The Small RNA Chaperone Hfq Is Required for the Virulence of Yersinia pseudotuberculosis

Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Avenue, Chicago, IL 60611, USA.
Infection and immunity (Impact Factor: 3.73). 03/2010; 78(5):2034-44. DOI: 10.1128/IAI.01046-09
Source: PubMed


Bacterial small, noncoding RNAs (sRNAs) participate in the posttranscriptional regulation of gene expression, often by affecting
protein translation, transcript stability, and/or protein activity. For proper function, many sRNAs rely on the chaperone
Hfq, which mediates the interaction of the sRNA with its target mRNA. Recent studies have demonstrated that Hfq contributes
to the pathogenesis of a number of bacterial species, suggesting that sRNAs play an essential role in the regulation of virulence.
The enteric pathogen Yersinia pseudotuberculosis causes the disease yersiniosis. Here we show that Hfq is required by Y. pseudotuberculosis to cause mortality in an intragastric mouse model of infection, and a strain lacking Hfq is attenuated 1,000-fold compared
to the wild type. Hfq is also required for virulence through the intraperitoneal route of infection and for persistence of
the bacterium in the Peyer's patches, mesenteric lymph nodes, and spleen, suggesting a role for Hfq in systemic infection.
Furthermore, the Δhfq mutant of Y. pseudotuberculosis is hypermotile and displays increased production of a biosurfactant-like substance, reduced intracellular survival in macrophages,
and decreased production of type III secretion effector proteins. Together, these data demonstrate that Hfq plays a critical
role in the virulence of Y. pseudotuberculosis by participating in the regulation of multiple steps in the pathogenic process and further highlight the unique role of Hfq
in the virulence of individual pathogens.

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    • "OMVs or lysates (20 µg each) were mixed with reducing sample buffer (10% glycerol, 100 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 0.02% bromophenol blue, 5% β-mercaptoethanol) and separated by SDS-PAGE. Proteins were transferred to nitrocellulose membranes for immunblot analyses with antibodies against Pla [74], Ail (Eric Krukonis, University of Detroit Mercy School of Dentistry), Hfq [53], Caf1 (Abcam), and RpoA (Melanie Marketon, Indiana University). "
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    ABSTRACT: Many Gram-negative bacteria produce outer membrane vesicles (OMVs) during cell growth and division, and some bacterial pathogens deliver virulence factors to the host via the release of OMVs during infection. Here we show that Yersinia pestis, the causative agent of the disease plague, produces and releases native OMVs under physiological conditions. These OMVs, approximately 100 nm in diameter, contain multiple virulence-associated outer membrane proteins including the adhesin Ail, the F1 outer fimbrial antigen, and the protease Pla. We found that OMVs released by Y. pestis contain catalytically active Pla that is competent for plasminogen activation and α2-antiplasmin degradation. The abundance of OMV-associated proteins released by Y. pestis is significantly elevated at 37°C compared to 26°C and is increased in response to membrane stress and mutations in RseA, Hfq, and the major Braun lipoprotein (Lpp). In addition, we show that Y. pestis OMVs are able to bind to components of the extracellular matrix such as fibronectin and laminin. These data suggest that Y. pestis may produce OMVs during mammalian infection and we propose that dispersal of Pla via OMV release may influence the outcome of infection through interactions with Pla substrates such as plasminogen and Fas ligand.
    Full-text · Article · Sep 2014 · PLoS ONE
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    • "The spectrum and severity of hfq mutant phenotypes can vary among the different pathogens. For example, Yersinia hfq mutant is hyper-motile [38] but hfq mutation impairs motility in Salmonella, P. aeruginosa and E. coli [8], [13], [30]. Besides, deletion of hfq does prevent RpoS production in Salmonella and E. coli but not in V. cholerae [8], [11]. "
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    ABSTRACT: Hfq is a bacterial RNA chaperone involved in the riboregulation of diverse genes via small noncoding RNAs. Here, we show that Hfq is critical for the uropathogenic Proteus mirabilis to effectively colonize the bladder and kidneys in a murine urinary tract infection (UTI) model and to establish burned wound infection of the rats. In this regard, we found the hfq mutant induced higher IL-8 and MIF levels of uroepithelial cells and displayed reduced intra-macrophage survival. The loss of hfq affected bacterial abilities to handle H2O2 and osmotic pressures and to grow at 50°C. Relative to wild-type, the hfq mutant had reduced motility, fewer flagella and less hemolysin expression and was less prone to form biofilm and to adhere to and invade uroepithelial cells. The MR/P fimbrial operon was almost switched to the off phase in the hfq mutant. In addition, we found the hfq mutant exhibited an altered outer membrane profile and had higher RpoE expression, which indicates the hfq mutant may encounter increased envelope stress. With the notion of envelope disturbance in the hfq mutant, we found increased membrane permeability and antibiotic susceptibilities in the hfq mutant. Finally, we showed that Hfq positively regulated the RpoS level and tolerance to H2O2 in the stationary phase seemed largely mediated through the Hfq-dependent RpoS expression. Together, our data indicate that Hfq plays a critical role in P. mirabilis to establish UTIs by modulating stress responses, surface structures and virulence factors. This study suggests Hfq may serve as a scaffold molecule for development of novel anti-P. mirabilis drugs and P. mirabilis hfq mutant is a vaccine candidate for preventing UTIs.
    Full-text · Article · Jan 2014 · PLoS ONE
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    • "Also, no flagella were present in an electron micrograph of an E. coli hfq mutant [20]. However, the species-specific role of Hfq in the regulation of motility is illustrated by the increased swarming motility of a Y. pseudotuberculosis hfq mutant as well as increased biosurfactant production [21]. Therefore, it will be interesting to explore further the role of Hfq in the regulation of flagellum production in S39006, and the cause of the decreased motility despite an increase in flagellar gene expression. "
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    ABSTRACT: Serratia sp. ATCC 39006 (S39006) is a Gram-negative enterobacterium that is virulent in plant and animal models. It produces a red-pigmented trypyrrole secondary metabolite, prodigiosin (Pig), and a carbapenem antibiotic (Car), as well as the exoenzymes, pectate lyase and cellulase. Secondary metabolite production in this strain is controlled by a complex regulatory network involving quorum sensing (QS). Hfq and RsmA (two RNA binding proteins and major post-transcriptional regulators of gene expression) play opposing roles in the regulation of several key phenotypes within S39006. Prodigiosin and carbapenem production was abolished, and virulence attenuated, in an S39006 [increment]hfq mutant, while the converse was observed in an S39006 rsmA transposon insertion mutant. In order to define the complete regulon of Hfq and RsmA, deep sequencing of cDNA libraries (RNA-seq) was used to analyse the whole transcriptome of S39006 [increment]hfq and rsmA::Tn mutants. Moreover, we investigated global changes in the proteome using an LC-MS/MS approach. Analysis of differential gene expression showed that Hfq and RsmA directly or indirectly regulate (at the level of RNA) 4% and 19% of the genome, respectively, with some correlation between RNA and protein expression. Pathways affected include those involved in antibiotic regulation, virulence, flagella synthesis, and surfactant production. Although Hfq and RsmA are reported to activate flagellum production in E. coli and an adherent-invasive E. coli hfq mutant was shown to have no flagella by electron microscopy, we found that flagellar production was increased in the S39006 rsmA and hfq mutants. Additionally, deletion of rsmA resulted in greater genomic flux with increased activity of two mobile genetic elements. This was confirmed by qPCR and analysis of rsmA culture supernatant revealed the presence of prophage DNA and phage particles. Finally, expression of a hypothetical protein containing DUF364 increased prodigiosin production and was controlled by a putative 5[prime] cis-acting regulatory RNA element. Using a combination of transcriptomics and proteomics this study provides a systems-level understanding of Hfq and RsmA regulation and identifies similarities and differences in the regulons of two major regulators. Additionally our study indicates that RsmA regulates both core and variable genome regions and contributes to genome stability.
    Full-text · Article · Nov 2013 · BMC Genomics
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