The Role Of AsnC Protein In Brucella Intracellular Survival And Its Molecular Mechanism

Brucellosis, a serious threat to human health and economic development, is one of the most important bacterial zoonoses endemic which is worldwide distributed to many areas. At present the most effectiveprocedure to prevent and control brucellos is depend on vaccination. Limited by vaccine,submited vaccine becomes the first choice for researchers because of its clear composition, weak toxic and side effect and high safety. In the present study, homologous recombination was used to construct mutant strain 16MAAsnC. And then, the ORF of AsnC gene was cloned into T vector to construct the complementary plasmid, which was then transformed into 16MAAsnC to generate the complementary strain 16MAAsnC-C.Results of experimental applications of mouse and murine macrophage-like RAW264.7 are given to illustrate that AsnC is closely associated with the virulence of Brucella. Brucella infection is the survival and reproduction in macrophages which involves multiple environmental stress factors. Therefore, we analyzed the viability of mutant in vitro under stress conditions,such as high osmolarity, oxidativestress, acid and hot. which are simulated as macrophage intracellular environment. Experimental results showed that, AsnC is conducive to the resistance of harsh environment pressure,which implies that AsnC is able to improve the ability of the survival in macrophage with the harsh environment. The relative B. melitensis 16M transcription of the AsnC was compared by quantitative RT-PCR in mouse,murine macrophage and stress conditions. The results show that AsnC was increased, which implies that AsnC is necessary for the Brucella’replication, the long-term survival, and the establishment of chronicinfection. We compared the transcription of the virulence genes such as vjbR,dnaK,htrA,gntR,omp25,Omp3lin infection of mouse and murine macrophage.The transcription of vjbR,dnaK,htrA, gntR was decreased when the AsnC gene deleted. It means that AsnC plays a role by regulating the expression of the target virulence gene. In summary,the role of B. melitensis AsnC in environmental stress, intracellular survival and its target genes regulated were investigated,which give a lot of valuable information about B. melitensis pathogenesis.