The RNA Chaperone Hfq Is Involved in Stress Response and Virulence in Neisseria meningitidis and Is a Pleiotropic Regulator of Protein Expression

Abstract

The well-conserved protein Hfq has emerged as the key modulator of riboregulation in bacteria. This protein is thought to function as an RNA chaperone and to facilitate base pairing between small regulatory RNA (sRNA) and mRNA targets, and many sRNAs are dependent on the Hfq protein for their regulatory functions. To address the possible role of Hfq in riboregulated circuits in Neisseria meningitidis, we generated an Hfq mutant of the MC58 strain, and the knockout mutant has pleiotropic phenotypes; it has a general growth phenotype in vitro in culture media, and it is sensitive to a wide range of stresses, including those that it may encounter in the host. Furthermore, the expression profile of a vast number of proteins is clearly altered in the mutant, and we have identified 27 proteins by proteomics. All of the phenotypes tested to date are also restored by complementation of Hfq expression in the mutant strain. Importantly, in ex vivo and in vivo models of infection the Hfq mutant is attenuated. These data indicate that Hfq plays a key role in stress response and virulence, and we propose a major role for Hfq in regulation of gene expression. Moreover, this study suggests that in meningococcus there is a large Hfq-mediated sRNA network which so far is largely unexplored.Hfq is a well-conserved RNA binding protein which was originally identified in Escherichia coli as a host factor required for the replication of Q␤ bacteriophage (14). It shares structural and functional homology with the Sm proteins in eukaryotes, which have central roles in RNA metabolism (42). It has more recently been described as a pleiotropic regulator that modulates the stability or translation of an increasing number of mRNAs (for reviews, see references 1, 6, and 62). Its role as a key mediator in many small regulatory RNA (sRNA) circuits has made it the focus of many well-studied systems. Most of our present knowledge of sRNAs has resulted from recent global search studies involving screening the genomes of certain organisms for novel sRNA genes through bioinformatic and comparative analyses and also experimental approaches (reviewed in references 2 and 64), and these studies have lead to the identification of over 80 new noncoding sRNAs in E. coli, many of which are conserved in closely related pathogens. Although the functional role of the majority of these sRNAs is unknown, those that have been characterized in detail regulate various cellular functions, including iron homeostasis, quorum sensing, virulence, metabolism, and adaptation to stresses such as envelope stress, oxidative stress, stationary phase, and other stresses (3,

Structure of Escherichia coli Hfq bound to polyriboadenylate RNA

“…Regardless, the significant gain in affinity of Hfq for longer A-R-N tracts can be attributed to an increase in the local concentration of these triplets, thus allowing the tract to zipper cooperatively into place, and the positive electrostatic nature of the distal face (56), which through electrostatic steering (41,42) can attract longer nucleotides more effectively. Although the distal face can accommodate 6 (A-R-N) triplets, once 5 A-R-E sites are filled, the presence of additional triplets does not affect binding affinity (compare A 16 and A 27 , the values of which parallel those reported previously) (refs. 35 and 40, and Table 1).…”

“…The poly(A) binding mechanism of Hfq also differs entirely from that used by the human poly(A) binding protein (hPABP), 6 160 Ϯ 30 A 16 1.4 Ϯ 0.9 A 27 1.6 Ϯ 0.9 (GGA) 2 88 Ϯ 28 (GGA) 9 16 Ϯ 1 (GCA) 9 70 Ϯ 4 (GCCA) 9 42 Ϯ 4 AU 5G 6 4 Ϯ 10 G 6 14,900 Ϯ 1,000 C 6 6,200 Ϯ 600 DNA-A 6 9,500 Ϯ 2,800 AAYAA element † 7.7 Ϯ 1 *Binding constants and errors are the average and standard deviation of at least 5 measurements. † The affinity of the AAYAA element has been measured only once, and the error is associated with the fit of the curve to the polarization data.…”

“…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). To combat phosphosugar toxicity, ptsG, which encodes a sugar transporter, is bound by the sRNA SgrS and degraded by the principal cellular endoribonuclease RNase E in an Hfqmediated process.…”

N. meningitidis1681 is a member of the FinO family of RNA chaperones

“…Recently, the Hfq protein in MC58 was examined and was shown to be important for both the response to stress and virulence with a potentially large network of sRNAs regulating these traits. 23 NMB1681 is positioned downstream from a putative gene for chorismate synthase and in the opposite orientation to a neighboring gene for a putative topoisomerase IV subunit B also called ParE; this synteny ©2 0 1 1 L a n d e s B i o s c i e n c e .…”

“…sRNA transactions are likely critical for Neisseria pathogenicity, as the Neisseria Hfq homolog is a major virulence factor in this organism. 23 Whereas Hfq chaperones act in a relatively non-specific manner, FinO appears to target only the FinP-traJ mRNA pair, but not other sRNAs. It is therefore possible that NMB1681 may also act in a highly selective manner.…”