Abstract
Brucella melitensis is a facultative intracellular bacterium that replicates within macrophages. The ability of brucellae to survive and multiply in the hostile environment of host macrophages is essential to its virulence. The RNA-binding protein Hfq is a global regulator that is involved in stress resistance and pathogenicity. Here we demonstrate that Hfq is essential for stress adaptation and intracellular survival in B. melitensis. A B. melitensis hfq deletion mutant exhibits reduced survival under environmental stresses and is attenuated in cultured macrophages and mice. Microarray-based transcriptome analyses revealed that 359 genes involved in numerous cellular processes were dysregulated in the hfq mutant. From these same samples the proteins were also prepared for proteomic analysis to directly identify Hfq-regulated proteins. Fifty-five proteins with significantly affected expression were identified in the hfq mutant. Our results demonstrate that Hfq regulates many genes and/or proteins involved in metabolism, virulence, and stress responses, including those potentially involved in the adaptation of Brucella to the oxidative, acid, heat stress, and antibacterial peptides encountered within the host. The dysregulation of such genes and/or proteins could contribute to the attenuated hfq mutant phenotype. These findings highlight the involvement of Hfq as a key regulator of Brucella gene expression and facilitate our understanding of the role of Hfq in environmental stress adaptation and intracellular survival of B. melitensis.
Highlights
Brucella spp. are gram-negative intracellular pathogens that belong to the a-2 subclass of proteobacteria, which live in close association with eukaryotic hosts [1]
The RNA-binding protein Hfq has emerged as a global posttranscriptional regulator of bacterial gene expression that participates in numerous regulatory pathways
We have demonstrated that Hfq modulates the expression of a wide range of genes and regulates the intracellular survival and virulence of B. melitensis
Summary
Brucella spp. are gram-negative intracellular pathogens that belong to the a-2 subclass of proteobacteria, which live in close association with eukaryotic hosts [1]. The intracellular environment of phagocytic cells is potentially hostile to microorganisms; intracellular pathogen can adapt to changes in their environment, avoiding degradation by host cell defense systems through the coordinated regulation of gene expression. Hfq is a bacterial Sm-like protein that acts as a posttranscriptional regulator of global gene expression [5,6]. The Hfq protein is highly conserved among bacteria, which was originally identified in Escherichia coli as a host factor essential for the replication of Qb RNA bacteriophage [7]. Hfq is required for the expression of some target genes in the absence of sRNA, by modulating the half-life of mRNAs directly or allowing the polyadenylation of mRNAs [5,9]. Robertson and Roop demonstrated that a Brucella abortus Dhfq mutant was defective in its ability to invade and survive inside animal cells and was more sensitive to stress environments, indicating the contribution of Hfq to the intracellular survival of B. abortus [11]
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