The Role Of Hfq Protein In Brucella Intracellular Survival

Brucellosis is a zoonotic disease caused by Brucella which brings serious harm to the health of livestock, wild animal and human. The disease distributed world wide is a serious threat to human health and economic development.Brucella, characterized with no virulence components plasmid and exotoxin, is an intracellular bacterium.The key of Brucella intracellular survival is to adapt to the the parasitic intracellular environment. Hfq (host factor for RNA phage Qβ replicase) encoded by perhaps half of all eubacterial species, RNA binding protein, is a key post transcriptional regulator of gene expression in bacteria. Hfq bounds RNA and displays its highest affinities for poly(A) and single-stranded AU-rich tracts.Hfq-dependent RNA can act on biological functions by binding to poly-riboadenylate RNA to affect its stability and assisting sRNA to pair with target mRNA for the regulation of gene expression. Previous studies have shown that Hfq protein is an important post transcriptional regulator,and the protein can regulate the expression of the target protein,so that it effects the global regulation,including pathogenicity and the other physiological processes in bacteria.Combinng the Hfq research about the other pathogens and the Brucella melitensis own characteristics, B. melitensis Hfq protein may play an important regulatory function to adapt to the harsh environment of the intracellular and intracellular survival,by affecting the expression of target genes.Therefore, The role of Brucella Hfq protein in intracellular survival will greatly facilitates the study of Brucella pathogenic mechanisms. In order to analyze the role of Brucella Hfq protein in intracellular survival and search target genes regulated by Hfq. Mutant strain16M-Ahfq and complementary strain16M-Ahfq-C were constructed firstly. The Hfq deletion strain16M△hfq was generated by Kan resistance gene replacement.Subsequently,the complementary strain was constructed with pUC19K plasmid which was already constructed in our laboratory.Then, hfq gene was ligated with pBBRl-MCS5plasmid which is able to replicate in Brucella, generating complementary plasmid cassettes which was electroporated into competent16M-△hfq. The deletion mutant and the complementary strain were further confirmed by RT-PCR,and the results showed that the mutant hfq had no transcriptional activity and hfq complemented strain of transcriptional activity is restored, indicating that the mutant and complementary strains were successfully constructed.After Hfq deletion mutant and the complementary strain were constructed.The related phenotype of wild-type strain, the Hfq mutant strain and the Hfq complementary strain were analyzed. In order to study the groth phenotype affected by Hfq in Brucella, the groth curve of16M,16M-△hfq and16M-△hfq-C were drew on the basis of cell optical density value. The curves had shown that the groth rate of16M-AHfq strain was drasticly lower and need more time to the platform than that of other strains whose groth curve showed consistend.The above curve trendency indicated that Hfq is associated with the groth of Brucella.In addition, The key about Brucella infection is the survival and reproduction in macrophages which involves multiple environmental stress factors.Therefore, we analyzed the viability of16MAhfq mutant in vitro under stress conditions,such as high osmolarity, oxidative stress, acid and hot,which are simulated as macrophage intracellular environment. The performance show that Hfq is conducive to the resistance of harsh environment pressure,which implies that Hfq is able to improve the ability of the survival in macrophage with the harsh environment. To understand fully the relation between survival capability and virulence, we also analyzed the viability of16M△hfq mutant in vivo,such as mouse and murine macrophage-like RAW264.7. Experimental results showed that, although16MAhfq mutants can invade macrophages and survival within the spleen cells, the viability of B. melitensis significantly decreased, suggesting that Hfq is necessary for the Brucella’replication in the host cell, the long-term survival and the establishment of chronicinfection,which Further confirmed the Hfq is closely related to Brucella intracellular survival and virulenceHfq, as a transcriptional regulatory factors, plays a role by regulating the expression of the target gene. Therefore, using DNA microarray expression profiling and comparative proteome analysis,Target genes were idendified and the function of these genes were elaborated, from the level of the whole genome and proteome, in chapters IV,so that Hfq target gene for research regulatory network were idendified. The relative B. melitensis16M transcription of the hfq was compared by quantitative RT-PCR in vitro under stress conditions which were simulated as macrophage intracellular environment. The results indicated that hfq was greatly activated in acidified minimum medium (GEM4.0).The transcriptome of the wild strains16M and the mutant strain16M-△hfq were compared by genome microarray expression profiling. After the Hfq gene was deleted, a total of359genes (approximately11%) showed at least2fold changes in expression levels,including genes involved in material transport and metabolism,transcription, translation, membrane function, intracellular transport and secretion. In addition, using comparative proteomics two-dimensional gel electrophoresis, the different proteins of whole cell protein between16M and16M-Ahfq were identified to search the proteins regulated by Hfq. The results of comparative proteomic indicated that after the hfq gene was deleted,the protein expression were changed in Brucella, significantly. These differentially expressed proteins involve in transporter, metabolism, post-translational modification,molecular chaperone, outer membrane, translation-associated protein.In summary, using comparative proteomics and comparative transcriptome expression profiling,the paper shows that a range of proteins and genes are regulated by Hfq,which ultimately leads to affect the Brucella’intracellular survival and virulence. These differentially expressed genes and proteins are associated with intracellular survival and virulence,such as metabolism and transport proteins, outer membrane protein, sigma factor, type IV secretion systems, quorum sensing system, flagellin, and the proteins or genes related to pressure (SodC、AhpC、OsmC、Dps、GroEL、GroES、HtrA、ClpP)In this study, the role of B. melitensis Hfq in environmental stress, intracellular survival and its target genes regulated were investigated,which give a lot of valuable information about B. melitensis pathogenesis.