The RNA Chaperone Hfq Promotes Fitness of Actinobacillus pleuropneumoniae during Porcine Pleuropneumonia

Abstract

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, an economically important disease of pigs. The hfq gene in A. pleuropneumoniae, encoding the RNA chaperone and posttranscriptional regulator Hfq, is upregulated during infection of porcine lungs. To investigate the role of this in vivo-induced gene in A. pleuropneumoniae, an hfq mutant strain was constructed. The hfq mutant was defective in biofilm formation on abiotic surfaces. The level of pgaC transcript, encoding the biosynthesis of poly-␤-1,6-N-acetylglucosamine (PNAG), a major biofilm matrix component, was lower and PNAG content was 10-fold lower in the hfq mutant than in the wild-type strain. When outer membrane proteins were examined, cysteine synthase, implicated in resistance to oxidative stress and tellurite, was not found at detectable levels in the absence of Hfq. The hfq mutant displayed enhanced sensitivity to superoxide generated by methyl viologen and tellurite. These phenotypes were readily reversed by complementation with the hfq gene expressed from its native promoter. The role of Hfq in the fitness of A. pleuropneumoniae was assessed in a natural host infection model. The hfq mutant failed to colonize porcine lungs and was outcompeted by the wild-type strain (median competitive index of 2 ؋ 10 ؊5). Our data demonstrate that the in vivo-induced gene hfq is involved in the regulation of PNAG-dependent biofilm formation, resistance to superoxide stress, and the fitness and virulence of A. pleuropneumoniae in pigs and begin to elucidate the role of an in vivo-induced gene in the pathogenesis of pleuropneumonia.

A computational strategy for the search of regulatory small RNAs in Actinobacillus pleuropneumoniae

“…Although the relevance of sRNAs in A. pleuropneumoniae pathogenesis is suggested by previous work with mutants for the sRNA molecular chaperone Hfq (Subashchandrabose et al 2013;Pereira et al 2015b), little information exists on abundance and possible roles.…”

“…We envisage that the Arccs discovered in our study are negative regulators, although further experiments, e.g., comparative RNA-seq Most sRNAs have as targets mRNAs directly implicated in either virulence or stress resistance. Although this was partially expected given the aforementioned phenotypes of reduced biofilm-forming capacity, sensitivity to oxidative stress (Subashchandrabose et al 2013), and reduced virulence in an alternative infection model (Pereira et al 2015b) displayed by A. pleuropneumoniae mutants for the hfq gene, new candidates for virulence determinants were defined by the present regulatory network. Two categories of potential targets that are part of extremely important systems for the establishment of A. pleuropneumoniae as a pig pathogen, and thus strong mutant candidates for live attenuated vaccines, are the iron-acquisition systems and Apx toxins.…”

“…The pathogenesis of pleuropneumonia is complex and involves many virulence factors, of which the Apx toxins, of the RTX family, are believed to play a central role (Frey 2011). Recently, A. pleuropneumoniae hfq mutants were shown to be defective in biofilm formation, displayed enhanced sensitivity to oxidative stress (Subashchandrabose et al 2013), and were attenuated in an alternative infection model (Pereira et al 2015b). Hfq is an RNA-binding protein that facilitates the pairing of sRNAs with their target mRNAs and affects gene expression (Vogel and Luisi 2011).…”

The RNA-Binding Chaperone Hfq Is an Important Global Regulator of Gene Expression in Pasteurella multocida and Plays a Crucial Role in Production of a Number of Virulence Factors, Including Hyaluronic Acid Capsule

“…Despite this normal in vitro growth rate, competition assays in mice revealed that the P. multocida hfq mutant showed significantly reduced fitness in vivo. Reduced in vivo fitness has also been demonstrated for hfq mutant strains of Y. pestis, Klebsiella pneumoniae, Francisella tularensis, and Actinobacillus pleuropneumoniae (20,(58)(59)(60).…”

Inhibition of Phagocytic Killing of Escherichia coli in Drosophila Hemocytes by RNA Chaperone Hfq

“…Hfq of Escherichia coli is a protein of 102 aa residues, and its orthologs exist in a number of Gram-negative and -positive bacteria constituting the Hfq family of proteins (14)(15)(16)(17). Hfq has been shown to play a role in the virulence of uropathogenic E. coli (18) and other bacterial species (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). However, the mechanism involved in Hfq-mediated changes in the virulence of bacteria remains unclear.…”