(Re)-definition of the holo- and apo-Fur direct regulons of Helicobacter pylori.

Iron homeostasis is a critical process for living organisms because this metal is an essential co-factor for fundamental biochemical activities, like energy production and detoxification, albeit its excess quickly leads to cell intoxication. The protein Fur (ferric uptake regulator) controls iron homeostasis in bacteria by switching from its apo- to holo-form as a function of the cytoplasmic level of ferrous ions, thereby modulating gene expression. The Helicobacter pylori HpFur protein has the rare ability to operate as a transcriptional commutator; apo- and holo-HpFur function as two different repressors with distinct DNA binding recognition properties for specific sets of target genes. Although the regulation of apo- and holo-HpFur in this bacterium has been extensively investigated, we propose a genome-wide redefinition of holo-HpFur direct regulon in H. pylori by integration of RNA-seq and ChIP-seq data, and a large extension of the apo-HpFur direct regulon. We show that in response to iron availability, new coding sequences, non-coding RNAs, toxin-antitoxin systems, and transcripts within open reading frames are directly regulated by apo- or holo-HpFur. These new targets and the more thorough validation and deeper characterization of those already known provide a complete and updated picture of the direct regulons of this two-faced transcriptional regulator.

Levofloxacin and Ciprofloxacin Co-Crystals with Flavonoids: Solid-State Investigation for a Multitarget Strategy against Helicobacter pylori.

In this paper, we address the problem of antimicrobial resistance in the case of Helicobacter pylori with a crystal engineering approach. Two antibiotics of the fluoroquinolone class, namely, levofloxacin (LEV) and ciprofloxacin (CIP), have been co-crystallized with the flavonoids quercetin (QUE), myricetin (MYR), and hesperetin (HES), resulting in the formation of four co-crystals, namely, LEV∙QUE, LEV∙MYR, LEV2∙HES, and CIP∙QUE. The co-crystals were obtained from solution, slurry, or mechanochemical mixing of the reactants. LEV∙QUE and LEV∙MYR were initially obtained as the ethanol solvates LEV∙QUE∙xEtOH and LEV∙MYR∙xEtOH, respectively, which upon thermal treatment yielded the unsolvated forms. All co-crystals were characterized by powder X-ray diffraction and thermal gravimetric analysis. The antibacterial performance of the four co-crystals LEV∙QUE, LEV∙MYR, LEV2∙HES, and CIP∙QUE in comparison with that of the physical mixtures of the separate components was tested via evaluation of the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). The results obtained indicate that the association with the co-formers, whether co-crystallized or forming a physical mixture with the active pharmaceutical ingredients (API), enhances the antimicrobial activity of the fluoroquinolones, allowing them to significantly reduce the amount of API otherwise required to display the same activity against H. pylori.

Insights into the Orchestration of Gene Transcription Regulators in Helicobacter pylori.

Bacterial pathogens employ a general strategy to overcome host defenses by coordinating the virulence gene expression using dedicated regulatory systems that could raise intricate networks. During the last twenty years, many studies of Helicobacter pylori, a human pathogen responsible for various stomach diseases, have mainly focused on elucidating the mechanisms and functions of virulence factors. In parallel, numerous studies have focused on the molecular mechanisms that regulate gene transcription to attempt to understand the physiological changes of the bacterium during infection and adaptation to the environmental conditions it encounters. The number of regulatory proteins deduced from the genome sequence analyses responsible for the correct orchestration of gene transcription appears limited to 14 regulators and three sigma factors. Furthermore, evidence is accumulating for new and complex circuits regulating gene transcription and H. pylori virulence. Here, we focus on the molecular mechanisms used by H. pylori to control gene transcription as a function of the principal environmental changes.

Targeting the Essential Transcription Factor HP1043 of Helicobacter pylori: A Drug Repositioning Study.

Antibiotic-resistant bacterial pathogens are a very challenging problem nowadays. Helicobacter pylori is one of the most widespread and successful human pathogens since it colonizes half of the world population causing chronic and atrophic gastritis, peptic ulcer, mucosa-associated lymphoid tissue-lymphoma, and even gastric adenocarcinoma. Moreover, it displays resistance to numerous antibiotics. One of the H. pylori pivotal transcription factors, HP1043, plays a fundamental role in regulating essential cellular processes. Like other bacterial transcription factors, HP1043 does not display a eukaryote homolog. These characteristics make HP1043 a promising candidate to develop novel antibacterial strategies. Drug repositioning is a relatively recent strategy employed in drug development; testing approved drugs on new targets considerably reduces the time and cost of this process. The combined computational and in vitro approach further reduces the number of compounds to be tested in vivo. Our aim was to identify a subset of known drugs able to prevent HP1043 binding to DNA promoters. This result was reached through evaluation by molecular docking the binding capacity of about 14,350 molecules on the HP1043 dimer in both conformations, bound and unbound to the DNA. Employing an ad hoc pipeline including MMGBSA molecular dynamics, a selection of seven drugs was obtained. These were tested in vitro by electrophoretic mobility shift assay to evaluate the HP1043-DNA interaction. Among these, three returned promising results showing an appreciable reduction of the DNA-binding activity of HP1043. Overall, we applied a computational methodology coupled with experimental validation of the results to screen a large number of known drugs on one of the H. pylori essential transcription factors. This methodology allowed a rapid reduction of the number of drugs to be tested, and the drug repositioning approach considerably reduced the drug design costs. Identified drugs do not belong to the same pharmaceutical category and, by computational analysis, bound different cavities, but all display a reduction of HP1043 binding activity on the DNA.

Multilayer Regulation of Neisseria meningitidis NHBA at Physiologically Relevant Temperatures.

Neisseria meningitidis colonizes the nasopharynx of humans, and pathogenic strains can disseminate into the bloodstream, causing septicemia and meningitis. NHBA is a surface-exposed lipoprotein expressed by all N. meningitidis strains in different isoforms. Diverse roles have been reported for NHBA in heparin-mediated serum resistance, biofilm formation, and adherence to host tissues. We determined that temperature controls the expression of NHBA in all strains tested, with increased levels at 30−32 °C compared to 37 °C. Higher NHBA expression at lower temperatures was measurable both at mRNA and protein levels, resulting in higher surface exposure. Detailed molecular analysis indicated that multiple molecular mechanisms are responsible for the thermoregulated NHBA expression. The comparison of mRNA steady-state levels and half-lives at 30 °C and 37 °C demonstrated an increased mRNA stability/translatability at lower temperatures. Protein stability was also impacted, resulting in higher NHBA stability at lower temperatures. Ultimately, increased NHBA expression resulted in higher susceptibility to complement-mediated killing. We propose that NHBA regulation in response to temperature downshift might be physiologically relevant during transmission and the initial step(s) of interaction within the host nasopharynx. Together these data describe the importance of NHBA both as a virulence factor and as a vaccine antigen during neisserial colonization and invasion.

Moraxella catarrhalis evades neutrophil oxidative stress responses providing a safer niche for nontypeable Haemophilus influenzae.

Moraxella catarrhalis and nontypeable Haemophilus influenzae (NTHi) are pathogenic bacteria frequently associated with exacerbation of chronic obstructive pulmonary disease (COPD), whose hallmark is inflammatory oxidative stress. Neutrophils produce reactive oxygen species (ROS) which can boost antimicrobial response by promoting neutrophil extracellular traps (NET) and autophagy. Here, we showed that M. catarrhalis induces less ROS and NET production in differentiated HL-60 cells compared to NTHi. It is also able to actively interfere with these responses in chemically activated cells in a phagocytosis and opsonin-independent and contact-dependent manner, possibly by engaging host immunosuppressive receptors. M. catarrhalis subverts the autophagic pathway of the phagocytic cells and survives intracellularly. It also promotes the survival of NTHi which is otherwise susceptible to the host antimicrobial arsenal. In-depth understanding of the immune evasion strategies exploited by these two human pathogens could suggest medical interventions to tackle COPD and potentially other diseases in which they co-exist.

Targeting of Regulators as a Promising Approach in the Search for Novel Antimicrobial Agents.

Since the discovery of penicillin in the first half of the last century, antibiotics have become the pillars of modern medicine for fighting bacterial infections. However, pathogens resistant to antibiotic treatment have increased in recent decades, and efforts to discover new antibiotics have decreased. As a result, it is becoming increasingly difficult to treat bacterial infections successfully, and we look forward to more significant efforts from both governments and the scientific community to research new antibacterial drugs. This perspective article highlights the high potential of bacterial transcriptional and posttranscriptional regulators as targets for developing new drugs. We highlight some recent advances in the search for new compounds that inhibit their biological activity and, as such, appear very promising for treating bacterial infections.

Feeling the Heat: The Campylobacter jejuni HrcA Transcriptional Repressor Is an Intrinsic Protein Thermosensor.

The heat-shock response, a universal protective mechanism consisting of a transcriptional reprogramming of the cellular transcriptome, results in the accumulation of proteins which counteract the deleterious effects of heat-stress on cellular polypeptides. To quickly respond to thermal stress and trigger the heat-shock response, bacteria rely on different mechanisms to detect temperature variations, which can involve nearly all classes of biological molecules. In Campylobacter jejuni the response to heat-shock is transcriptionally controlled by a regulatory circuit involving two repressors, HspR and HrcA. In the present work we show that the heat-shock repressor HrcA acts as an intrinsic protein thermometer. We report that a temperature upshift up to 42 °C negatively affects HrcA DNA-binding activity to a target promoter, a condition required for de-repression of regulated genes. Furthermore, we show that this impairment of HrcA binding at 42 °C is irreversible in vitro, as DNA-binding was still not restored by reversing the incubation temperature to 37 °C. On the other hand, we demonstrate that the DNA-binding activity of HspR, which controls, in combination with HrcA, the transcription of chaperones' genes, is unaffected by heat-stress up to 45 °C, portraying this master repressor as a rather stable protein. Additionally, we show that HrcA binding activity is enhanced by the chaperonin GroE, upon direct protein-protein interaction. In conclusion, the results presented in this work establish HrcA as a novel example of intrinsic heat-sensing transcriptional regulator, whose DNA-binding activity is positively modulated by the GroE chaperonin.

Definition of the Binding Architecture to a Target Promoter of HP1043, the Essential Master Regulator of Helicobacter pylori.

HP1043 is an essential orphan response regulator of Helicobacter pylori orchestrating multiple crucial cellular processes. Classified as a member of the OmpR/PhoB family of two-component systems, HP1043 exhibits a highly degenerate receiver domain and evolved to function independently of phosphorylation. Here, we investigated the HP1043 binding mode to a target sequence in the hp1227 promoter (Php1227). Scanning mutagenesis of HP1043 DNA-binding domain and consensus sequence led to the identification of residues relevant for the interaction of the protein with a target DNA. These determinants were used as restraints to guide a data-driven protein-DNA docking. Results suggested that, differently from most other response regulators of the same family, HP1043 binds in a head-to-head conformation to the Php1227 target promoter. HP1043 interacts with DNA largely through charged residues and contacts with both major and minor grooves of the DNA are required for a stable binding. Computational alanine scanning on molecular dynamics trajectory was performed to corroborate our findings. Additionally, in vitro transcription assays confirmed that HP1043 positively stimulates the activity of RNA polymerase.

Deconvolution of intergenic polymorphisms determining high expression of Factor H binding protein in meningococcus and their association with invasive disease.

Neisseria meningitidis is a strictly human pathogen and is the major cause of septicemia and meningitis worldwide. Factor H binding protein (fHbp) is a meningococcal surface-exposed lipoprotein that binds the human Complement factor H allowing the bacterium to evade the host innate immune response. FHbp is also a key antigen in two vaccines against N. meningitidis serogroup B. Although the fHbp gene is present in most circulating meningococcal strains, level of fHbp expression varies among isolates and has been correlated to differences in promoter sequences upstream of the gene. Here we elucidated the sequence determinants that control fHbp expression in globally circulating strains. We analyzed the upstream fHbp intergenic region (fIR) of more than 5800 strains representative of the UK circulating isolates and we identified eleven fIR sequence alleles which represent 88% of meningococcal strains. By engineering isogenic recombinant strains where fHbp expression was under the control of each of the eleven fIR alleles, we confirmed that the fIR sequence determines a specific and distinct level of expression. Moreover, we identified the molecular basis for variation in expression through polymorphisms within key regulatory regions that are known to affect fHbp expression. We experimentally established three expression groups, high-medium-low, that correlated directly with the susceptibility to killing mediated by anti-fHbp antibodies and the ability of the meningococcal strain to survive within human serum. By using this sequence classification and information about the variant, we predicted fHbp expression in the panel of UK strains and we observed that strains with higher expressing fIR alleles are more likely associated with invasive disease. Overall, our findings can contribute to understand and predict vaccine coverage mediated by fHbp as well as to shed light on the role of this virulence factor in determining an invasive phenotype.

Cooperative Regulation of Campylobacter jejuni Heat-Shock Genes by HspR and HrcA.

The heat-shock response is defined by the transient gene-expression program that leads to the rapid accumulation of heat-shock proteins. This evolutionary conserved response aims at the preservation of the intracellular environment and represents a crucial pathway during the establishment of host-pathogen interaction. In the food-borne pathogen Campylobacter jejuni two transcriptional repressors, named HspR and HrcA, are involved in the regulation of the major heat-shock genes. However, the molecular mechanism underpinning HspR and HrcA regulatory function has not been defined yet. In the present work, we assayed and mapped the HspR and HrcA interactions on heat-shock promoters by high-resolution DNase I footprintings, defining their regulatory circuit, which governs C. jejuni heat-shock response. We found that, while DNA-binding of HrcA covers a compact region enclosing a single inverted repeat similar to the so-called Controlling Inverted Repeat of Chaperone Expression (CIRCE) sequence, HspR interacts with multiple high- and low-affinity binding sites, which contain HspR Associated Inverted Repeat (HAIR)-like sequences. We also explored the DNA-binding properties of the two repressors competitively on their common targets and observed, for the first time, that HrcA and HspR can directly interact and their binding on co-regulated promoters occurs in a cooperative manner. This mutual cooperative mechanism of DNA binding could explain the synergic repressive effect of HspR and HrcA observed in vivo on co-regulated promoters. Peculiarities of the molecular mechanisms exerted by HspR and HrcA in C. jejuni are compared to the closely related bacterium H. pylori that uses homologues of the two regulators.

The Helicobacter pylori HspR-Modulator CbpA Is a Multifunctional Heat-Shock Protein.

The medically important human pathogen Helicobacter pylori relies on a collection of highly conserved heat-shock and chaperone proteins to preserve the integrity of cellular polypeptides and to control their homeostasis in response to external stress and changing environmental conditions. Among this set of chaperones, the CbpA protein has been shown to play a regulatory role in heat-shock gene regulation by directly interacting with the master stress-responsive repressor HspR. Apart from this regulatory role, little is known so far about CbpA functional activities. Using biochemistry and molecular biology approaches, we have started the in vitro functional characterization of H. pylori CbpA. Specifically, we show that CbpA is a multifunctional protein, being able to bind DNA and to stimulate the ATPase activity of the major chaperone DnaK. In addition, we report a preliminary observation suggesting that CbpA DNA-binding activity can be affected by the direct interaction with the heat-shock master repressor HspR, supporting the hypothesis of a reciprocal crosstalk between these two proteins. Thus, our work defines novel functions for H. pylori CbpA and stimulates further studies aimed at the comprehension of the complex regulatory interplay among chaperones and heat-shock transcriptional regulators.

Helicobacter pylori Stress-Response: Definition of the HrcA Regulon.

Bacteria respond to different environmental stresses by reprogramming the transcription of specific genes whose proper expression is critical for their survival. In this regard, the heat-shock response, a widespread protective mechanism, triggers a sudden increase in the cellular concentration of different proteins, including molecular chaperones and proteases, to preserve protein folding and maintain cellular homeostasis. In the medically important gastric pathogen Helicobacter pylori the regulation of the principal heat-shock genes is under the transcriptional control of two repressor proteins named HspR and HrcA. To define the HrcA regulon, we carried out whole transcriptome analysis through RNA-sequencing, comparing the transcriptome of the H. pylori G27 wild type strain to that of the isogenic hrcA-knockout strain. Overall, differential gene expression analysis outlined 49 genes to be deregulated upon hrcA gene inactivation. Interestingly, besides controlling the transcription of genes coding for molecular chaperones and stress-related mediators, HrcA is involved in regulating the expression of proteins whose function is linked to several cellular processes crucial for bacterial survival and virulence. These include cell motility, membrane transporters, Lipopolysaccharide modifiers and adhesins. The role of HrcA as a central regulator of H. pylori transcriptome, as well as its interconnections with the HspR regulon are here analyzed and discussed. As the HrcA protein acts as a pleiotropic regulator, influencing the expression of several stress-unrelated genes, it may be considered a promising target for the design of new antimicrobial strategies.

Absence of Protein A Expression Is Associated With Higher Capsule Production in Staphylococcal Isolates.

Staphylococcus aureus is a major human pathogen, and a leading cause of soft tissue and blood stream infections. One of the causes of its success as a pathogen is the peculiar array of immune evasion factors through which the bacterium avoids host defenses, where the staphylococcal protein A (SpA) plays a major role thanks to its IgG binding activities. Moreover, SpA has recently been proposed as a promising vaccine antigen. In this study, we evaluated the expression of SpA in a collection of staphylococcal strains, about 7% of which did not express SpA (SpA- strains), despite the presence of the gene. By a comparative genomic analysis, we identified that a mutation in the spa 5' UTR sequence affecting the RBS is responsible for the loss of SpA in a subset of SpA- strains. Using a high-throughput qRT-PCR approach on a selected panel of virulence-related genes, we identified that the SpA- phenotype is associated with lower spa transcript levels and increased expression and production of capsule as well as other changes in the transcription of several key virulence factors. Our data suggest that the SpA- phenotype has occurred in geographically distinct strains through different molecular mechanisms including both mutation, leading likely to translation alterations, and transcriptional deregulation. Furthermore, we provide evidence that SpA- strains are highly susceptible to phagocytic uptake mediated by anti-capsule antibodies. These data suggest that S. aureus may alter its virulence factor expression pattern as an adaptation to the host or environment. Vaccination strategies targeting both SpA and capsule could therefore result in broader coverage against staphylococcal isolates than SpA alone.

The Helicobacter pylori Heat-Shock Repressor HspR: Definition of Its Direct Regulon and Characterization of the Cooperative DNA-Binding Mechanism on Its Own Promoter.

The ability of pathogens to perceive environmental conditions and modulate gene expression accordingly is a crucial feature for bacterial survival. In this respect, the heat-shock response, a universal cellular response, allows cells to adapt to hostile environmental conditions and to survive during stress. In the major human pathogen Helicobacter pylori the expression of chaperone-encoding operons is under control of two auto-regulated transcriptional repressors, HrcA and HspR, with the latter acting as the master regulator of the regulatory circuit. To further characterize the HspR regulon in H. pylori, we used global transcriptome analysis (RNA-sequencing) in combination with Chromatin Immunoprecipitation coupled with deep sequencing (ChIP-sequencing) of HspR genomic binding sites. Intriguingly, these analyses showed that HspR is involved in the regulation of different crucial cellular functions through a limited number of genomic binding sites. Moreover, we further characterized HspR-DNA interactions through hydroxyl-radical footprinting assays. This analysis in combination with a nucleotide sequence alignment of HspR binding sites, revealed a peculiar pattern of DNA protection and highlighted sequence conservation with the HAIR motif (an HspR-associated inverted repeat of Streptomyces spp.). Site-directed mutagenesis demonstrated that the HAIR motif is fundamental for HspR binding and that additional nucleotide determinants flanking the HAIR motif are required for complete binding of HspR to its operator sequence spanning over 70 bp of DNA. This finding is compatible with a model in which possibly a dimer of HspR recognizes the HAIR motif overlapping its promoter for binding and in turn cooperatively recruits two additional dimers on both sides of the HAIR motif.

The Interplay between Two Transcriptional Repressors and Chaperones Orchestrates Helicobacter pylori Heat-Shock Response.

The ability to gauge the surroundings and modulate gene expression accordingly is a crucial feature for the survival bacterial pathogens. In this respect, the heat-shock response, a universally conserved mechanism of protection, allows bacterial cells to adapt rapidly to hostile conditions and to survive during environmental stresses. The important and widespread human pathogen Helicobacter pylori enrolls a collection of highly conserved heat-shock proteins to preserve cellular proteins and to maintain their homeostasis, allowing the pathogen to adapt and survive in the hostile niche of the human stomach. Moreover, various evidences suggest that some chaperones of H. pylori may play also non-canonical roles as, for example, in the interaction with the extracellular environment. In H. pylori, two dedicated transcriptional repressors, named HspR and HrcA, homologues to well-characterized regulators found in many other bacterial species, orchestrate the regulation of heat-shock proteins expression. Following twenty years of intense research, characterized by molecular, as well as genome-wide, approaches, it is nowadays possible to appreciate the complex picture representing the heat-shock regulation in H. pylori. Specifically, the HspR and HrcA repressors combine to control the transcription of target genes in a way that the HrcA regulon results embedded within the HspR regulon. Moreover, an additional level of control of heat-shock genes' expression is exerted by a posttranscriptional feedback regulatory circuit in which chaperones interact and modulate HspR and HrcA DNA-binding activity. This review recapitulates our understanding of the roles and regulation of the most important heat-shock proteins of H. pylori, which represent a crucial virulence factor for bacterial infection and persistence in the human host.

Regulation of heat-shock genes in bacteria: from signal sensing to gene expression output.

The heat-shock response is a mechanism of cellular protection against sudden adverse environmental growth conditions and results in the prompt production of various heat-shock proteins. In bacteria, specific sensory biomolecules sense temperature fluctuations and transduce intercellular signals that coordinate gene expression outputs. Sensory biomolecules, also known as thermosensors, include nucleic acids (DNA or RNA) and proteins. Once a stress signal is perceived, it is transduced to invoke specific molecular mechanisms controlling transcription of genes coding for heat-shock proteins. Transcriptional regulation of heat-shock genes can be under either positive or negative control mediated by dedicated regulatory proteins. Positive regulation exploits specific alternative sigma factors to redirect the RNA polymerase enzyme to a subset of selected promoters, while negative regulation is mediated by transcriptional repressors. Interestingly, while various bacteria adopt either exclusively positive or negative mechanisms, in some microorganisms these two opposite strategies coexist, establishing complex networks regulating heat-shock genes. Here, we comprehensively summarize molecular mechanisms that microorganisms have adopted to finely control transcription of heat-shock genes.

Insight into the essential role of the Helicobacter pylori HP1043 orphan response regulator: genome-wide identification and characterization of the DNA-binding sites.

Many bacterial regulatory genes appear to be dispensable, as they can be deleted from the genome without loss of bacterial functionalities. In Helicobacter pylori, the hp1043 gene, also known as hsrA, is one of the transcriptional regulator that is essential for cell viability. This gene could not be deleted, nor the amount of protein modulated, supporting the hypothesis that HP1043 could be involved in the regulation of crucial cellular processes. Even though detailed structural data are available for the HP1043 protein, its targets are still ill-defined. Using Chromatin Immunoprecipitation-sequencing (ChIP-seq), one of the most powerful approaches to characterize protein-DNA interactions in vivo, we were able to identify genome-wide several new HP1043 binding sites. Moreover, in vitro DNA binding assays enabled precise mapping of the HP1043 binding sites on the new targets, revealing the presence of a conserved nucleotide sequence motif. Intriguingly, a significant fraction of the newly identified binding sites overlaps promoter regions controlling the expression of genes involved in translation. Accordingly, when protein translation was blocked, a significant induction of almost all HP1043 target genes was detected. These observations prompted us to propose HP1043 as a key regulator in H. pylori, likely involved in sensing and in coordinating the response to environmental conditions that provoke an arrest of protein synthesis. The essential role of HP1043 in coordinating central cellular processes is discussed.

HexR Controls Glucose-Responsive Genes and Central Carbon Metabolism in Neisseria meningitidis.

UNLABELLED: Neisseria meningitidis, an exclusively human pathogen and the leading cause of bacterial meningitis, must adapt to different host niches during human infection. N. meningitidis can utilize a restricted range of carbon sources, including lactate, glucose, and pyruvate, whose concentrations vary in host niches. Microarray analysis of N. meningitidis grown in a chemically defined medium in the presence or absence of glucose allowed us to identify genes regulated by carbon source availability. Most such genes are implicated in energy metabolism and transport, and some are implicated in virulence. In particular, genes involved in glucose catabolism were upregulated, whereas genes involved in the tricarboxylic acid cycle were downregulated. Several genes encoding surface-exposed proteins, including the MafA adhesins and Neisseria surface protein A, were upregulated in the presence of glucose. Our microarray analysis led to the identification of a glucose-responsive hexR-like transcriptional regulator that controls genes of the central carbon metabolism of N. meningitidis in response to glucose. We characterized the HexR regulon and showed that the hexR gene is accountable for some of the glucose-responsive regulation; in vitro assays with the purified protein showed that HexR binds to the promoters of the central metabolic operons of the bacterium. Based on DNA sequence alignment of the target sites, we propose a 17-bp pseudopalindromic consensus HexR binding motif. Furthermore, N. meningitidis strains lacking hexR expression were deficient in establishing successful bacteremia in an infant rat model of infection, indicating the importance of this regulator for the survival of this pathogen in vivo. IMPORTANCE: Neisseria meningitidis grows on a limited range of nutrients during infection. We analyzed the gene expression of N. meningitidis in response to glucose, the main energy source available in human blood, and we found that glucose regulates many genes implicated in energy metabolism and nutrient transport, as well as some implicated in virulence. We identified and characterized a transcriptional regulator (HexR) that controls metabolic genes of N. meningitidis in response to glucose. We generated a mutant lacking HexR and found that the mutant was impaired in causing systemic infection in animal models. Since N. meningitidis lacks known bacterial regulators of energy metabolism, our findings suggest that HexR plays a major role in its biology by regulating metabolism in response to environmental signals.

Global transcriptome analysis reveals small RNAs affecting Neisseria meningitidis bacteremia.

Most bacterial small RNAs (sRNAs) are post-transcriptional regulators involved in adaptive responses, controlling gene expression by modulating translation or stability of their target mRNAs often in concert with the RNA chaperone Hfq. Neisseria meningitides, the leading cause of bacterial meningitis, is able to adapt to different host niches during human infection. However, only a few sRNAs and their functions have been fully described to date. Recently, transcriptional expression profiling of N. meningitides in human blood ex vivo revealed 91 differentially expressed putative sRNAs. Here we expanded this analysis by performing a global transcriptome study after exposure of N. meningitides to physiologically relevant stress signals (e.g. heat shock, oxidative stress, iron and carbon source limitation). and we identified putative sRNAs that were differentially expressed in vitro. A set of 98 putative sRNAs was obtained by analyzing transcriptome data and 8 new sRNAs were validated, both by Northern blot and by primer extension techniques. Deletion of selected sRNAs caused attenuation of N. meningitides infection in the in vivo infant rat model, leading to the identification of the first sRNAs influencing meningococcal bacteremia. Further analysis indicated that one of the sRNAs affecting bacteremia responded to carbon source availability through repression by a GntR-like transcriptional regulator. Both the sRNA and the GntR-like regulator are implicated in the control of gene expression from a common network involved in energy metabolism.