Comparative Proteomics Analyses Reveal the virB of B. melitensis Affects Expression of Intracellular Survival Related Proteins

Zhoujia Wang,Tianyi Ying,Jin Zhao,Ruifu Yang,Xitong Yuan,Zeliang Chen,Feng Qiao,Shicun Dong,Yansong Sun,Jing Yuan,Leili Jia,Xinying Du,Zhijun Zhong,Yufei Wang,Liuyu Huang,David M. Ojcius,Lei Zhou

doi:10.1371/journal.pone.0005368

Abstract

Backgound Brucella melitensis is a facultative, intracellular, pathogenic bacterium that replicates within macrophages. The type IV secretion system encoded by the virB operon (virB) is involved in Brucella intracellular survival. However, the underlying molecular mechanisms, especially the target proteins affected by the virB, remain largely unclear.Methodology/Principal FindingsIn order to define the proteins affected by virB, the proteomes of wild-type and the virB mutant were compared under in vitro conditions where virB was highly activated. The differentially expressed proteins were identified by MALDI-TOF-MS. Forty-four down-regulated and eighteen up-regulated proteins which exhibited a 2-fold or greater change were identified. These proteins included those involved in amino acid transport and metabolism, lipid metabolism, energy production, cell membrane biogenesis, translation, post-translational modifications and protein turnover, as well as unknown proteins. Interestingly, several important virulence related proteins involved in intracellular survival, including VjbR, DnaK, HtrA, Omp25, and GntR, were down-regulated in the virB mutant. Transcription analysis of virB and vjbR at different growth phase showed that virB positively affect transcription of vjbR in a growth phase dependent manner. Quantitative RT-PCR showed that transcription of these genes was also affected by virB during macrophage cell infection, consistent with the observed decreased survival of the virB mutant in macrophage.Conclusions/SignificanceThese data indicated that the virB operon may control the intracellular survival of Brucella by affecting the expression of relevant proteins.

Highlights

Brucellosis, known as undulant or Malta fever, is one of the most common bacterial zoonoses endemic in many countries, developing ones [1]
Mutant strains that lose intracellular survival cannot carry out infection of their host; the virulence of Brucella depends upon its ability to survive and replicate within host cells
PCR verification and DNA sequencing showed that the virB operon was correctly rescued and cloned in pBBR-IVGT

Summary

Introduction

Brucellosis, known as undulant or Malta fever, is one of the most common bacterial zoonoses endemic in many countries, developing ones [1]. Brucellosis is caused by the genus Brucella, which consists of seven species according to antigenic variation and primary host. Humans can be infected by B. melitensis, B. abortus, and B. suis. The pathological manifestations of Brucellosis in humans include meningitis, endocarditis, spondylitis, and arthritis. Brucella infection occurs through inhalation or ingestion of the organisms. Following penetration of the epithelium, the bacteria are transported, either free or within phagocytes, to the regional lymph nodes and to different tissues [2]. Brucella species can survive within professional and nonprofessional phagocytes. Mutant strains that lose intracellular survival cannot carry out infection of their host; the virulence of Brucella depends upon its ability to survive and replicate within host cells

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