Hfq, a Novel Pleiotropic Regulator of Virulence-Associated Genes in Francisella tularensis

Abstract

Francisella tularensis is a highly infectious pathogen that infects animals and humans, causing tularemia. The ability to replicate within macrophages is central for virulence and relies on expression of genes located in the Francisella pathogenicity island (FPI), as well as expression of other genes. Regulation of FPI-encoded virulence gene expression in F. tularensis involves at least four regulatory proteins and is not fully understood. Here we studied the RNA-binding protein Hfq in F. tularensis and particularly the role that it plays as a global regulator of gene expression in stress tolerance and pathogenesis. We demonstrate that Hfq promotes resistance to several cellular stresses (including osmotic and membrane stresses). Furthermore, we show that Hfq is important for the ability of the F. tularensis vaccine strain LVS to induce disease and persist in organs of infected mice. We also demonstrate that Hfq is important for stress tolerance and full virulence in a virulent clinical isolate of F. tularensis, FSC200. Finally, microarray analyses revealed that Hfq regulates expression of numerous genes, including genes located in the FPI. Strikingly, Hfq negatively regulates only one of two divergently expressed putative operons in the FPI, in contrast to the other known regulators, which regulate the entire FPI. Hfq thus appears to be a new pleiotropic regulator of virulence in F. tularensis, acting mostly as a repressor, in contrast to the other regulators identified so far. Moreover, the results obtained suggest a novel regulatory mechanism for a subset of FPI genes.Regulation of gene expression by noncoding or small RNAs (sRNAs) is prevalent in both prokaryotes and eukaryotes, and recent studies have identified numerous sRNAs in different organisms. Most sRNAs encoded by bacterial chromosomes act by base pairing with mRNA targets that are encoded in trans, and these sRNAs commonly require Hfq in order to function (47). Hfq is a bacterial RNA-binding protein that was initially recognized as a host factor for replication of the Q␤ RNA phage in Escherichia coli (17). The Hfq protein is very abundant, and it belongs to the eukaryotic and archaeal families of Sm and Sm-like proteins, respectively, that form homohexameric structures (for reviews, see references 5 and 53). The importance of Hfq became clear when an E. coli hfq null mutant was created. This mutant had pleiotropic phenotypes, such as a decreased growth rate, increased sensitivity to cellular stresses, and increased cell length (51). Hfq is a posttranscriptional regulator that binds sRNAs and mRNA and facilitates RNA-RNA interaction (1,18,23,32,35,55). For the most part, sRNA-mRNA interactions result in mRNA degradation and/or inhibition of translation, but they can also increase translation (for reviews, see references 1, 20, and 48). As might be expected from the pleiotropic phenotypes of an hfq mutant, deletion of hfq has been correlated with consider-

Structure of Escherichia coli Hfq bound to polyriboadenylate RNA

“…A C-terminal truncation mutant of Ec Hfq, lacking residues 70-102, was crystallized with the oligoribonucleotide A 15 . This truncation, although reported to disrupt rpoS binding (37), has little effect on poly(A) binding affinity but moderately affects the stability of Ec Hfq (38).…”

“…Hfq is also involved in the regulation of a quorum sensing in Vibrio cholerae and Vibrio harveyi by eliciting the sRNAmediated destabilization of the mRNAs that encode the quorum-sensing master regulators hapR and luxR, respectively (15). Further, Hfq plays a key role in the cellular response to phosphosugar toxicity and low cellular iron concentrations (16)(17)(18)(19)(20)(21)(22).…”

Pivotal role of the Francisella tularensis heat-shock sigma factor RpoH

“…The plasmid used for overexpression of rpoH, pFNLTP6gro-0851, was constructed by amplifying a 957 bp fragment (corresponding to the sequence 78 bp upstream of the rpoH gene and the entire gene) using primers 0851_F and 0851_R (Supplementary Table S1), followed by digestion with NdeI and NotI and cloning into plasmid pFNLTP6gro (Maier et al, 2004;Meibom et al, 2009) downstream of the groE operon promoter of F. tularensis. pFNLTP6gro-0851 was introduced into the LVS strain by electroporation.…”

Insights into virulence factors determining the pathogenicity ofCronobacter sakazakii

“…Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, Yersinia pseudotuberculosis, and Francisella tularensis have also expressed the importance of Hfq in the pathogenesis. [119][120][121][122][123] Limited studies regarding the effect of Cronobacter invasion on immune response have been done. The pathogen is reported to persist within human macrophages indicating that the Cronobacter possessed virulence properties that make it allows to tolerate the intracellular environment of macrophages.…”