The RNA chaperone Hfq has a multifaceted role in Edwardsiella ictaluri.

Edwardsiella ictaluri is a Gram-negative, facultative intracellular bacterium that causes enteric septicemia in catfish (ESC). The RNA chaperone Hfq (host factor for phage Qß replication) facilitates gene regulation via small RNAs (sRNAs) in various pathogenic bacteria. Despite its significance in other bacterial species, the role of hfq in E. ictaluri remains unexplored. This study aimed to elucidate the role of hfq in E. ictaluri by creating an hfq mutant (EiΔhfq) through in-frame gene deletion and characterization. Our findings revealed that the Hfq protein is highly conserved within the genus Edwardsiella. The deletion of hfq resulted in a significantly reduced growth rate during the late exponential phase. Additionally, EiΔhfq displayed a diminished capacity for biofilm formation and exhibited increased motility. Under acidic and oxidative stress conditions, EiΔhfq demonstrated impaired growth, and we observed elevated hfq expression when subjected to in vitro and in vivo stress conditions. EiΔhfq exhibited reduced survival within catfish peritoneal macrophages, although it had no discernible effect on the adherence and invasion of epithelial cells. The infection model revealed that hfq is needed for bacterial persistence in catfish, and its absence caused significant virulence attenuation in catfish. Finally, the EiΔhfq vaccination completely protected catfish against subsequent EiWT infection. In summary, these results underscore the pivotal role of hfq in E. ictaluri, affecting its growth, motility, biofilm formation, stress response, and virulence in macrophages and within catfish host.

Genome-wide profiling of Hfq-bound RNAs reveals the iron-responsive small RNA RusT in Caulobacter crescentus.

The alphaproteobacterium Caulobacter crescentus thrives in oligotrophic environments and is able to optimally exploit minimal resources by entertaining an intricate network of gene expression control mechanisms. Numerous transcriptional activators and repressors have been reported to contribute to these processes, but only few studies have focused on regulation at the post-transcriptional level in C. crescentus. Small RNAs (sRNAs) are a prominent class of regulators of bacterial gene expression, and most sRNAs characterized today engage in direct base-pairing interactions to modulate the translation and/or stability of target mRNAs. In many cases, the ubiquitous RNA chaperone, Hfq, contributes to the establishment of RNA-RNA interactions. Although the deletion of the hfq gene is associated with a severe loss of fitness in C. crescentus, the RNA ligands of the chaperone have remained largely unexplored. Here we report on the identification of coding and non-coding transcripts associated with Hfq in C. crescentus and demonstrate Hfq-dependent post-transcriptional regulation in this organism. We show that the Hfq-bound sRNA RusT is transcriptionally controlled by the NtrYX two-component system and induced in response to iron starvation. By combining RusT pulse expression with whole-genome transcriptome analysis, we determine 16 candidate target transcripts that are deregulated, many of which encode outer membrane transporters. We hence suggest RusT to support remodeling of the C. crescentus cell surface when iron supplies are limited.IMPORTANCEThe conserved RNA-binding protein Hfq contributes significantly to the adaptation of bacteria to different environmental conditions. Hfq not only stabilizes associated sRNAs but also promotes inter-molecular base-pairing interactions with target transcripts. Hfq plays a pivotal role for growth and survival, controlling central metabolism and cell wall synthesis in the oligotroph Caulobacter crescentus. However, direct evidence for Hfq-dependent post-transcriptional regulation and potential oligotrophy in C. crescentus has been lacking. Here, we identified sRNAs and mRNAs associated with Hfq in vivo, and demonstrated the requirement of Hfq for sRNA-mediated regulation, particularly of outer membrane transporters in C. crescentus.

Multifaceted Interplay between Hfq and the Small RNA GssA in Pseudomonas aeruginosa.

Behind the pathogenic lifestyle of Pseudomonas aeruginosa exists a complex regulatory network of intertwined switches at both the transcriptional and posttranscriptional levels. Major players that mediate translation regulation of several genes involved in host-P. aeruginosa interaction are small RNAs (sRNAs) and the Hfq protein. The canonical role of Hfq in sRNA-driven regulation is to act as a matchmaker between sRNAs and target mRNAs. Besides, the sRNA CrcZ is known to sequester Hfq and abrogate its function of translation repression of target mRNAs. In this study, we describe the novel sRNA GssA in the strain PA14 and its multifaceted interplay with Hfq. We show that GssA is multiresponsive to environmental and physiological signals and acts as an apical repressor of key bacterial functions in the human host such as the production of pyocyanin, utilization of glucose, and secretion of exotoxin A. We suggest that the main role of Hfq is not to directly assist GssA in its regulatory role but to repress GssA expression. In the case of pyocyanin production, we suggest that Hfq interplays with GssA also by converging a positive effect on this pathway. Furthermore, our results indicate that both Hfq and GssA play a positive role in anaerobic growth, possibly by regulating the respiratory chain. On the other hand, we show that GssA can modulate not only Hfq expression at both transcriptional and posttranscriptional levels but also that of CrcZ, thus potentially influencing the pleiotropic role of Hfq. IMPORTANCE The pathogenic lifestyle of the bacterium Pseudomonas aeruginosa, a leading cause of life-threatening infections in the airways of cystic fibrosis patients, is based on the fine regulation of virulence-associated factors. Regulatory small RNAs (sRNAs) and the RNA-binding protein Hfq are recognized key components within the P. aeruginosa regulatory networks involved in host-pathogen interaction. In this study, we characterized in the highly virulent P. aeruginosa strain PA14 the novel sRNA GssA. We found that it can establish a many-sided reciprocal interplay with Hfq which goes beyond the canonical mechanism of direct physical interaction that had previously been characterized for other sRNAs. Given that the Hfq-driven regulatory network of virulence factors is very broad and important for the progression of infection, we consider GssA as a new RNA target that can potentially be used to develop new antibacterial drugs.

Expanding the genetic toolkit helps dissect a global stress response in the early-branching species Fusobacterium nucleatum.

Fusobacterium nucleatum, long known as a common oral microbe, has recently garnered attention for its ability to colonize tissues and tumors elsewhere in the human body. Clinical and epidemiological research has now firmly established F. nucleatum as an oncomicrobe associated with several major cancer types. However, with the current research focus on host associations, little is known about gene regulation in F. nucleatum itself, including global stress-response pathways that typically ensure the survival of bacteria outside their primary niche. This is due to the phylogenetic distance of Fusobacteriota to most model bacteria, their limited genetic tractability, and paucity of known gene functions. Here, we characterize a global transcriptional stress-response network governed by the extracytoplasmic function sigma factor, σE. To this aim, we developed several genetic tools for this anaerobic bacterium, including four different fluorescent marker proteins, inducible gene expression, scarless gene deletion, and transcriptional and translational reporter systems. Using these tools, we identified a σE response partly reminiscent of phylogenetically distant Proteobacteria but induced by exposure to oxygen. Although F. nucleatum lacks canonical RNA chaperones, such as Hfq, we uncovered conservation of the noncoding arm of the σE response in form of the noncoding RNA FoxI. This regulatory small RNA acts as an mRNA repressor of several membrane proteins, thereby supporting the function of σE. In addition to the characterization of a global stress response in F. nucleatum, the genetic tools developed here will enable further discoveries and dissection of regulatory networks in this early-branching bacterium.

The Role and Targets of the RNA-Binding Protein ProQ in the Gram-Negative Bacterial Pathogen Pasteurella multocida.

The Gram-negative pathogen Pasteurella multocida is the causative agent of many important animal diseases. While a number of P. multocida virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. One mechanism by which bacteria regulate transcript abundance and protein production is riboregulation, which involves the interaction of a small RNA (sRNA) with a target mRNA to alter transcript stability and/or translational efficiency. This interaction often requires stabilization by an RNA-binding protein such as ProQ or Hfq. In Escherichia coli and a small number of other species, ProQ has been shown to play a critical role in stabilizing sRNA-mRNA interactions and preferentially binds to the 3' stem-loop regions of the mRNA transcripts, characteristic of intrinsic transcriptional terminators. The aim of this study was to determine the role of ProQ in regulating P. multocida transcript abundance and identify the RNA targets to which it binds. We assessed differentially expressed transcripts in a proQ mutant and identified sites of direct ProQ-RNA interaction using in vivo UV-cross-linking and analysis of cDNA (CRAC). These analyses demonstrated that ProQ binds to, and stabilizes, ProQ-dependent sRNAs and transfer RNAs in P. multocida via adenosine-enriched, highly structured sequences. The binding of ProQ to two RNA molecules was characterized, and these analyses showed that ProQ bound within the coding sequence of the transcript PmVP161_1121, encoding an uncharacterized protein, and within the 3' region of the putative sRNA Prrc13. IMPORTANCE Regulation in P. multocida involving the RNA-binding protein Hfq is required for hyaluronic acid capsule production and virulence. This study further expands our understanding of riboregulation by examining the role of a second RNA-binding protein, ProQ, in transcript regulation and abundance in P. multocida.

Hfq and sRNA 179 Inhibit Expression of the Pseudomonas aeruginosa cAMP-Vfr and Type III Secretion Regulons.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen causing skin and soft tissue, respiratory, and bloodstream infections. The type III secretion system (T3SS) is one important virulence factor. Production of the T3SS is controlled by ExsA, a transcription factor that activates expression of the entire T3SS regulon. Global regulators including Vfr, RsmA, and Hfq also contribute to regulation of the T3SS. Vfr is a cAMP-responsive transcription factor that activates exsA transcription. RsmA, an RNA-binding protein, inversely controls expression of the T3SS and the type VI secretion system (T6SS). Hfq is an RNA chaperone that functions by stabilizing small noncoding RNAs (sRNAs) and/or facilitating base pairing between sRNAs and mRNA targets. A previous study identified sRNA 1061, which directly targets the exsA mRNA and likely inhibits ExsA synthesis. In this study, we screened an sRNA expression library and identified sRNA 179 as an Hfq-dependent inhibitor of T3SS gene expression. Further characterization revealed that sRNA 179 inhibits the synthesis of both ExsA and Vfr. The previous finding that RsmA stimulates ExsA and Vfr synthesis suggested that sRNA 179 impacts the Gac/Rsm system. Consistent with that idea, the inhibitory activity of sRNA 179 is suppressed in a mutant lacking rsmY and rsmZ, and sRNA 179 expression stimulates rsmY transcription. RsmY and RsmZ are small noncoding RNAs that sequester RsmA from target mRNAs. Our combined findings show that Hfq and sRNA 179 indirectly regulate ExsA and Vfr synthesis by reducing the available pool of RsmA, leading to reduced expression of the T3SS and cAMP-Vfr regulons.IMPORTANCE Control of gene expression by small noncoding RNA (sRNA) is well documented but underappreciated. Deep sequencing of mRNA preparations from Pseudomonas aeruginosa suggests that >500 sRNAs are generated. Few of those sRNAs have defined roles in gene expression. To address that knowledge gap, we constructed an sRNA expression library and identified sRNA 179 as a regulator of the type III secretion system (T3SS) and the cAMP-Vfr regulons. The T3SS- and cAMP-Vfr-controlled genes are critical virulence factors. Increased understanding of the signals and regulatory mechanisms that control these important factors will enhance our understanding of disease progression and reveal potential approaches for therapeutic intervention.

Loss of RNA Chaperone Hfq Unveils a Toxic Pathway in Pseudomonas aeruginosa.

Hfq is an RNA chaperone that serves as a master regulator of bacterial physiology. Here we show that in the opportunistic pathogen Pseudomonas aeruginosa, the loss of Hfq can result in a dramatic reduction in growth in a manner that is dependent upon MexT, a transcription regulator that governs antibiotic resistance in this organism. Using a combination of chromatin immunoprecipitation with high-throughput sequencing and transposon insertion sequencing, we identify the MexT-activated genes responsible for mediating the growth defect of hfq mutant cells. These include a newly identified MexT-controlled gene that we call hilR We demonstrate that hilR encodes a small protein that is acutely toxic to wild-type cells when produced ectopically. Furthermore, we show that hilR expression is negatively regulated by Hfq, offering a possible explanation for the growth defect of hfq mutant cells. Finally, we present evidence that the expression of MexT-activated genes is dependent upon GshA, an enzyme involved in the synthesis of glutathione. Our findings suggest that Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of specific MexT-regulated genes. Moreover, our results identify glutathione to be a factor important for the in vivo activity of MexT.IMPORTANCE Here we show that the conserved RNA chaperone Hfq is important for the growth of the opportunistic pathogen Pseudomonas aeruginosa We found that the growth defect of hfq mutant cells is dependent upon the expression of genes that are under the control of the transcription regulator MexT. These include a gene that we refer to as hilR, which we show is negatively regulated by Hfq and encodes a small protein that can be toxic when ectopically produced in wild-type cells. Thus, Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of MexT-regulated genes, including one encoding a previously unrecognized small protein. We also show that MexT activity depends on an enzyme that synthesizes glutathione.

The RNase YbeY Is Vital for Ribosome Maturation, Stress Resistance, and Virulence of the Natural Genetic Engineer Agrobacterium tumefaciens.

Riboregulation involving regulatory RNAs, RNA chaperones, and ribonucleases is fundamental for the rapid adaptation of gene expression to changing environmental conditions. The gene coding for the RNase YbeY belongs to the minimal prokaryotic genome set and has a profound impact on physiology in a wide range of bacteria. Here, we show that the Agrobacterium tumefaciensybeY gene is not essential. Deletion of the gene in the plant pathogen reduced growth, motility, and stress tolerance. Most interestingly, YbeY is crucial for A. tumefaciens-mediated T-DNA transfer and tumor formation. Comparative proteomics by using isobaric tags for relative and absolute quantitation (iTRAQ) revealed dysregulation of 59 proteins, many of which have previously been found to be dependent on the RNA chaperone Hfq. YbeY and Hfq have opposing effects on production of these proteins. Accumulation of a 16S rRNA precursor in the ybeY mutant suggests that A. tumefaciens YbeY is involved in rRNA processing. RNA coimmunoprecipitation-sequencing (RIP-Seq) showed binding of YbeY to the region immediately upstream of the 16S rRNA. Purified YbeY is an oligomer with RNase activity. It does not physically interact with Hfq and thus plays a partially overlapping but distinct role in the riboregulatory network of the plant pathogen.IMPORTANCE Although ybeY gene belongs to the universal bacterial core genome, its biological function is incompletely understood. Here, we show that YbeY is critical for fitness and host-microbe interaction in the plant pathogen Agrobacterium tumefaciens Consistent with the reported endoribonuclease activity of YbeY, A. tumefaciens YbeY acts as a RNase involved in maturation of 16S rRNA. This report adds a worldwide plant pathogen and natural genetic engineer of plants to the growing list of bacteria that require the conserved YbeY protein for host-microbe interaction.

Hfq, a RNA Chaperone, Contributes to Virulence by Regulating Plant Cell Wall-Degrading Enzyme Production, Type VI Secretion System Expression, Bacterial Competition, and Suppressing Host Defense Response in Pectobacterium carotovorum.

Hfq is a RNA chaperone and participates in a wide range of cellular processes and pathways. In this study, mutation of hfq gene from Pectobacterium carotovorum subsp. carotovorum PccS1 led to significantly reduced virulence and plant cell wall-degrading enzyme (PCWDE) activities. In addition, the mutant exhibited decreased biofilm formation and motility and greatly attenuated carbapenem production as well as secretion of hemolysin coregulated protein (Hcp) as compared with wild-type strain PccS1. Moreover, a higher level of callose deposition was induced in Nicotiana benthamiana leaves when infiltrated with the mutant. A total of 26 small (s)RNA deletion mutants were obtained among a predicted 27 sRNAs, and three mutants exhibited reduced virulence in the host plant. These results suggest that hfq plays a key role in Pectobacterium virulence by positively impacting PCWDE production, secretion of the type VI secretion system, bacterial competition, and suppression of host plant responses.

Functional analyses of putative PalS (Palindromic Self-recognition) motifs in bacterial Hfq.

The bacterial protein Hfq has been linked to nucleic acid metabolism and signaling, however its explicit role has been elusive. Recently it was proposed that the C-termini of Hfq subunits in Hfq6 complexes could be involved in functional interactions with other Hfq hexamers and/or nucleic acids. To test the proposed model of the native Hfq complex experimentally, we genetically engineered chimeric Hfq6 complexes, in which C-termini of bacterial Hfq subunits were substituted with a sequence derived from human histone H2B (hH2B) that includes multiple functionally significant amino acids whose modifications have been linked to carcinogenesis. We demonstrate that this substitution results in an enhanced formation of dodecameric assemblies by the Hfq-hH2B hybrid - a result pointing to the possibility of a (functional) homology between these motifs in proteins from distant kingdoms. We hypothesize that these putative Palindromic Self-recognition (PalS) motifs could act as proteins' 'cohesive ends' that could allow the protein complexes carrying such motifs to interact dynamically and dissociate-reassociate in response to stress and/or growth phase-specific changes. We provide experimental support to the latter hypothesis and demonstrate that in E. coli the dodecameric Hfq assemblies are formed in a growth stage-specific manner. We describe a refined system - consisting solely of purified Hfq, polynucleotide phosporylase (PNP) and ADP - that allows reconstitution in vitro of characteristic 'SDS-insensitive' Hfq6-Hfq6 assemblies observed in experiments with whole-cell extracts obtained from exponentially-growing cells. We also optimized conditions for the extraction of intact native dodecameric Hfq complexes.

The Small RNA GcvB Promotes Mutagenic Break Repair by Opposing the Membrane Stress Response.

Microbes and human cells possess mechanisms of mutagenesis activated by stress responses. Stress-inducible mutagenesis mechanisms may provide important models for mutagenesis that drives host-pathogen interactions, antibiotic resistance, and possibly much of evolution generally. In Escherichia coli, repair of DNA double-strand breaks is switched to a mutagenic mode, using error-prone DNA polymerases, via the SOS DNA damage and general (σS) stress responses. We investigated small RNA (sRNA) clients of Hfq, an RNA chaperone that promotes mutagenic break repair (MBR), and found that GcvB promotes MBR by allowing a robust σS response, achieved via opposing the membrane stress (σE) response. Cells that lack gcvB were MBR deficient and displayed reduced σS-dependent transcription but not reduced σS protein levels. The defects in MBR and σS-dependent transcription in ΔgcvB cells were alleviated by artificially increasing σS levels, implying that GcvB promotes mutagenesis by allowing a normal σS response. ΔgcvB cells were highly induced for the σE response, and blocking σE response induction restored both mutagenesis and σS-promoted transcription. We suggest that GcvB may promote the σS response and mutagenesis indirectly, by promoting membrane integrity, which keeps σE levels lower. At high levels, σE might outcompete σS for binding RNA polymerase and so reduce the σS response and mutagenesis. The data show the delicate balance of stress response modulation of mutagenesis. IMPORTANCE: Mutagenesis mechanisms upregulated by stress responses promote de novo antibiotic resistance and cross-resistance in bacteria, antifungal drug resistance in yeasts, and genome instability in cancer cells under hypoxic stress. This paper describes the role of a small RNA (sRNA) in promoting a stress-inducible-mutagenesis mechanism, mutagenic DNA break repair in Escherichia coli The roles of many sRNAs in E. coli remain unknown. This study shows that ΔgcvB cells, which lack the GcvB sRNA, display a hyperactivated membrane stress response and reduced general stress response, possibly because of sigma factor competition for RNA polymerase. This results in a mutagenic break repair defect. The data illuminate a function of GcvB sRNA in opposing the membrane stress response, and thus indirectly upregulating mutagenesis.

HCF1 and OCT2 Cooperate with EBNA1 To Enhance OriP-Dependent Transcription and Episome Maintenance of Latent Epstein-Barr Virus.

UNLABELLED: Epstein-Barr virus (EBV) establishes latent infections as multicopy episomes with complex patterns of viral gene transcription and chromatin structure. The EBV origin of plasmid replication (OriP) has been implicated as a critical control element for viral transcription, as well as viral DNA replication and episome maintenance. Here, we examine cellular factors that bind OriP and regulate histone modification, transcription regulation, and episome maintenance. We found that OriP is enriched for histone H3 lysine 4 (H3K4) methylation in multiple cell types and latency types. Host cell factor 1 (HCF1), a component of the mixed-lineage leukemia (MLL) histone methyltransferase complex, and transcription factor OCT2 (octamer-binding transcription factor 2) bound cooperatively with EBNA1 (Epstein-Barr virus nuclear antigen 1) at OriP. Depletion of OCT2 or HCF1 deregulated latency transcription and histone modifications at OriP, as well as the OriP-regulated latency type-dependent C promoter (Cp) and Q promoter (Qp). HCF1 depletion led to a loss of histone H3K4me3 (trimethylation of histone H3 at lysine 4) and H3 acetylation at Cp in type III latency and Qp in type I latency, as well as an increase in heterochromatic H3K9me3 at these sites. HCF1 depletion resulted in the loss of EBV episomes from Burkitt's lymphoma cells with type I latency and reactivation from lymphoblastoid cells (LCLs) with type III latency. These findings indicate that HCF1 and OCT2 function at OriP to regulate viral transcription, histone modifications, and episome maintenance. As HCF1 is best known for its function in herpes simplex virus 1 (HSV-1) immediate early gene transcription, our findings suggest that EBV latency transcription shares unexpected features with HSV gene regulation. IMPORTANCE: EBV latency is associated with several human cancers. Viral latent cycle gene expression is regulated by the epigenetic control of the OriP enhancer region. Here, we show that cellular factors OCT2 and HCF1 bind OriP in association with EBNA1 to maintain elevated histone H3K4me3 and transcriptional enhancer function. HCF1 is known as a transcriptional coactivator of herpes simplex virus (HSV) immediate early (IE) transcription, suggesting that OriP enhancer shares aspects of HSV IE transcription control.

Changing the Codon Usage of hfq Gene has Profound Effect on Phenotype and Pathogenicity of Salmonella Typhimurium.

Genome recoding with bias codons (synonymous rare codons) or codon pair bias is being used as a method to attenuate virulence mostly in viruses. The target gene chosen for attenuation in general in bacteria is mostly toxin or virulence gene. We have used RNA chaperone hfq, a global post-transcriptional regulator of bacterial gene expression that regulates about 20 % genes in Salmonella, as the target of recoding. The hfq gene was recoded by replacing the codons of hfq gene with synonymous rare codons. Recoding decreased the expression of Hfq protein about two-fold in the mutant as compared to the parent strain. Recoding did not affect growth kinetics, but in growth competition the mutant strain was outcompeted by the parent strain. There was significant decrease in survivability of mutant strain in macrophage as compared to the parent strain. The biofilm formation was significantly impaired in case of recoded mutant. The mutants were also less motile as compared to the parent strain. Intraperitoneal infection of mice with the mutant strain had shown better survival as compared to parent strain. The results show that recoding is an effective method of reducing virulence.

Hfq plays important roles in virulence and stress adaptation in Cronobacter sakazakii ATCC 29544.

Cronobacter spp. are opportunistic pathogens that cause neonatal meningitis and sepsis with high mortality in neonates. Despite the peril associated with Cronobacter infection, the mechanisms of pathogenesis are still being unraveled. Hfq, which is known as an RNA chaperone, participates in the interaction with bacterial small RNAs (sRNAs) to regulate posttranscriptionally the expression of various genes. Recent studies have demonstrated that Hfq contributes to the pathogenesis of numerous species of bacteria, and its roles are varied between bacterial species. Here, we tried to elucidate the role of Hfq in C. sakazakii virulence. In the absence of hfq, C. sakazakii was highly attenuated in dissemination in vivo, showed defects in invasion (3-fold) into animal cells and survival (10(3)-fold) within host cells, and exhibited low resistance to hydrogen peroxide (10(2)-fold). Remarkably, the loss of hfq led to hypermotility on soft agar, which is contrary to what has been observed in other pathogenic bacteria. The hyperflagellated bacteria were likely to be attributable to the increased transcription of genes associated with flagellar biosynthesis in a strain lacking hfq. Together, these data strongly suggest that hfq plays important roles in the virulence of C. sakazakii by participating in the regulation of multiple genes.

Transcriptional profiling of Bordetella pertussis reveals requirement of RNA chaperone Hfq for Type III secretion system functionality.

Bordetella pertussis, the causative agent of human whooping cough (pertussis) produces a complex array of virulence factors in order to establish efficient infection in the host. The RNA chaperone Hfq and small regulatory RNAs are key players in posttranscriptional regulation in bacteria and have been shown to play an essential role in virulence of a broad spectrum of bacterial pathogens. This study represents the first attempt to characterize the Hfq regulon of the human pathogen B. pertussis under laboratory conditions as well as upon passage in the host and indicates that loss of Hfq has a profound effect on gene expression in B. pertussis. Comparative transcriptional profiling revealed that Hfq is required for expression of several virulence factors in B. pertussis cells including the Type III secretion system (T3SS). In striking contrast to the wt strain, T3SS did not become operational in the hfq mutant passaged either through mice or macrophages thereby proving that Hfq is required for the functionality of the B. pertussis T3SS. Likewise, expression of virulence factors vag8 and tcfA encoding autotransporter and tracheal colonization factor, respectively, was strongly reduced in the hfq mutant. Importantly, for the first time we demonstrate that B. pertussis T3SS can be activated upon contact with macrophage cells in vitro.

Structure of bacterial regulatory RNAs determines their performance in competition for the chaperone protein Hfq.

Bacterial regulatory RNAs require the chaperone protein Hfq to enable their pairing to mRNAs. Recent data showed that there is a hierarchy among sRNAs in the competition for access to Hfq, which could be important for the tuning of sRNA-dependent translation regulation. Here, seven structurally different sRNAs were compared using filter-based competition assays. Moreover, chimeric sRNA constructs were designed to identify structure elements important for competition performance. The data showed that besides the 3'-terminal oligouridine sequences also the 5'-terminal structure elements of sRNAs were essential for their competition performance. When the binding of sRNAs to Hfq mutants was compared, the data showed the important role of the proximal and rim sites of Hfq for the binding of six out of seven sRNAs. However, ChiX sRNA, which was the most efficient competitor, bound Hfq in a unique way using the opposite-distal and proximal-faces of this ring-shaped protein. The data indicated that the simultaneous binding to the opposite faces of Hfq was enabled by separate adenosine-rich and uridine-rich sequences in the long, single-stranded region of ChiX. Overall, the results suggest that the individual structural composition of sRNAs serves to tune their performance to different levels resulting in a hierarchy of sRNAs in the competition for access to the Hfq protein.

Binding of the RNA chaperone Hfq to the type IV pilus base is crucial for its function in Synechocystis sp. PCC 6803.

The bacterial RNA-binding protein Hfq functions in post-transcriptional regulation of gene expression. There is evidence in a range of bacteria for specific subcellular localization of Hfq; however, the mechanism and role of Hfq localization remain unclear. Cyanobacteria harbour a subfamily of Hfq that is structurally conserved but exhibits divergent RNA binding sites. Mutational analysis in the cyanobacterium Synechocystis sp. PCC 6803 revealed that several conserved amino acids on the proximal side of the Hfq hexamer are crucial not only for Hfq-dependent RNA accumulation but also for phototaxis, the latter of which depends on type IV pili. Co-immunoprecipitation and yeast two-hybrid analysis show that the secretion ATPase PilB1 (a component of the type IV pilus base) is an interaction partner of Hfq. Fluorescence microscopy revealed that Hfq is localized to the cytoplasmic membrane in a PilB1-dependent manner. Concomitantly, Hfq-dependent RNA accumulation is abrogated in a ΔpilB1 mutant, indicating that localization to the pilus base via interaction with PilB1 is essential for Hfq function in cyanobacteria.

The RNA chaperone Hfq regulates expression of fimbrial-related genes and virulence of Salmonella enterica serovar Enteritidis.

Salmonella Enteritidis is an intracellular pathogen that causes enteritis and systemic disease in humans and other animals. The RNA chaperone protein Hfq mediates the binding of small noncoding RNAs to target mRNA and assists in post-transcriptional gene regulation in bacteria. In this study, we constructed an hfq deletion mutant in S. Enteritidis SE50336 and analyzed the expression of major fimbrial subunits sefA, bcfA, fimA, safA, stbA, sthA, csgA, csgD, and pegA using quantitative real-time PCR. The gene expression of sefA increased about 14-fold in the hfq mutant, as compared with its expression in the wild-type strain. The expression of fimA and pegA did not change significantly, while the expression of the other fimbrial genes was significantly down-regulated in the hfq mutant. The ability of SE50336Δhfq adhering to Caco-2 cells was also reduced as compared with wild-type adherence. The virulence of the hfq mutant was significantly reduced in a 1-day-old chicken model of S. Enteritidis disease, as determined by quantifying the lethal dose 50% of the bacterial strains. We conclude that Hfq critically contributes to S. Enteritidis virulence, likely partially affected by regulating fimbrial gene expression.

The RNA chaperone Hfq promotes fitness of Actinobacillus pleuropneumoniae during porcine pleuropneumonia.

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-ß-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.

Brucella melitensis 16MΔhfq attenuation confers protection against wild-type challenge in BALB/c mice.

Brucellosis is a globally distributed zoonotic disease that causes animal and human diseases. Although effective, the current Brucella vaccines (Rev.1 and M5-90) have several drawbacks. The first involves residual virulence for animals and humans and the second is the inability to differentiate natural infection from that caused by vaccination. Therefore, Brucella melitensis 16M hfq mutant (16MΔhfq) was constructed to overcome these drawbacks. Similarly to Rev.1 and M5-90, 16MΔhfq reduces survival in macrophages and mice and induces strong protective immunity in BALB/c mice. Moreover, these vaccines elicit anti-Brucella-specific IgG1 and IgG2a subtype responses and induce secretion of gamma interferon and interleukin-4. The Hfq antigen also allows serological differentiation between infected and vaccinated animals. These results show that 16MΔhfq is an ideal live attenuated vaccine candidate against virulent Brucella melitensis 16M infection. It will be further evaluated in sheep.