BASI74, a Virulence-Related sRNA in Brucella abortus

Hao Dong,Jiabo Ding,Yanyan De,Qingmin Wu,Xiaowei Peng,Xiaolei Wang,Chuanbin Wang,Yufu Liu,Tao Han,Lin Yuan,Tonglei Wu

doi:10.3389/fmicb.2018.02173

Abstract

Brucella spp. are intracellular pathogens that infect a wide variety of mammals including humans, posing threats to the livestock industry and human health in developing countries. A number of genes associated with the intracellular trafficking and multiplication have so far been identified in Brucella spp. However, the sophisticated post-transcriptional regulation and coordination of gene expression that enable Brucella spp. to adapt to changes in environment and to evade host cell defenses are not fully understood. Bacteria small RNAs (sRNAs) play a significant role in post-transcriptional regulation, which has already been confirmed in a number of bacteria but the role of sRNAs in Brucella remains elusive. In this study, we identified several different sRNAs in Brucella spp., and found that over-expression of a sRNA, tentatively termed BASI74, led to alternation in virulence of Brucella in macrophage infection model. The expression level of BASI74 increased while Brucella abortus 2308 was grown in acidic media. In addition, BASI74 affected the growth ratio of the Brucella cells in minimal media and iron limiting medium. Using a two-plasmid reporter system, we identified four genes as the target of BASI74. One target gene, BABI1154, was predicted to encode a cytosine-N4-specific DNA methyltransferase, which protects cellular DNA from the restriction endonuclease in Brucella. These results show that BASI74 plays an important role in Brucella survival in macrophage infection model, speculatively by its connection with stress response or impact on restriction-modification system. Our study promotes the understanding of Brucella sRNAs, as well as the mechanism by which sRNAs use to influence Brucella physiology and pathogenesis.

Highlights

Brucella spp. as well as other bacteria are capable of quickly adapting to changing conditions to survive
Two small RNAs (sRNAs) (AbcR1 and AbcR2) regulating Brucella virulence were identified, and AbcR1 and AbcR2 double mutant was defective in both macrophage infection model and mice chronic infection model (Caswell et al, 2012; Sheehan and Caswell, 2017)
Additional 43 sRNA from 109 remaining candidates were detected by RT-PCR and the role of all verified sRNAs in virulence of Brucella was examined by over-expression in the wild type strain B. abortus 2308

Summary

INTRODUCTION

Brucella spp. as well as other bacteria are capable of quickly adapting to changing conditions to survive. Based on the results of strand-specific RNA deep-sequencing approach, 1321 sRNAs were found in B. melitensis 16 M, and one sRNA, BSR0441, involved in bacterial virulence in both macrophages and mice infection models was found (Zhong et al, 2016). Additional 43 sRNA from 109 remaining candidates were detected by RT-PCR and the role of all verified sRNAs in virulence of Brucella was examined by over-expression in the wild type strain B. abortus 2308. We identified and characterized one sRNA (BASI74) that significantly changed Brucella virulence in macrophage infection model. For construction of sRNA over-expression strains, the pBBR1MCS6 plasmid with putative sRNA encoding sequence was electroporated into B. abortus 2308, and cells were plated onto TSA containing chloramphenicol for selection of positive clones. The over-expression strains were further verified by PCR using universal primers

MATERIALS AND METHODS

RESULTS

DISCUSSION