RNA-seq reveals the critical role of OtpR in regulating Brucella melitensis metabolism and virulence under acidic stress.

Wenxiao Liu,Xiaolei Wang,Jing Li,Zuoshuang Xiang,Yongqun He,Qixing Ou,Qingmin Wu,Yujin Lv,Hao Dong

doi:10.1038/srep10864

Abstract

The response regulator OtpR is critical for the growth, morphology and virulence of Brucella melitensis. Compared to its wild type strain 16 M, B. melitensis 16 MΔotpR mutant has decreased tolerance to acid stress. To analyze the genes regulated by OtpR under acid stress, we performed RNA-seq whole transcriptome analysis of 16 MΔotpR and 16 M. In total, 501 differentially expressed genes were identified, including 390 down-regulated and 111 up-regulated genes. Among these genes, 209 were associated with bacterial metabolism, including 54 genes involving carbohydrate metabolism, 13 genes associated with nitrogen metabolism, and seven genes associated with iron metabolism. The 16 MΔotpR also decreased capacity to utilize different carbon sources and to tolerate iron limitation in culture experiments. Notably, OtpR regulated many Brucella virulence factors essential for B. melitensis intracellular survival. For instance, the virB operon encoding type IV secretion system was significantly down-regulated, and 36 known transcriptional regulators (e.g., vjbR and blxR) were differentially expressed in 16 MΔotpR. Selected RNA-seq results were experimentally confirmed by RT-PCR and RT-qPCR. Overall, these results deciphered differential phenomena associated with virulence, environmental stresses and cell morphology in 16 MΔotpR and 16 M, which provided important information for understanding the detailed OtpR-regulated interaction networks and Brucella pathogenesis.

Highlights

Domain with a phosphoacceptor site and an effector domain with DNA-binding activity[9]
To detect all the possible genes regulated by OtpR during acid stress, the Next-Generation Sequencing (NGS) technology was used to sequence the whole transcriptomic profiles of 16 MΔ otpR and its wild-type strain 16 M
Our RNA-seq study found that under acidic stress, OtpR regulated 501 genes associated with many important functions, including metabolism, membrane transport, transcription, regulation, translation, and DNA replication and repair[51,52,53,54]

Summary

Introduction

Domain with a phosphoacceptor site and an effector domain with DNA-binding activity[9]. Salmonella typhimurium activates virulence gene transcription within acidified macrophages[16] These studies indicate that low pH acts as an intracellular signal on the regulation genes involved in survival and multiplication within phagocytic cells[4,16]. To further define the OtpR-regulated Brucella pathogenesis mechanism, we hypothesized that OtpR play a major role as an important transcription regulator in regulating Brucella genes critical for intracellular survival under an acidic condition. To address this hypothesis, it would be ideal to use a high throughput technology to detect and compare the gene expression profiles of 16 MΔ otpR and its parental strain 16 M under an acid stress. The results provided fundamental gene-level evidence and detailed gene expression profiles regulated by OtpR in Brucella

Methods

Results

Conclusion