Screening Of SRNAs Related To Brucella Intracellular Survival And Function Study Of BSR0441

Brucellosis is a serious zoonotic diseases caused by Brucella.lt is a worldwide epidemic zoonosis has brought great harm to human health and the development of animal husbandry. Brucella is an intracellular gram-negative bacterium which survives and replicates in mononuclear cells (mainly macrophages). So the most important virulence characteristics of Brucella are its intracellular survival and replication capability. Non-coding small RNAs are recently discovered RNA regulators of bacterial metabolism, virulence and adaptation to the environment in bacteria, playing important roles in the regulation of gene expression mainly in response to environmental changes. They mainly interact with their target mRNAs to affect mRNA stability and translation, in order to regulate gene expression at the transcriptional level. In this study, using a strand-specific RNA deep-sequencing approach, we identified a global set of sRNAs expressed by B. melitensis 16M. Selected sRNAs were then verified by RT-PCR for their transcription under stress conditions that simulating intracellular environments. The role of BSR0441, one of these sRNAs in the virulence of B. melitensis 16M was further characterized. And the target genes of BSR0441 were predicted and confirmed.Brucella melitensis 16M were cultured under normal culture TSB7.0, and then performed RNA extraction purification and strand-specific dUTP library preparation for Illumina sequencing. The generated reads were mapped to genome sequence of 16M and transcriptome datas were analyzed to identify sRNAs. The results analysis showed that genome sequence was covered with the reads with perfect quality. In total,1321 sRNAs were identified ranging from 100 to 600 nucleotides. These sRNAs differ in their expression levels and strand and chromosomal distributions. RT-PCR analysis showed that selected candidate sRNAs have different transcription profiles under the different stress conditions, implying that they may be related to intracellular survival. Using TargetRNA2, we predict target genes of sRNAs founding that target genes of BSR0441 encoded important transcriptional regulators related to the virulence of B. melitensis 16M.Using Northern blot, we confirmed the transcription of BSR0441. Quantitative RT-PCR showed that BSR0441 was highly induced during the infection of macrophages and mice. To further analyze the roles of BSR0441 in Brucella intracellular survival, we constructed the deletion mutant and its overexpression strains. The virulence related phenotypes of the mutant and overexpression strains was analyzed. The results of the growth curve showed that BSR0441 has little influence on the growth of Brucella. Meanwhile, the survival experiments of Brucella under heat stress, oxidative stress, hyperhaline stress, osmotic stress and acid stress conditions in vitro revealed that the survival capacity of the mutant and/or overexpression strains was reduced, significantly under heat stress or acid stress. Macrophage infection assays showed that the deletion or overexpression of BSR0441 made the survival capacity of Brucella reduced during macrophage infection. Mice infection assays showed that the spleen colonization of the deletion mutant was significantly reduced in the middle and late phases of infection, indicating that BSR0441 is involved in Brucella survival in mice. To further understand the regulation of BSR0441 to its target genes, we performed qRT-PCR assays to test the transcriptional level of five putative mRNA targets of BSR0441 in wild strains, mutant and overexpression strains during macrophage and mice infection. The results showed that the expression of BSR0441 target genes was also altered in the BSR0441 mutant strain during macrophage and mice infection, which is consistent with its reduced intracellular survival capacity. And it can interact with several target mRNAs.