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

Abstract

cThe Gram-negative bacterium Pasteurella multocida is the causative agent of a number of economically important animal diseases, including avian fowl cholera. Numerous P. multocida virulence factors have been identified, including capsule, lipopolysaccharide (LPS), and filamentous hemagglutinin, but little is known about how the expression of these virulence factors is regulated. Hfq is an RNA-binding protein that facilitates riboregulation via interaction with small noncoding RNA (sRNA) molecules and their mRNA targets. Here, we show that a P. multocida hfq mutant produces significantly less hyaluronic acid capsule during all growth phases and displays reduced in vivo fitness. Transcriptional and proteomic analyses of the hfq mutant during midexponential-phase growth revealed altered transcript levels for 128 genes and altered protein levels for 78 proteins. Further proteomic analyses of the hfq mutant during the early exponential growth phase identified 106 proteins that were produced at altered levels. Both the transcript and protein levels for genes/proteins involved in capsule biosynthesis were reduced in the hfq mutant, as were the levels of the filamentous hemagglutinin protein PfhB2 and its secretion partner LspB2. In contrast, there were increased expression levels of three LPS biosynthesis genes, encoding proteins involved in phosphocholine and phosphoethanolamine addition to LPS, suggesting that these genes are negatively regulated by Hfq-dependent mechanisms. Taken together, these data provide the first evidence that Hfq plays a crucial role in regulating the global expression of P. multocida genes, including the regulation of key P. multocida virulence factors, capsule, LPS, and filamentous hemagglutinin. S mall noncoding RNA (sRNA) molecules play essential roles in regulating the production of a wide range of proteins, including those involved in virulence; quorum sensing; and the metabolism of carbon, amino acids, and iron (1-5). Typically, sRNA molecules are between 50 and 400 nucleotides long and act primarily by interacting with one or more target mRNAs via short imperfect base pairing (6, 7). Protein production can be controlled by sRNAs via multiple mechanisms, including inhibition of translation, activation of translation, and alteration of transcript degradation rates (8, 9). The regulation of bacterial protein expression via sRNAs often requires the activity of Hfq, an RNAbinding chaperone protein that belongs to the Sm-like RNA-binding protein family and displays a highly conserved core sequence (6, 10). Hfq monomers form a homohexameric ring-shaped structure that preferentially binds to A/U-rich sequences on target RNA molecules and mediates the various posttranscriptional regulatory mechanisms of sRNAs (11-13). Hfq can also directly protect sRNA molecules from RNase E-mediated degradation (14) and can have an sRNA-independent role in the regulation of mRNA decay, via direct interaction with poly(A) polymerase (15).Hfq homologues have been identified in a range of Gram-positive and Gram-...

Characterization of Two Novel Lipopolysaccharide Phosphoethanolamine Transferases in Pasteurella multocida and Their Role in Resistance to Cathelicidin-2

“…Moreover, growth-phase-dependent changes in petL expression correlate strongly with the abundance of the global regulatory protein Fis, expression of which is greatest during early log-phase growth (7.2-fold higher in early log phase compared to late log phase). Transcriptomic analyses from two P. multocida VP161 regulatory mutants revealed that petL expression was dramatically (48-fold) decreased following fis inactivation but increased when hfq was inactivated (37), suggesting that Fis and Hfq are involved in the control of PEtn decoration of the P. multocida lipid A molecule. Together, the data indicate that the relative positions of the PEtn residues on the P. multocida LPS molecule may change during different growth phases.…”

“…Each strain was grown in HI broth (in duplicate), and samples were taken for RNA extraction during the early exponential growth phase (OD 600 ϭ 0.2), the mid-exponential growth phase (OD 600 nm ϭ 0.4), and the late-exponential growth phase (OD 600 ϭ 0.6). RNA was extracted and purified, rRNA was depleted, and cDNA libraries were prepared, validated, and normalized as described previously (37). Libraries were sequenced and mapped to the draft VP161 genome as described previously (53).…”

“…To identify if any of the PEtn genes were differentially expressed during growth of VP161 in the early exponential, mid-exponential, and late exponential growth phases, available transcriptomic data for each gene were examined. Also included in the analysis were transcrip- tomic data obtained from a VP161 strain with an inactivating mutation in fis (unpublished data), encoding the growth-phase-dependent transcriptional regulator Fis and data from a VP161 hfq mutant unable to produce Hfq, a protein essential for the activity of many small RNA (sRNA) regulatory molecules (37). The expression values for the four LPS PEtn genes, petL, petK, lpt3, and petG, and for the two regulatory genes fis and hfq during early exponential growth of the wild-type strain VP161 were compared to the expression values determined during late-exponential growth ( Table 4).…”

Determination of the small RNA GcvB regulon in the Gram-negative bacterial pathogen Pasteurella multocida and identification of the GcvB seed binding region

“…Recently, we showed that the Hfq protein was essential for the appropriate expression of a range of proteins in the P. multocida serogroup A strain VP161, including those required for the biosynthesis of hyaluronic acid capsule which is a primary virulence factor (Mégroz et al 2016). The Hfq protein is an RNA chaperone that directly interacts with particular small regulatory RNA (sRNA) molecules to facilitate their binding to specific mRNA targets.…”

“…Comparative global transcriptomic and proteomic analyses of the P. multocida strain VP161 and an isogenic hfq mutant revealed that many genes displayed altered transcript expression, and/or altered protein production, when hfq was inactivated (Mégroz et al 2016). Analysis of the transcriptional data also allowed for the identification of a number of intergenic regions encoding putative sRNAs (M Mégroz, unpubl.).…”