Large-scale identification of small noncoding RNA with strand-specific deep sequencing and characterization of a novel virulence-related sRNA in Brucella melitensis.

Shuangshuang Lei,Xinying Du,Yuehua Ke,Zhijun Zhong,Zeliang Chen,Shiwei Liu,Jiuxuan Yu,Guangneng Peng,Xinran Li,Xiaoyang Xu,Jiuyun Yuan,Yufei Wang,Mingjuan Yang,Zhoujia Wang,Zhihua Ren

doi:10.1038/srep25123

Abstract

Brucella is the causative agent of brucellosis, a worldwide epidemic zoonosis. Small noncoding RNAs (sRNAs) are important modulators of gene expression and involved in pathogenesis and stress adaptation of Brucella. In this study, using a strand-specific RNA deep-sequencing approach, we identified a global set of sRNAs expressed by B. melitensis 16M. In total, 1321 sRNAs were identified, ranging from 100 to 600 nucleotides. These sRNAs differ in their expression levels and strand and chromosomal distributions. The role of BSR0441, one of these sRNAs, in the virulence of B. melitensis 16M was further characterized. BSR0441 was highly induced during the infection of macrophages and mice. The deletion mutant of BSR0441 showed significantly reduced spleen colonization in the middle and late phases of infection. The expression of the BSR0441 target mRNA genes was also altered in the BSR0441 mutant strain during macrophage and mice infection, which is consistent with its reduced intracellular survival capacity. In summary, Brucella encodes a large number of sRNAs, which may be involved in the stress adaptation and virulence of Brucella. Further investigation of these regulators will extend our understanding of the Brucella pathogenesis mechanism and the interactions between Brucella and its hosts.

Highlights

Brucella is the causative agent of brucellosis, a worldwide epidemic zoonosis
After the ribosomal RNA was removed, a strand-specific library was constructed with the deoxyuridine triphosphate second-strand marking procedure and sequenced with the Illumina HiSeq 2000 system
After the data were filtered, 13,249,202 cDNA reads were obtained: 97.29% of the reads were mapped to the B. melitensis 16M genome and 65.79% were mapped to annotated genes (Figure S1); 94.24% and 63.72% of the reads were uniquely mapped to the genome and genes, respectively

Summary

Introduction

Brucella is the causative agent of brucellosis, a worldwide epidemic zoonosis. Small noncoding RNAs (sRNAs) are important modulators of gene expression and involved in pathogenesis and stress adaptation of Brucella. Brucella invades and replicates in host phagocytes, where it is subject to hostile environmental conditions and encounters a variety of environmental stressors, including low pH, oxidative stresses, and nutrient deprivation[3] This process requires the complex and multifactorial regulation of the gene expression associated with stress adaptation and intracellular survival. A large number of sRNAs have been identified in various bacteria, including Escherichia coli[6,7], Pseudomonas aeruginosa[8], Pyrococcus abyssi[9], Neisseria meningitides[10], human-avirulent Yersinia pestis[11], and mycobacterial species[12] Many of these sRNAs are induced by stress and are associated with bacterial adaptation to the host and with bacterial virulence. We investigated the role of a novel sRNA, BSR0441, in the intracellular survival of B. melitensis

Methods

Results

Conclusion