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Traditional antimicrobial treatments consist of drugs which target different essential functions in pathogens. Nevertheless, bacteria continue to evolve new mechanisms to evade this drug-mediated killing with surprising speed on the deployment of each new drug and antibiotic worldwide, a phenomenon called antimicrobial resistance (AMR). Nowadays, AMR represents a critical health threat, for which new medical interventions are urgently needed. By 2050, it is estimated that the leading cause of death will be through untreatable AMR pathogens. Although antibiotics remain a first-line treatment, non-antibiotic therapies such as prophylactic vaccines and therapeutic monoclonal antibodies (mAbs) are increasingly interesting alternatives to limit the spread of such antibiotic resistant microorganisms. For the discovery of new vaccines and mAbs, the search for effective antigens that are able to raise protective immune responses is a challenging undertaking. In this context, outer membrane vesicles (OMV) represent a promising approach, as they recapitulate the complete antigen repertoire that occurs on the surface of Gram-negative bacteria. In this review, we present Escherichia coli and Pseudomonas aeruginosa as specific examples of key AMR threats caused by Gram-negative bacteria and we discuss the current status of mAbs and vaccine approaches under development as well as how knowledge on OMV could benefit antigen discovery strategies.

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 the antimicrobial response by promoting neutrophil extracellular traps (NET) and autophagy. Here, we showed that M. catarrhalis induces ROS and NET production in differentiated HL-60 cells to a lesser extent compared to NTHi. It is also able to actively interfere with these responses in chemically-activated cells in a phagocytosis-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. Therefore, 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-infect.

Background The display of recombinant proteins on cell surfaces has a plethora of applications including vaccine development, screening of peptide libraries, whole-cell biocatalysts and biosensor development for diagnostic, industrial or environmental purposes. In the last decades, a wide variety of surface display systems have been developed for the exposure of recombinant proteins on the surface of Escherichia coli, such as autotransporters and outer membrane proteins. Results In this study, we assess three approaches for the surface display of a panel of heterologous and homologous mature lipoproteins in E. coli: four from Neisseria meningitidis and four from the host strain that are known to be localised in the inner leaflet of the outer membrane. Constructs were made carrying the sequences coding for eight mature lipoproteins, each fused to the delivery portion of three different systems: the autotransporter adhesin involved in diffuse adherence-I (AIDA-I) from enteropathogenic E. coli, the Lpp’OmpA chimaera and a truncated form of the ice nucleation protein (INP), InaK-NC (N-terminal domain fused with C-terminal one) from Pseudomonas syringae. In contrast to what was observed for the INP constructs, when fused to the AIDA-I or Lpp’OmpA, most of the mature lipoproteins were displayed on the bacterial surface both at 37 and 25 °C as demonstrated by FACS analysis, confocal and transmission electron microscopy. Conclusions To our knowledge this is the first study that compares surface display systems using a number of passenger proteins. We have shown that the experimental conditions, including the choice of the carrier protein and the growth temperature, play an important role in the translocation of mature lipoproteins onto the bacterial surface. Despite all the optimization steps performed with the InaK-NC anchor motif, surface exposure of the passenger proteins used in this study was not achieved. For our experimental conditions, Lpp’OmpA chimaera has proved to be an efficient surface display system for the homologous passenger proteins although cell lysis and phenotype heterogeneity were observed. Finally, AIDA-I was found to be the best surface display system for mature lipoproteins (especially heterologous ones) in the E. coli host strain with no inhibition of growth and only limited phenotype heterogeneity.

Despite high vaccination coverage world-wide, whooping cough, a highly contagious disease caused by Bordetella pertussis, is recently increasing in occurrence suggesting that novel vaccine formulations targeted at the prevention of colonization and transmission should be investigated. To identify new candidates for inclusion in the acellular formulation, we used spontaneously released outer membrane vesicles (OMV)1 as a potential source of key adhesins. The enrichment of Bvg+ OMV with adhesins and the ability of anti-OMV serum to inhibit the adhesion of B. pertussis to lung epithelial cells in vitro were demonstrated. We employed a proteomic approach to identify the differentially expressed proteins in OMV purified from bacteria in the Bvg+ and Bvg− virulence phases, thus comparing the outer membrane protein pattern of this pathogen in its virulent or avirulent state. Six of the most abundant outer membrane proteins were selected as candidates to be evaluated for their adhesive properties and vaccine potential. We generated E. coli strains singularly expressing the selected proteins and assessed their ability to adhere to lung epithelial cells in vitro. Four out of the selected proteins conferred adhesive ability to E. coli. Three of the candidates were specifically detected by anti-OMV mouse serum suggesting that these proteins are immunogenic antigens able to elicit an antibody response when displayed on the OMV. Anti-OMV serum was able to inhibit only BrkA-expressing E. coli adhesion to lung epithelial cells. Finally, stand-alone immunization of mice with recombinant BrkA resulted in significant protection against infection of the lower respiratory tract after challenge with B. pertussis. Taken together, these data support the inclusion of BrkA and possibly further adhesins to the current acellular pertussis vaccines to improve the impact of vaccination on the bacterial clearance.

K.L.S. was supported by the Australian National Health and Medical Research Council (NHMRC) C. J. Martin Fellowship and Career Development Fellowship. A.F.H. was supported by a Marie Curie Fellowship (PIEF-GA-2012-328377). F.O., L.F., and S.B. were recipients of Novartis fellowships from the Ph.D. program of the University of Siena (Siena, Italy) and University of Bologna (Bologna, Italy), respectively. GNA2091 is one of the components of the 4-component meningococcal serogroup B vaccine (4CMenB) vaccine and is highly conserved in all meningococcal strains. However, its functional role has not been fully characterized. Here we show that nmb2091 is part of an operon and is cotranscribed with the nmb2089, nmb2090, and nmb2092 adjacent genes, and a similar but reduced operon arrangement is conserved in many other gram-negative bacteria. Deletion of the nmb2091 gene causes an aggregative phenotype with a mild defect in cell separation; differences in the outer membrane composition and phospholipid profile, in particular in the phosphoethanolamine levels; an increased level of outer membrane vesicles; and deregulation of the zinc-responsive genes such as znuD. Finally, the Δ2091 strain is attenuated with respect to the wild-type strain in competitive index experiments in the infant rat model of meningococcal infection. Altogether these data suggest that GNA2091 plays important roles in outer membrane architecture, biogenesis, homeostasis, and in meningococcal survival in vivo, and amodel for its role is discussed. These findings highlight the importance of GNA2091 as a vaccine component. Postprint

Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.

In vivo imaging of bioluminescent bacteria permits their visualization in infected mice, allowing spatial and temporal evaluation of infection progression. Most available bioluminescent strains were obtained by integration of the luciferase genes into the bacterial chromosome, a challenging and time-consuming approach. Recently, episomal plasmids were used, which were introduced in bacteria and expressed all genes required for bioluminescence emission. However, the plasmid was progressively lost in vitro and in vivo, if bacteria were not maintained under antibiotic selective pressure. Increased stability could be obtained inserting into the plasmid backbone sequences that assured plasmid partition between daughter bacterial cells, or caused death of bacteria that had lost the plasmid. So far, no detailed analysis was performed of either plasmid stability in vivo or contribution of different stabilizing sequence types. Here we report the construction of a plasmid, which includes the Photorhabdus luminescens lux cassette expressed under the control of a Staphylococcus aureus specific gene promoter, and toxin/antitoxin (T/A) and partition sequences (Par) conferring stability and transmissibility of the plasmid. Following infection of mice with S. aureus carrying this plasmid, we demonstrated that the promoter-lux fusion was functional in vivo, that the plasmid was retained by 70–100 % of bacterial cells 7 days post-infection, and that both stabilizing sequence types were required to maximize plasmid retention. These data suggest that the plasmid can be a valuable tool to study gene expression and bacterial spread in small laboratory animals infected with S. aureus or possibly other Gram-positive human pathogens.

Bexsero, a new vaccine against Neisseria meningitidis serogroup B (MenB), is composed of 3 main recombinant proteins and an outer membrane vesicle component. One of the main bactericidal antigens, neisseria heparin binding antigen (NHBA), is present as a fusion protein with the accessory protein genome-derived neisserial antigen (GNA) 1030 to further increase its immunogenicity. The gene encoding for GNA1030 is present and highly conserved in all Neisseria strains, and although orthologs are present in numerous species, its biologic function is unknown. Native mass spectrometry was used to demonstrate that GNA1030 forms a homodimer associated with 2 molecules of ubiquinone-8 (Ub8), a cofactor mainly involved in the electron transport chain and with antioxidant properties. Disc diffusion assays on the wild-type and knockout mutant of GNA1030, in the presence of various compounds, suggested that GNA1030 is not involved in oxidative stress or electron chain transport per se, although it contributes to constitutive refilling of the inner membrane with Ub8. These studies shed light on an accessory protein present in Bexsero and reveal functional insights into the family of related proteins. On the basis of our findings, we propose to name the protein neisseria ubiquinone binding protein (NUbp).

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein specific for Neisseria and constitutes one of the three main protein antigens of the Bexsero vaccine. Meningococcal and human proteases, cleave NHBA protein upstream or downstream of a conserved Arg-rich region, respectively. The cleavage results in the release of the C-terminal portion of the protein. The C-terminal fragment originating from the processing of meningococcal proteases, referred to as C2 fragment, exerts a toxic effect on endothelial cells altering the endothelial permeability. In this work, we reported that recombinant C2 fragment has no influence on the integrity of human airway epithelial cell monolayers, consistent with previous findings showing that Neisseria meningitidis traverses the epithelial barrier without disrupting the junctional structures. We showed that epithelial cells constantly secrete proteases responsible for a rapid processing of C2 fragment, generating a new fragment that does not contain the Arg-rich region, a putative docking domain reported to be essential for C2-mediated toxic effect. Moreover, we found that the C3-convertase of the alternative complement pathway is one of the proteases responsible for this processing. Overall, our data provide new insights on the cleavage of NHBA protein during meningococcal infection. NHBA cleavage may occur at different stages of the infection, and it likely has a different role depending on the environment the bacterium is interacting with.

Staphylococcus aureus is an opportunistic human pathogen and a major cause of invasive infections such as bacteremia, endocarditis, pneumonia, and wound infections. FhuD2 is a staphylococcal lipoprotein involved in the uptake of iron-hydroxymate and is under the control of the iron uptake regulator Fur. This protein is part of an investigational multicomponent vaccine formulation that has shown protective efficacy in several murine models of infection. Even though fhuD2 expression has been shown to be upregulated in murine kidneys infected with S. aureus , it is not known whether the bacterium undergoes increased iron deprivation during prolonged infection. Furthermore, different S. aureus infection niches might provide different environments and levels of iron availability, resulting in different fhuD2 expression patterns among organs of the same host. To address these questions, we characterized the in vitro expression of the fhuD2 gene and confirmed Fur-dependent regulation of its expression. We further investigated its expression in mice infected with a bioluminescent reporter strain of S. aureus expressing the luciferase operon under the control of the fhuD2 promoter. The emission of bioluminescence in different organs was followed over a 7-day time course, and quantitative real-time PCR analysis of the RNA transcribed from the endogenous fhuD2 gene was performed. Using this approach, we were able to show that fhuD2 expression was induced during infection in all organs analyzed and that differences in expression were observed at different time points and in different infected organs. Our data suggest that S. aureus undergoes increased iron deprivation during the progression of infection in diverse host organs and accordingly induces dedicated iron acquisition mechanisms. Since FhuD2 plays a central role in providing the pathogen with the required iron, further knowledge of the patterns of fhuD2 expression in vivo during infection will be instrumental in better defining the role of this antigen in S. aureus pathogenesis and as a vaccine antigen.

Factor H-binding protein (fHbp) is an important meningococcal vaccine antigen. Native outer membrane vesicles with over-expressed fHbp (NOMV OE fHbp) have been shown to induce antibodies with broader functional activity than recombinant fHbp (rfHbp). Improved understanding of this broad coverage would facilitate rational vaccine design. We performed a pair-wise analysis of 48 surface-exposed amino acids involved in interacting with factor H, among 383 fHbp variant group 1 sequences. We generated isogenic NOMV-producing meningococcal strains from an African serogroup W isolate, each over-expressing one of four fHbp variant group 1 sequences (ID 1, 5, 9, or 74), including those most common among invasive African meningococcal isolates. Mice were immunised with each NOMV, and sera tested for IgG levels against each of the rfHbp ID and for ability to kill a panel of heterologous meningococcal isolates. At the fH-binding site, ID pairs differed by a maximum of 13 (27%) amino acids. ID 9 shared an amino acid sequence common to 83 ID types. The selected ID types differed by up to 6 amino acids, in the fH-binding site. All NOMV and rfHbp induced high IgG levels against each rfHbp. Serum killing from mice immunised with rfHbp was generally less efficient and more restricted compared to NOMV, which induced antibodies that killed most meningococci tested, with decreased stringency for ID type differences. Breadth of killing was mostly due to anti-fHbp antibodies, with some restriction according to ID type sequence differences. Nevertheless, under our experimental conditions, no relationship between antibody cross-reactivity and variation fH-binding site sequence was identified. NOMV over-expressing different fHbp IDs belonging to variant group 1 induce antibodies with fine specificities against fHbp, and ability to kill broadly meningococci expressing heterologous fHbp IDs. The work reinforces that meningococcal NOMV with OE fHbp is a promising vaccine strategy, and provides a basis for rational selection of antigen sequence types for over-expression on NOMV.

Reverse vaccinology defines the process of antigen discovery starting from genome information. From its first application to Neisseria meningitidis group B, this approach has gradually evolved and is now accepted as a successful method of vaccine discovery, as it can be exploited to develop vaccines against many types of pathogens. Current reverse vaccinology approaches include comparative in silico analyses of multiple genome sequences in order to identify conserved antigens within a heterogeneous pathogen population and identification of antigens that are unique to pathogenic isolates but not present in commensal strains. In addition, transcriptomic and proteomic data sets are integrated into a selection process that yields a short list of candidate antigens to be tested in animal models, thus reducing the costs and time of downstream analyses. In this chapter, we will review the past and present applications of reverse vaccinology and describe the advantages, challenges and opportunities for this evolving discipline in the broader context of vaccine development.

Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis., Author Summary Serogroup B meningococcus (MenB) causes fatal sepsis and invasive meningococcal disease, particularly in young children and adolescents, as highlighted by recent MenB outbreaks in universities of the United States and Canada. The Bexsero vaccine protects against MenB and has recently been approved in > 35 countries worldwide. Neisseria adhesin A (NadA) present on the meningococcal surface can mediate binding to human cells and is one of the three MenB vaccine protein antigens. The amount of NadA exposed on the meningococcal surface also influences the antibody-mediated serum bactericidal response measured in vitro. A deep understanding of nadA expression is therefore important, otherwise the contribution of NadA to vaccine-induced protection against meningococcal meningitis may be underestimated. The abundance of surface-exposed NadA is regulated by the ligand-responsive transcriptional repressor NadR. Here, we present functional, biochemical and high-resolution structural data on NadR. Our studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. These findings shed light on the regulation of NadR, a key MarR-family virulence factor of this important human pathogen.

The Neisseria meningitidis regulator NadR was shown to repress expression of the NadA adhesin and play a major role in NadA phase-variable expression. In this study, we identified through microarray analysis over 30 genes coregulated with nadA in the NadR mutant and defined members of the NadR regulon through in vitro DNA-binding assays. Two distinct types of promoter architectures (I and II) were identified for NadR targets, differing in both the number and position of NadR-binding sites. All NadR-regulated genes investigated were found to respond to 4-hydroxyphenylacetic acid (4HPA), a small molecule secreted in human saliva, which was previously demonstrated to induce nadA expression by alleviating NadR-dependent repression. Interestingly, two types of NadR 4HPA responsive activities were found on different NadR targets corresponding to the two types of genes identified by different promoter architectures: while NadA and the majority of NadR targets (type I) are induced, only the MafA adhesins (type II) are corepressed in response to the same 4HPA signal. This alternate behavior of NadR was confirmed in a panel of strains in response to 4HPA and after incubation in saliva. The in vitro NadR binding activity at type I and type II promoter regions is differentially affected by 4HPA, suggesting that the nature of the NadR binding sites may define the regulation to which they will be subjected. We conclude that NadR coordinates a broad transcriptional response to signals present in human saliva, mimicked in vitro by 4HPA, enabling the meningococcus to adapt to the relevant host niche.

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by Neisseria meningitidis strains and an antigen of the Bexsero® vaccine. NHBA binds heparin through a conserved Arg-rich region that is the target of two proteases, the meningococcal NalP and human lactoferrin (hLf). In this work, in vitro studies showed that recombinant NHBA protein was able to bind epithelial cells and mutations of the Arg-rich tract abrogated this binding. All N-terminal and C-terminal fragments generated by NalP or hLf cleavage, regardless of the presence or absence of the Arg-rich region, did not bind to cells, indicating that a correct positioning of the Arg-rich region within the full length protein is crucial. Moreover, binding was abolished when cells were treated with heparinase III, suggesting that this interaction is mediated by heparan sulfate proteoglycans (HSPGs). N. meningitidis nhba knockout strains showed a significant reduction in adhesion to epithelial cells with respect to isogenic wild-type strains and adhesion of the wild-type strain was inhibited by anti-NHBA antibodies in a dose-dependent manner. Overall, the results demonstrate that NHBA contributes to meningococcal adhesion to epithelial cells through binding to HSPGs and suggest a possible role of anti-Bexsero® antibodies in the prevention of colonization.

Small non-coding RNAs (sRNA) are emerging as key elements of post-transcriptional gene regulation in bacteria. The conserved Hfq protein is thought to function as an RNA chaperone and facilitate base-pairing between sRNAs and mRNA targets. In this study we identify a novel sRNA of Neisseria meningitidis through global gene expression studies of regulated transcripts in the Hfq mutant. The synthesis of this sRNA, named AniS, is anaerobically induced through activation of its promoter by the FNR global regulator. Whole-genome expression analyses led to the identification of putative mRNA targets, two of which are predicted to base pair with AniS. We show that Hfq binds the AniS transcript in vitro and is necessary for the downregulation of the identified target mRNAs in vivo. Contrary to many Hfq-dependent sRNA of the Enterobacteriaceae, Hfq promotes decay of AniS in N. meningitidis. Our analysis shows that the AniS regulator is part of the FNR regulon and may be responsible for the downregulation of FNR-repressed genes. Furthermore the presence of similar conserved regulatory sequences in all Neisseria spp. to date suggests that an analogous FNR-regulated sRNA, with a variable 5' sequence, may be ubiquitous to all commensals and pathogens of the Genus.

Native outer membrane vesicles (NOMV) (not detergent treated), which are prepared from recombinant strains with attenuated endotoxin activity and overexpressed factor H binding protein (fHbp), elicited broad serum bactericidal antibody responses in mice. The amount of overexpressed fHbp required for optimal immunogenicity is not known. In this study we prepared NOMV vaccines from LpxL1 knockout (ΔLpxL1) mutants with penta-acylated lipooligosaccharide and attenuated endotoxin activity. The recombinant strains had wild-type (1×) fHbp expression or were engineered for 3-fold- or 10-fold-increased fHbp expression (3× or 10× fHbp). Control vaccines included NOMV from ΔLpxL1/ΔfHbp mutants or recombinant fHbp. In mice, only the 10× fHbp NOMV vaccine elicited significantly higher serum IgG anti-fHbp antibody titers than the corresponding 1× fHbp NOMV or recombinant fHbp vaccine. The 10× fHbp NOMV vaccine also elicited higher bactericidal responses (P< 0.05) against five group B strains with heterologous PorA than the recombinant fHbp or 1× fHbp NOMV vaccine. The 3× fHbp NOMV vaccine gave higher bactericidal titers against only one strain. Serum bactericidal titers in mice immunized with the control ΔfHbp NOMV vaccines were

Factor H binding protein (fHBP) is a surface-exposed lipoprotein in Neisseria meningitidis , which is a component of several investigational vaccines against serogroup B meningococcus (MenB) currently in development. fHBP enables the bacterium to evade complement-mediated killing by binding factor H, a key downregulator of the complement alternative pathway, and, in addition, fHBP is important for meningococcal survival in the presence of the antimicrobial peptide LL-37. In this study, we investigate the molecular mechanisms involved in transcription and regulation of the fHBP-encoding gene, fhbp . We show that the fHBP protein is expressed from two independent transcripts: one bicistronic transcript that includes the upstream gene and a second shorter monocistronic transcript from its own dedicated promoter, P fhbp . Transcription from the promoter P fhbp responds to oxygen limitation in an FNR-dependent manner, and, accordingly, the FNR protein binds to a P fhbp probe in vitro . Furthermore, expression in meningococci of a constitutively active FNR mutant results in the overexpression of the fHBP protein. Finally, the analysis of fHBP regulation was extended to a panel of strains expressing different fHBP allelic variants at different levels, and we demonstrate that FNR is involved in the regulation of this antigen in all but one of the strains tested. Our data suggest that oxygen limitation may play an important role in inducing the expression of fHBP from a dedicated FNR-regulated promoter. This implies a role for this protein in microenvironments lacking oxygen, for instance in the submucosa or intracellularly, in addition to its demonstrated role in serum resistance in the blood.

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.

Previous microarray studies have suggested that an indirect mechanism of Fur regulation may be present in meningococcus at the posttranscriptional level through a small regulatory RNA (sRNA) system analogous to that of Escherichia coli and Pseudomonas aeruginosa . Recently, a Fur-regulated sRNA, NrrF, was identified that is involved in the iron regulation of the sdhA and sdhC succinate dehydrogenase genes. Here we report a detailed transcriptional analysis of the nrrF gene and show that NrrF is a Hfq-dependent sRNA. The Hfq protein mediates nrrF downregulation and Fur-dependent upregulation of the sdhCDAB operon, the major in vivo NrrF-regulated operon. NrrF forms a duplex in vitro with a region of complementarity overlapping the sdhDA mRNA junction. Furthermore, Hfq binds to NrrF in vitro and considerably enhances the efficiency of the interaction of the sRNA with the identified target. Our data suggest that Hfq-meditated binding of NrrF to the in vivo target in the sdhCDAB mRNA may cause the rapid degradation of the transcript, resulting in Fur-dependent positive regulation of succinate dehydrogenase. In addition, while the upregulation of sodB and fumB by Fur is dependent on the Hfq protein, it is unaffected in the nrrF knockout, which suggests that there is more than one sRNA regulator involved in iron homeostasis in meningococcus.

The hrcA and hspR genes of Helicobacter pylori encode two transcriptional repressor proteins that negatively regulate expression of the groES-groEL and hrcA-grpE-dnaK operons. While HspR was previously shown to bind far upstream of the promoters transcribing these operons, the binding sites of HrcA were not identified. Here, we demonstrate by footprinting analysis that HrcA binds to operator elements similar to the so-called CIRCE sequences overlapping both promoters. Binding of HspR and HrcA to their respective operators occurs in an independent manner, but the DNA binding activity of HrcA is increased in the presence of GroESL, suggesting that the GroE chaperonin system corepresses transcription together with HrcA. Comparative transcriptome analysis of the wild-type strain and hspR and hrcA singly and doubly deficient strains revealed that a set of 14 genes is negatively regulated by the action of one or both regulators, while a set of 29 genes is positively regulated. While both positive and negative regulation of transcription by HspR and/or HrcA could be confirmed by RNA primer extension analyses for two representative genes, binding of either regulator to the promoters could not be detected, indicating that transcriptional regulation at these promoters involves indirect mechanisms. Strikingly, 14 of the 29 genes which were found to be positively regulated by HspR or HrcA code for proteins involved in flagellar biosynthesis. Accordingly, loss of motility functions was observed for HspR and HrcA single or double mutants. The possible regulatory intersections of the heat shock response and flagellar assembly are discussed.

Helicobacter pylori, a microaerophilic, gram-negative bacterium is a human pathogen that colonizes the gastric niche and is associated with several acute and chronic stomach diseases. In order to survive in the gastric environment and become pathogenic, the bacterium relies on a plethora of virulence factors, which also include heat shock proteins. We previously showed that two out of the three operons encoding the major cellular chaperone machineries are transcriptionally repressed by two regulators, HrcA and HspR. Till now, molecular studies aimed at the understanding of the role of each protein in controlling transcription was hampered by toxicity and insolubility of HrcA in heterologous expression systems. Similar problems were encountered by many other groups studying HrcA from different bacteria. In this study, we analyzed the amino acid sequence of HrcA that predicted association of this protein to the inner membrane, which was experimentally verified. Subsequently, we implemented a dedicated induction protocol which enabled the overexpression of the recombinant His-HrcA protein in the soluble fraction of Escherichia coli cells. Moreover, we developed a purification procedure for His-HrcA that allowed us to obtain highly pure preparation of the protein. The functionality of the purified protein was then confirmed with an in vitro DNA-binding assay.

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.

Iron homeostasis is particularly important in pathogenic bacteria, which need to compete with the host for this essential cofactor. In Helicobacter pylori , a causative agent of several gastric pathologies, iron uptake and storage genes are regulated at the transcriptional level by the ferric uptake regulator Fur. The regulatory circuit of Fur has recently come under focus because of an intimate interlink with a broader regulatory network governing metal homeostasis, acidic response, and virulence. To dissect the Fur regulatory circuit and identify in vivo targets of regulation, we developed a genome-wide location analysis protocol which allowed the identification of 200 genomic loci bound by Fur as well as the investigation of the binding efficiency of the protein to these loci in response to iron. Comparative analysis with transcriptomes of wild-type and fur deletion mutant strains allowed the distinction between targets associated with Fur regulation and genes indirectly influenced by the fur mutation. The Fur regulon includes 59 genes, 25 of which appear to be positively regulated. A case study conducted by primer extension analysis of two oppositely regulated genes, hpn2 and flaB , suggests that negative regulation as well as positive regulation occurs at the transcriptional level. Furthermore, the results revealed the existence of 13 Fur targeted loci within polycistronic operons, which were associated with transcript deregulation in the fur mutant strain. This study provides a systematic insight of Fur regulation at the genome-wide level in H. pylori and points to regulatory functions extending beyond the classical Fur repression paradigm.

The ferric uptake regulator Fur is a well-known iron-responsive repressor of gene transcription, which is used by many bacteria to respond to the low-iron environment that pathogens encounter during infection. In this study we used comparative transcriptome analysis to define the role of the Fur protein in the global control of gene transcription and iron regulation in Neisseria meningitidis . By using the Fur-null mutant and its complemented derivative, we identified 83 genes whose transcription is controlled by Fur. We report that Fur may control differential expression of these genes by binding directly to their promoters or through indirect mechanisms. In addition, mutation of the fur gene resulted in the induction of the heat shock response, and transcription of these genes does not respond to iron limitation. Furthermore, analysis of the iron starvation stimulon in the Fur-null mutant provided evidences of iron-responsive regulation that is independent of Fur. We began to dissect the regulatory networks of Fur and the heat shock (stress) response in N. meningitidis , and the observed interlink between the two circuits is discussed.

Two important metal-responsive regulators, NikR and Fur, are involved in nickel and iron homeostasis and controlling gene expression in Helicobacter pylori. To date, they have been implicated in the regulation of sets of overlapping genes. We have attempted here dissection of the molecular mechanisms involved in transcriptional regulation of the NikR and Fur proteins, and we investigated protein-promoter interactions of the regulators with known target genes. We show that H. pylori NikR is a tetrameric protein and, through DNase I footprinting analysis, we have identified operators for NikR to which it binds with different affinities in a metal-responsive way. Mapping of the NikR binding site upstream of the urease promoter established a direct role for NikR as a positive regulator of transcription and, through scanning mutagenesis of this binding site, we have determined two subsites that are important for the binding of the protein to its target sequence. Furthermore, by alignment of the operators for NikR, we have shown that the H. pylori protein recognizes a sequence that is distinct from its well-studied orthologue in Escherichia coli. Moreover, we show that NikR and Fur can bind independently at distinct operators and also compete for overlapping operators in some coregulated gene promoters, adding another dimension to the previous suggested link between iron and nickel regulation. Finally, the importance of an interconnection between metal-responsive gene networks for homeostasis is discussed.

The nickel-containing enzyme urease is an essential colonization factor of the human gastric pathogen Helicobacter pylori which enables the bacteria to survive the low-pH conditions of the stomach. Transcription of the urease genes is positively controlled in response to increasing concentrations of nickel ions and acidic pH. Here we demonstrate that acid-induced transcription of the urease genes is mediated directly by the ArsRS two-component system. Footprint analyses identify binding sites of the phosphorylated ArsR response regulator within the ureA and ureI promoters. Furthermore, deletion of a distal upstream ArsR binding site of the ureA promoter demonstrates its role in acid-dependent activation of the promoter. In addition, acid-induced transcription of the ureA gene is unaltered in a nikR mutant, providing evidence that pH-responsive regulation and nickel-responsive regulation of the ureA promoter are mediated by independent mechanisms involving the ArsR response regulator and the NikR protein.

It is well established that motility is an essential virulence trait of the human gastric pathogenHelicobacter pylori. Accordingly, chemotaxis contributes to the ability ofH. pylorito colonize animal infection models. Chemotactic signal transduction inH. pyloridiffers from the enterobacterial paradigm in several respects. In addition to a separate CheY response regulator protein (CheY1),H. pyloricontains a CheY-like receiver domain (CheY2) which is C-terminally fused to the histidine kinase CheA. Furthermore, the genome ofH. pyloriencodes three CheV proteins consisting of an N-terminal CheW-like domain and a C-terminal receiver domain, while there are no orthologues of the chemotaxis genescheB,cheRandcheZ. To obtain insight into the mechanisms controlling the chemotactic response ofH. pylori, we investigated the phosphotransfer reactions between the purified two-component signalling modulesin vitro. We demonstrate that both CheY1 and CheY2 are phosphorylated by CheA∼P and that the three CheV proteins mediate the dephosphorylation of CheA∼P, but with a clearly reduced efficiency as compared to CheY1 and CheY2. Furthermore, our data indicate retrophosphorylation of CheAY2 by CheY1∼P, suggesting a role of CheY2 as a phosphate sink to modulate the half-life of CheY1∼P.

We have previously shown that in the human pathogen Neisseria meningitidis group B (MenB) more than 200 genes are regulated in response to growth with iron. Among the Fur-dependent, upregulated genes identified by microarray analysis was a putative operon constituted by three genes, annotated as NMB1436, NMB1437 and NMB1438 and encoding proteins with so far unknown function. The operon was remarkably upregulated in the presence of iron and, on the basis of gel retardation analysis, its regulation was Fur dependent. In this study, we have further characterized the role of iron and Fur in the regulation of the NMB1436-38 operon and we have mapped the promoter and the Fur binding site. We also demonstrate by mutant analysis that the NMB1436-38 operon is required for protection of MenB to hydrogen peroxide-mediated killing. By using both microarray analysis and S1 mapping, we demonstrate that the operon is not regulated by oxidative stress signals. We also show that the deletion of the NMB1436-38 operon results in an impaired capacity of MenB to survive in the blood of mice using an adult mouse model of MenB infection. Finally, we show that the NMB1436-38 deletion mutant exhibits increased susceptibility to the killing activity of polymorphonuclears (PMNs), suggesting that the 'attenuated' phenotype is mediated in part by the increased sensitivity to reactive oxygen species-producing cells. This study represents one of the first examples of the use of DNA microarray to assign a biological role to hypothetical genes in bacteria.

The HspR repressor regulates transcription of the groESL , hrcA-grpE-dnaK , and cbpA-hspR-orf operons of Helicobacter pylori . Here we show that two of the HspR-regulated operons, namely, the groESL and dnaK operons, encoding the major cellular chaperone machineries are also regulated by the H. pylori homologue of the HrcA repressor. Similarly to the hspR mutation, deletion of the hrcA gene also leads to complete derepression of the P gro and P hrc promoters. The presence of both HspR and HrcA is therefore necessary for regulated transcription from these promoters. HrcA binds directly to P gro and P hrc , likely contacting two inverted repeats with similarity to the CIRCE motif, which are present on both promoters. HrcA regulation is, however, shown to depend on binding of the HspR protein, since deletion of the HspR-binding site of the P gro promoter leads to loss of heat inducibility of this promoter. In contrast, transcription from the P cbp promoter is regulated solely by HspR. HspR is also shown to form oligomers in vivo through a stretch of hydrophobic repeats between amino acid positions 66 and 97. The implications of these findings for the elucidation of the networks regulating heat shock gene expression in H. pylori are discussed.