect the expression of SUMO-1/2/3 and Ubc-9 after 4、8、12 and 24 h by the infection;(2)Interfering and overexpressing the expression of SUMO-1/2/3 and Ubc-9 in mice macrophage RAW264.7,and then brucella melitensis 16 M infect the cells to detective CFU, percentage of apoptotic cells;(3) Extracted the lipopolysaccharide LPS of Brucella 16 M, and then infected murine macrophages at a concentration of 0.1, 1 and 10 ng / μl gradient, we collected cells after 30 min, 1 and 3 h;While brucella 16M-△Vir B2 infected cells, the cells were collected after 1, 3 and 5 h, used q RT-PCR and Western bloting to detect the expression levels of SUMO-1 and Ubc-9.Results:(1)SUMO-1/2/3 and the conjugation enzyme Ubc-9 in mice macrophages showed a regular change after brucella infection. As time goes on, the expression of SUMO-1 began to rise, the highest was in 8 h, then began to decline, it was increased in 12 h,in the whole process, the expression was under normal levels, the difference was significant(P<0.05). SUMO-2/3 and Ubc9 had a similar trend.After infection, the expression of two gens were higher than normal levels, the difference was significant(P <0.05), the highest expression at 8 h, and then begin to decline, it reached lowest at 12h;(2)Pick one better from each two interfering models,which percentage of interfering is 38% for p LL3.7-S1 and 48.3% for p LL3.7-U1.And the percentage of overexpressing is 183% for p LEX-SUMO-1 and 223% for p LEX-SUMO-2/3.CFU experiment shows that the log CFU of p LEX-SUMO-2/3 was remarkable lower than control groups.Differently, PLL3.7-S1 and PLL3.7-U1 were higher than control groups. The percentage of apoptotic cells experiment shows that p LEX-SUMO-2/3 was rised,and p LL3.7-S1 and p LL-U1 was lower.As infective time went on, the apoptotic of each groups were still raised.(3)The lipopolysaccharide LPS of brucella 16 M had no effect on SUMO-1 and Ubc-9. However, after brucella 16M-△Vir B2 infected,compared with the control group, the expression of SUMO-1 and Ubc-9 were significantly increased after mutant strain infected, and the difference was significant( P <0.01).Conclusion: All of the above showed that brucella infection had effect on the murine macrophage SUMO system, and this effection was connected with T4 SS.Construction p LEX-SUMO-1、p LEX-SUMO-2/3 through lentivirus expression plasmid,and then was packaged by 293 FT cells,after infected by lentivirus, the expression of SUMO-1 and SUMO-2/3 were raised, SUMO reduced the number of brucella in mouse macrophages, and increased the number of apoptotic cells, in order to facilitate the body’s immune system to kill other intracellular brucella. In contrast, when SUMO-1 and Ubc-9 were silented, the viability of brucella had been improved, while reduced the percentage of apoptotic, which was beneficial to persistent exist for brucella; purified brucella LPS had no effect on the expression of SUMO-1 and Ubc-9; After brucella 16M-△Vir B2 infected, SUMO-1 and Ubc-9 both increased, therefore,we speculated, brucella into a host cell,and then secreted proteins by IV secretion system, interfered SUMO system.To sum up,SUMO-1/2/3 and Ubc-9 were important for brucella to intracellular survival and apoptosis induced by brucella among brucella infecting.