Secretion Proteins VceA And VceC Of Brucella Effects On Autophagy And Apoptosis

Brucellosis is an anthropozoonose caused by brucella.Brucella can survival and reproduce in host body by escape the host body's immune system.Body's innate immune and adaptive immunity can monitor the invasion of pathogens,defensive and clear it by anti-infection immune.Pathogen generally have three ways to escape host immunity,they are respectively concealment,mutagenesis and inhibition.First one refer to Brucella are secrete in autophagosomes and prevent fusion of autophagy and lysosome,then avoid are killed by host cells.Second one,mutagenesis refers to the fact that pathogens escape has been induced immune response by altering their antigenic characteristics.Last one refer to Brucella inhibition of host cell apoptosis occur,thus avoiding Brucella be cleaned up by body's humoral immunity and cellular immunity when cell death and disintegration,so that protecting it parasite in the host cell in elong-term and causing persistent infection.Type IV secretion system(T4SS)is one of the main virulence factors of Brucella.It can transfer its secretory protein to host cells during infection of host cells,and it helps Brucella invasion and colonization in host cells,but this mechanism is incomplete unclear.This paper mainly studies the effect of secretory proteins of T4 SS on autophagy and apoptosis.The main research work is as follows:(1)Construction of VceA and VceC gene deletion mutants of B.abortus 2308(S2308).The upstream,downstream homologous arms and kanna gene were amplified by PCR technology respectively.The fusion PCR technology was used to fuse the three genes and connected PMD18-T.The target genes were exchanged by homologous recombination principle in the genome of the parent strain and detect its growth characteristics.Using two deletion strains respectively infected embryonic trophoblast cells(HPT-8)and female mouses and detected its viability in the cell.The results showed that the deletion strains ?VceA and ?VceC were successfully constructed and stable at least 10 generations.The growth curve reached the logarithmic growth phase at 12 h,when grow 30 h entered the platform period and there was no difference between their parent strain.They survival in HPT-8 cells and mouse spleen was significantly lower than parent strain S2308.(2)Expression and purification of Brucella VceA and VceC protein and analyzed effect of it on cells.They were constructed prokaryotic expression vector of VceA and VceC genes and acquired purified recombinant protein.It is detected of protein reactionogenicity and the production of cytokines when protein act on cells.The results showed that VceA and VceC prokaryotic expression vectors were successfully constructed.Western Blot detected purified protein was apper hybridization band and certificated they have good reactionogenicity.The secretion of TNF-?,IL-1? and TGF-?1 increased significantly after VceA protein added in cells,and the secretion of IL-18,TNF-? and TGF-?1 significantly decreased in VceC-treated cells.(3)To investigate the effects of gene VceA and VceC on autophagy.The mRNA expression of autophagy-related genes ATG5,p62 and LC3 was detected.TheIV expression of p62 protein was detected by Western blot,confocal microscopy and mouse uterine immunohistochemistry,using transmission electron microscopy to detect autophagosome.Result showed that ?VceA was significantly lower than S2308 group the expression of p62 mRNA,and ?VceC significantly higher S2308 group after infected mouse 1d and 7d.There was no difference in VceC protein group with S2308.?VceA infected cells in 3h,24 h and infected mouse 1d,56 d were significantly higher than S2308 group in the expression of ATG5,and ?VceC group was significantly lower than S2308 group at infected HPT-8 cells 24 h and infected mouse28 d,VceA protein group is significantly lower than NC group at 3h and 12 h protein,but VceC protein group no significant difference with NC group.The ratio of LC3-II / LC3-I in ?VceA group was significantly higher than S2308 group at infected cells 12 h,24h and infected mouse 1d and 7d,but ?VceC was significantly lower than S2308 group.The WB experiments showed that ?VceA group had no significant change in infected cells but was significantly lower than S2308 group when infected mouse,?VceC group was higher than control group and VceA protein group was significantly higher than normal group in expression of p62 proteinWB.In the expression of ATG5 protein,the ?VceA group was significantly higher than S2308 group,but ?VceC infection group contrary with it,and the VceC protein group is higher than the normal group.Laser confocal showed that ?VceA group was lower than S2308 group,whereas ?VceC was higher than S2308 group.Immunohistochemical experiments of mouse uterine deteced p62 protein showed that?VceA group was less than S2308 group of the brown point in infected 1d,7d and14 d,but ?VceC was more than S2308 group.Autophagosome were found in ?VceA infection group under electron microscope.Results show that ?VceA can promote the occurrence of autophagy in different degrees,VceA protein can inhibit autophagy,and ?VceC is inhibited cell autophagy by different degrees and the VceC protein promotes autophagy.(4)To investigate the effect of VceA and VceC gene on cell apoptosis.The mRNA expression of Caspase-3,Bcl-2 and Bax was detected.The expression of Caspase-3,Bcl-2 and intracellular cytochrome C(Cyt-C)protein detected by Western blot.The apoptosis rate of HPT-8 cells was detected by flow cytometry.The release of Cyt-C was detected by laser confocal microscopy and mouse uterus immunohistochemistry assay.Test result showed that the expression level of Caspase-3 mRNA ?VceA group were significantly lower than S2308 group after infected 24 h and 1d.The results of Bax / Bcl-2 showed that ?VceA group less than 1 after infected3 h,24h and 7d,and ?VceC group less than 1 when infected 24 h,7d,14 d,however VceA protein group was greater than 1 at 12 h and 24 h and VceC protein was greater than 1 at 24 h.The expression of Caspase-3 and localization and expression of Cyt-C protein the two deletion mutants were lower than S2308 group at 24 h,while the expression of Bcl-2 protein that two deletion mutants was higher than S2308 group and both of protein groups were lower than control group.Flow cytometry detection show that ?VceC was lower than S2308 group at 12 h,two deletion strains were lowerthan the parental strain at 24 h.The immunohistochemical staining of the mouse showed that two mutant groups were less than the control group.Results show that?VceA and ?VceC all inhibit the occurrence of apoptosis in different degrees,while VceA and VceC proteins promote apoptosis.