Factor H-binding protein (fHBP; GNA1870) is one of the antigens of the recombinant vaccine against serogroup B Neisseria meningitidis, which has been developed using reverse vaccinology and is the basis of a meningococcal B vaccine entering phase III clinical trials. Binding of factor H (fH), an inhibitor of the complement alternative pathway, to fHBP enables N. meningitidis to evade killing by the innate immune system. All fHBP null mutant strains analyzed were sensitive to killing in ex vivo human whole blood and serum models of meningococcal bacteremia with respect to the isogenic wild-type strains. The fHBP mutant strains of MC58 and BZ83 (high fHBP expressors) survived in human blood and serum for less than 60 min (decrease of > 2 log(10) CFU), while NZ98/254 (intermediate fHBP expressor) and 67/00 (low fHBP expressor) showed decreases of > 1 log10 CFU after 60 to 120 min of incubation. In addition, fHBP is important for survival in the presence of the antimicrobial peptide LL-37 (decrease of > 3 log10 CFU after 2 h of incubation), most likely due to electrostatic interactions between fHBP and the cationic LL-37 molecule. Hence, the expression of fHBP by N. meningitidis strains is important for survival in human blood and human serum and in the presence of LL-37, even at low levels. The functional significance of fHBP in mediating resistance to the human immune response, in addition to its widespread distribution and its ability to induce bactericidal antibodies, indicates that it is an important component of the serogroup B meningococcal vaccine.

GNA 1870 is a novel surface-exposed lipoprotein, identified by genome analysis of Neisseria meningitidis strain MC58, which induces bactericidal antibodies. Three sequence variants of the protein were shown to be sufficient to induce bactericidal antibodies against a panel of strains representative of the diversity of serogroup B meningococci. Here, we studied the antigenic and immunogenic properties of GNA 1870. which for convenience was divided into domains A, B, and C. The immune responses of mice immunized,with each of the three variants were tested using overlapping peptides scanning the entire protein length and using recombinant fragments. We found that while most of the linear epitopes are located in the A domain. the bactericidal antibodies are directed against conformational epitopes located in the BC domain. This was also confirmed by the isolation of a bactericidal murine monoclonal antibody, which failed to recognize linear peptides on the A. B, and C domains separately but recognized a conformational epitope formed only by the combination of the B and C domains. Arginine in position 204 was identified as important for binding of the monoclonal antibody. The identification of the region containing bactericidal epitopes is an important step in the design of new vaccines against meningococci.

Neisseria meningitidis is a human pathogen, which is a major cause of sepsis and meningitis. The bacterium colonizes the upper respiratory tract of approximately 10% of humans where it lives as a commensal. On rare occasions, it crosses the epithelium and reaches the bloodstream causing sepsis. From the bloodstream it translocates the blood-brain barrier, causing meningitis. Although all strains have the potential to cause disease, a subset of them, which belongs to hypervirulent lineages, causes disease more frequently than others. Recently, we described NadA, a novel antigen of N. meningitidis, present in three of the four known hypervirulent lineages. Here we show that NadA is a novel bacterial invasin which, when expressed on the surface of Escherichia coli, promotes adhesion to and invasion into Chang epithelial cells. Deletion of the N-terminal globular domain of recombinant NadA or pronase treatment of human cells abrogated the adhesive phenotype. A hypervirulent strain of N. meningitidis where the nad A gene was inactivated had a reduced ability to adhere to and invade into epithelial cells in vitro. NadA is likely to improve the fitness of N. meningitidis contributing to the increased virulence of strains that belong to the hypervirulent lineages.

Sepsis and meningitis caused by serogroup B meningococcus are devastating diseases of infants and young adults, which cannot yet be prevented by vaccination. By genome mining, we discovered GNA1870, a new surface-exposed lipoprotein of Neisseria meningitidis that induces high levels of bactericidal antibodies. The antigen is expressed by all strains of N. meningitidis tested. Sequencing of the gene in 71 strains representative of the genetic and geographic diversity of the N. meningitidis population, showed that the protein can be divided into three variants. Conservation within each variant ranges between 91.6 to 100%, while between the variants the conservation can be as low as 62.8%. The level of expression varies between strains, which can be classified as high, intermediate, and low expressors. Antibodies against a recombinant form of the protein elicit complement-mediated killing of the strains that carry the same variant and induce passive protection in the infant rat model. Bactericidal titers are highest against those strains expressing high yields of the protein; however, even the very low expressors are efficiently killed. The novel antigen is a top candidate for the development of a new vaccine against meningococcus.

Bordetella pertussis undergoes phenotypic changes modulated by the bvgAS locus, which regulates the expression of many genes related to virulence and immunogenicity. We previously reported the N-terminal sequence of a 90 kDa bvg-regulated outer membrane protein (OMP) of B. pertussis (SWISS-PROT accession No. p81549), a novel potential virulence factor that we named Vir90. The open reading frames (ORFs) which potentially code for Vir90 in B. pertussis, B. parapertussis and B. bronchiseptica were identified by computer analysis of the genomic sequences available for the three Bordetella species. Nucleotide sequence analysis of the vir90 upstream region revealed the presence of a putative promoter, a BvgA binding site and a putative Fur binding site. The B. pertussis Vir90 protein showed significant homology with ferrisiderophore receptors from Gram-negative bacteria. An antiserum raised against Vir90His recombinant protein recognized the 90-kDa protein in immunoblots of OMPs from these three virulent Bordetella species. The accumulation of the Vir90 protein increased 4-fold under low iron growth conditions. Therefore, the vir90 gene is expressed in the tested species and its expression is regulated positively by the BvgAS system and negatively under high iron concentration, likely by Fur.

Neisseria meningitidis is a Gram-negative bacterium which colonizes the human upper respiratory tract. Occasionally, it translocates to the bloodstream causing sepsis and from there it can cross the blood-brain barrier and cause meningitis. Many of the molecules, which mediate the interaction of N. meningitidis to host cells, are still unknown. Recently, App (Adhesion and penetration protein) was described as a member of the autotransporter family and a homologue to the Hap (Haemophilus adhesion and penetration) protein of Haemophilus influenzae, a molecule that plays a role in the interaction with human epithelial cells. In this study we expressed app in Escherichia coli in order to analyse the functional properties of the protein. We show that the protein is exported to the E. coli surface, processed by an endogenous serine-protease activity and released in the culture supernatant. Escherichia coli expressing app adhere to Chang epithelial cells, showing that App is able to mediate bacterial adhesion to host cells. The serine protease activity is localized at the amino-terminal domain, whereas the binding domain is in the carboxy-terminal region. The role of App in adhesion was confirmed also in N. meningitidis.

Mono ADP-ribosyltransferases (ADPRTs) are a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. In bacteria, these enzymes often act as potent toxins and play an important role in pathogenesis. Here we report a profile-based computational approach that, assisted by secondary structure predictions, has allowed the identification of a previously undiscovered ADP-ribosyltransferase in Neisseria meningitidis (NarE). NarE shows structural homologies with E. coli heat-labile enterotoxin (LT) and cholera toxin (CT) and possesses ADP-ribosylating and NAD-glycohydrolase activities. As in the case of LT and CT, NarE catalyses the transfer of the ADP-ribose moiety to arginine residues. Despite the absence of a signal peptide, the protein is efficiently exported into the periplasm of Neisseria. The narE gene is present in 25 out of 43 strains analysed, is always present in ET-5 and Lineage 3 but absent in ET-37 and Cluster A4 hypervirulent lineages. When present, the gene is 100% conserved in sequence and is inserted upstream of and co-transcribed with the lipoamide dehydrogenase E3 gene. Possible roles in the pathogenesis of N. meningitidis are discussed.

In a previous study, we used the genome of serogroup B Meningococcus to identify novel vaccine candidates. One of these molecules, GNA33, is well conserved among Meningococcus B strains, other Meningococcus serogroups and Gonococcus and induces bactericidal antibodies as a result of being a mimetic antigen of the PorA epitope P1.2. GNA33 encodes a 48-kDa lipoprotein that is 34.5% identical with membrane-bound lytic transglycosylase A (MltA) from Escherichia coli. In this study, we expressed GNA33, i.e. Meningococcus MltA, as a lipoprotein in E. coli. The lipoprotein nature of recombinant MltA was demonstrated by incorporation of [3H]palmitate. MltA lipoprotein was purified to homogeneity from E. coli membranes by cation-exchange chromatography. Muramidase activity was confirmed when MltA was shown to degrade insoluble murein sacculi and unsubstituted glycan strands. HPLC analysis demonstrated the formation of 1,6-anhydrodisaccharide tripeptide and tetrapeptide reaction products, confirming that the protein is a lytic transglycosylase. Optimal muramidase activity was observed at pH 5.5 and 37 degrees C and enhanced by Mg2+, Mn2+ and Ca2+. The addition of Ni2+ and EDTA had no significant effect on activity, whereas Zn2+ inhibited activity. Triton X-100 stimulated activity 5.1-fold. Affinity chromatography indicated that MltA interacts with penicillin-binding protein 2 from Meningococcus B, and, like MltA from E. coli, may form part of a multienzyme complex.

Neisseria meningitidis is a human pathogen, which, in spite of antibiotic therapy, is still a major cause of mortality due to sepsis and meningitis. Here we describe NadA, a novel surface antigen of N. meningitidis that is present in 52 out of 53 strains of hypervirulent lineages electrophoretic types (ET) ET37, ET5, and cluster A4. The gene is absent in the hypervirulent lineage III, in N. gonorrhoeae and in the commensal species N. lactamica and N. cinerea. The guanine/cytosine content, lower than the chromosome, suggests acquisition by horizontal gene transfer and subsequent limited evolution to generate three well-conserved alleles. NadA has a predicted molecular structure strikingly similar to a novel class of adhesins (YadA and UspA2), forms high molecular weight oligomers, and binds to epithelial cells in vitro supporting the hypothesis that NadA is important for host cell interaction. NadA induces strong bactericidal antibodies and is protective in the infant rat model suggesting that this protein may represent a novel antigen for a vaccine able to control meningococcal disease caused by three hypervirulent lineages.

Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence bf a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified. and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.

Cells treated with the VacA toxin fromHelicobacter pylori develop large membrane-bound vacuoles that originate from the late endocytotic pathway. Using different experimental approaches, we showed that VacA can induce vacuoles by acting within the cell cytosol. Moreover, separation of VacA-induced vacuoles at an early stage of formation, using a novel isopycnic density ultracentrifugation method, allowed us to show that they resemble a hybrid compartment, containing elements of both late endosomes and lysosomes. Functional defects of the endocytotic pathway were also studied before any macroscopic vacuolation is evident. VacA-intoxicated cells degrade extracellular ligands with reduced efficiency and, at the same time, they secrete acidic hydrolases into the extracellular medium, normally sorted to lysosomes. All these findings indicate that VacA translocates into the cell cytosol where it causes a lesion of the late endosomal/lysosomal compartments, such that protein trafficking across this crucial cross-point is altered with consequences that may be relevant to the pathogenesis of gastroduodenal ulcers.

Cells exposed to Helicobacter pylori toxin VacA develop large vacuoles that originate from massive swelling of membranous compartments of late stages of the endocytic pathway. To determine if the toxin is active from the cell cytosol, cells were either microinjected with toxin or transfected with plasmids encoding VacA. Both procedures cause formation of intracellular vacuoles. Cytosolic localization of the toxin was assessed by indirect immunofluorescence with specific antibodies and by expression of an active green fluorescence protein (GFP)–VacA chimera. Vacuoles induced by internally produced VacA are morphologically and functionally identical to those induced by externally added toxin. It is concluded that VacA is a toxin acting intracellularly by altering a cytosol‐exposed target, possibly involved in the control of membrane trafficking.

Bordetella pertussis produces a number of virulence factors whose expression is coordinately regulated by the bvgAS locus. Transcription of virulence genes is repressed by environmental factors such as low temperature (25 degrees C) and chemical stimuli. Temperature shift of bacterial cultures from 25 degrees C to 37 degrees C activates two classes of bvg-regulated virulence genes: the early genes, which are activated within 10 min, and late genes, which require 2-4 h for activation. During the interval between the activation of the early and late genes, the intracellular concentration of BvgA increases 50-fold. It has been proposed that this increased concentration may be required for the activation of the late genes. Here we have analysed the response of the bvg locus to intermediate temperature and to repeated temperature shifts. Temperature shifts of B. pertussis cultures from 22 degrees C to 28 degrees C or 35 degrees C resulted in the synthesis of low, intermediate, and high amounts of BvgA. This implied that the intracellular concentration of BvgA is temperature-dependent. We have also observed that the amount of virulence factors produced correlates with the BvgA concentration. When bacteria grown at 37 degrees C were shifted to 22 degrees C, transcription from the adenylate cyclase toxin haemolysis promoter (PAC) was repressed after 30 min, while transcription from the bvg (P1) and filamentous haemagglutinin (PFHA) promoters was repressed after 2 h. During this time, the amount of BvgA did not decrease.(ABSTRACT TRUNCATED AT 250 WORDS)

The human pathogen Helicobacter pylori is associated with gastritis, peptic ulcer disease, and gastric cancer. The pathogenesis of H. pylori infection in vivo was studied by adapting fresh clinical isolates of bacteria to colonize the stomachs of mice. A gastric pathology resembling human disease was observed in infections With cytotoxin-producing strains but not with noncytotoxic strains. Oral immunization with purified H. pylori antigens protected mice from bacterial infection. This mouse model will allow the development of therapeutic agents and vaccines against H. pylori infection in humans.

We report the identification of a protein homologous to a histone H1 in Bordetella pertussis. The B. pertussis histone homologue, BpH1, varies in size in different strains from 182 to 206 amino acids. The variability of the size of the protein is due to gene variability by insertion or deletion of DNA modules. Insertion of a kanamycin cassette into the bpH1 gene generates a BpH1 null mutant with phenotypic properties and growth rate similar to those of the wild-type strain, showing that this gene is dispensable. In vitro, the BpH1 protein prevents chromosomal DNA degradation from DNase I and constrains supercoiled DNA. Transcription of the bpH1 gene is activated during exponential growth of the bacteria, whereas it is repressed during the stationary phase of growth. It is proposed that BpH1 plays a role in chromatin formation and condensation during DNA replication and that repression of transcription depends upon a reduced rate of DNA replication.

Expression of virulence factors of Bordetella pertussis is coordinately regulated by the products of the bvg locus, which codes for a sensory protein (BvgS) and a positive regulator of transcription (BvgA), a pair in the family of bacterial 'two-component' regulators. Transcription of the bvg-regulated promoters is repressed by modulating environmental factors such as 50 mM MgSO4, 10 mM nicotinic acid (NA) or low temperature (25 degrees C). We have isolated a spontaneous mutant (SK170) which expresses virulence genes at either 25 degrees C, or in the presence of 1-5 mM NA, or 10-50 mM MgSO4. Virulence factors in strain SK170 are still repressed by higher concentrations of NA (10 mM), or by a combination of low temperature (25 degrees C) and one of the other modulating agents. From this strain, we have isolated a second mutant (SK180) that showed constitutive synthesis of the virulence factors under any growth regime. Nucleotide (nt) and deduced amino acid (aa) sequence analysis showed that SK170 contains a substitution at aa(570) of BvgS and SK180 contains an additional substitution at aa(680). These substitutions are confined to a 161-aa sequence that links the transmembrane (TM) and kinase domains of BvgS. These mutations also alter the transcriptional autoregulation of the P-1 and P-2 promoters of the bvg locus. P-1, which in the wild-type (wt) strain is repressed by modulating agents, is constitutively active in the mutant strains. On the contrary, P-2, which is normally induced by all three modulating agents, is active in strain SK170 only in the presence of MgSO4 or NA, while in strain Sk180 this promoter is repressed by modulating agents. The mutants exhibit elevated levels of the BvgA regulatory protein and have a virulent phenotype also in the presence of modulating agents.

Bordetella pertussis, the human pathogen of whooping cough, when grown at 22 degrees C is nonvirulent and unable to bind eukaryotic cells. In response to a temperature shift to 37 degrees C, the bacterium acquires the ability to bind eukaryotic cells in a time-dependent fashion. By studying in vitro the temperature-induced transition, from the nonvirulent to the virulent state, we found that binding to CHO cells is mediated by the Arg-Gly-Asp-containing domain of filamentous hemagglutinin (FHA), a protein with multiple binding specificities. This protein is synthesized as a 367-kDa polypeptide within 10 min after temperature shift, but requires 2 hr before it is detected on the bacterial cell surface and starts to bind CHO cells. Mutations affecting the cell surface export of FHA abolish bacterial adhesion to CHO cells, while mutations in the outer membrane protein pertactin strongly reduce binding. This suggests that multiple chaperon proteins are required for a correct function of FHA. Finally, several hours after maximum binding efficiency is achieved, the N-terminal 220-kDa portion of FHA that contains the binding regions is cleaved off, possibly to release the bacteria from the bound cells and facilitate spreading. The different forms of FHA may play different roles during bacterial infection.

The bvg locus of Bordetella pertussis encodes an environmentally inducible operon essential for the expression of virulence genes. We show that in Escherichia coli, the P(TOX) promoter cloned in cis of the bvg locus is activated and environmentally regulated. Cotransformation of E. coli with the bvg locus cloned in a low-copy-number plasmid and with the P(TOX) promoter cloned in a high-copy-number plasmid can give rise to two different results. If the P(TOX) promoter is cloned in the pGem-3 vector, transcription is absent. If the P(TOX) promoter is cloned in the plasmid pKK232, containing the P(TOX) promoter between two ribosomal gene terminators of transcription, transcription occurs, although regulation of transcription is abolished. Under these conditions, the intracellular amount of RNA transcripts is increased by adding to the culture medium novobiocin, an inhibitor of bacterial gyrases. In vitro, the transcription of the P(TOX) promoter is activated on E. coli RNA polymerase supplemented with cell extracts from wild-type B. pertussis. Addition of DNA gyrase to the mixture dramatically reduces the amount of RNA synthesized. Our data show that the products of the bvg locus, BvgA and BvgS, are directly involved in the regulation of the P(TOX) promoter in E. coli and that DNA topology may play a role in the induction of transcription.