Factor H-binding protein is important for meningococcal survival in human whole blood and serum and in the presence of the antimicrobial peptide LL-37.

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 log(10) 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 log(10) 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.

Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing.

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 of 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.

Action site and cellular effects of cytotoxin VacA produced by Helicobacter pylori.

Cells treated with the VacA toxin from Helicobacter 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.

Helicobacter pylori toxin VacA induces vacuole formation by acting in the cell cytosol.

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.

Response of the bvg regulon of Bordetella pertussis to different temperatures and short-term temperature shifts.

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)

Development of a mouse model of Helicobacter pylori infection that mimics human disease.

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.

A novel chromatin-forming histone H1 homologue is encoded by a dispensable and growth-regulated gene in Bordetella pertussis.

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.

Mutations in the linker region of BvgS abolish response to environmental signals for the regulation of the virulence factors in Bordetella pertussis.

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 aa570 of BvgS and SK180 contains an additional substitution at aa680. 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 P1 and P2 promoters of the bvg locus. P1, which in the wild-type (wt) strain is repressed by modulating agents, is constitutively active in the mutant strains. On the contrary, P2, 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.

Adhesion of Bordetella pertussis to eukaryotic cells requires a time-dependent export and maturation of filamentous hemagglutinin.

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.

DNA topology affects transcriptional regulation of the pertussis toxin gene of Bordetella pertussis in Escherichia coli and in vitro.

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 PTOX 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 PTOX promoter cloned in a high-copy-number plasmid can give rise to two different results. If the PTOX promoter is cloned in the pGem-3 vector, transcription is absent. If the PTOX promoter is cloned in the plasmid pKK232, containing the PTOX 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 PTOX 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 PTOX promoter in E. coli and that DNA topology may play a role in the induction of transcription.

Environmental regulation of virulence factors in Bordetella species.

Many bacteria respond in a coordinate manner to environmental changes. External stimuli, sensed by receptors, are transduced to regulatory proteins which participate in well defined pathways of gene expression by varying their structure and mode of action. The network of environmental signal transduction is responsible for a fine and continuous communication between the host and the pathogenic bacteria. As a result, the gene expression machinery of the pathogen is modified continuously, in order to establish the optimal conditions for bacterial survival and multiplication.

Sequential activation and environmental regulation of virulence genes in Bordetella pertussis.

Bacterial pathogens undergo profound physiological changes when they infect their host and require co-ordinated regulation of gene expression in response to the stress encountered during infection. In Bordetella pertussis, the human pathogen which causes whooping cough, virulence factors are synthesized in response to environmental signals under the control of the bvg regulatory locus. Here we demonstrate that the bvg locus is responsible for two events of gene activation. In the first step the bvg locus transactivates its own autoregulated promoter (P1) and the promoter of the adherence factor filamentous haemagglutinin (PFHA). The second step occurs several hours later and consists of the transactivation of adenylate cyclase and pertussis toxin genes. We provide evidence that the second step of transactivation requires overexpression of regulatory proteins. Our results imply that bacterial adhesion and tissue colonization--intoxication are two separate steps at the molecular level.

Structural and genetic analysis of the bvg locus in Bordetella species.

The bvg locus contains two genes, bvgA and bvgS, which control the expression of the virulence-associated genes in Bordetella species by a system similar to the two-component systems used by a variety of bacterial species to respond to environmental stimuli. We determined the nucleotide sequence of the bvg loci of Bordetella parapertussis and Bordetella bronchiseptica and compared them with the previously determined sequence of Bordetella pertussis. The nucleotide and amino acid sequences of the bvg loci of these species are well conserved in those regions coding for the protein domains which have putative kinase and DNA-binding activities. In marked contrast, the region of BvgS that codes for the protein domain with putative sensor activity shows a high degree of variability. In total, we find 198 base-pair changes in the bvg loci of B. parapertussis and B. bronchiseptica relative to the bvg locus of B. pertussis. One hundred and seventy-three of these base-pair changes are identical in B. parapertussis and B. bronchiseptica. This confirms our previous observation that B. parapertussis and B. bronchiseptica are more related to each other than to B. pertussis. We have mapped the mutations that cause phase changes in B. bronchiseptica and we have found that in three cases these are due to spontaneous deletions in the bvgS gene. The wild-type bvg locus present on a multicopy plasmid cannot complement avirulent derivatives of B. bronchiseptica to wild-type levels, but it can do so when the bvgA gene on the plasmid is inactivated. This suggests that hyperexpression of bvgA down-regulates the bvg system.

The bvg-dependent promoters show similar behaviour in different Bordetella species and share sequence homologies.

The expression of the virulence-associated genes in Bordetella species is co-ordinately regulated by the gene products encoded by the bvg locus. In Bordetella pertussis the expression of this locus is regulated by the P1, P2, P3 and P4 promoters which are located in a 350 bp DNA fragment also containing the PFHA promoter. Here we report the transcriptional regulation of the bvg locus and the fha gene in Bordetella parapertussis and a sequence analysis of the bvg-regulated promoters. The Pp1, Pp2, Pp4 and PpFHA promoters are indistinguishable, both in transcription initiation sites and environmental regulation, from the corresponding promoters of B. pertussis, while the Pp3 promoter is not active. Sequence homologies from nine bvg-regulated promoters show a conserved dinucleotide, 5'-TG-3', at approximately one turn of helix upstream of the -10 5'-A.AaTat-3' region, and a 5'-TTTCC-3' sequence in the -90 region. Since the nucleotide sequence of the inactive Pp3 promoter shows several base substitutions with respect to the found sequence homologies, it is likely that some of these bases play an essential role in promoter activity.

Positive transcriptional feedback at the bvg locus controls expression of virulence factors in Bordetella pertussis.

Regulation of the genes coding for virulence factors in Bordetella pertussis is controlled by the bvg locus, which encodes one putative sensory protein (BvgS) and one positive regulator of transcription (BvgA). We have studied the transcription of the bvg locus and found that this is controlled by a 350-base-pair DNA fragment, which contains five promoters, three of which transcribe the bvg locus, one transcribes an antisense RNA, and one transcribes a virulence-associated gene. Under noninducing conditions, only the promoter P2 is active and this is responsible for the production of low amounts of regulatory proteins. Upon induction, the other four promoters become active and, by a mechanism that may involve transcriptional and translational regulation, cause a 50-fold increase of the transcriptional activator BvgA. A model of the autoregulation of the bvg locus is presented.

The human genome contains a single processed pseudogene for alpha enolase located on chromosome 1.

We have isolated and characterized human genomic clones containing an alpha enolase pseudogene which lacks introns and has the hallmarks of having been generated by reverse transcription. Two in-frame termination codons renders its coding region incapable of producing a functional protein. An Alu-like sequence is present in the region homologous to the 3' untranslated of the alpha enolase mRNA. Comparison of the two sequences shows that the pseudogene diverged from its functional counterpart about 14 million years ago and interestingly it is the only alpha enolase pseudogene present in the human genome. Chromosomal mapping locates the processed pseudogene to human chromosome 1, the same chromosome where the functional gene has been mapped.

Phase variants of Bordetella bronchiseptica arise by spontaneous deletions in the vir locus.

Bordetella bronchiseptica is a common respiratory tract pathogen of many mammalian species. Nucleotide sequences from the locus involved in coordinate regulation of B. pertussis virulence factors, vir, were shown to have a high degree of homology to chromosomal DNA from virulent (Vir+) and avirulent (Vir-) strains of B. bronchiseptica. Small deletions, 50 bp to 500 bp, within the vir locus were found in some of the Vir- phase variants. The vir locus and the adjacent 5' portion of the fhaB structural gene were cloned from the parental Vir+ B. bronchiseptica strain on a 23.5 kb BamHI fragment. Restriction enzyme mapping of the cloned B. bronchiseptica vir locus revealed similarities with and differences from the previously cloned B. pertussis vir locus. The cloned B. bronchiseptica vir locus complemented spontaneous Vir- variants of Bordetella pertussis and B. bronchiseptica as well as vir::Tn5 mutants of B. pertussis. Comparison of various functions of the vir loci of B. bronchiseptica and B. pertussis revealed some interesting differences in the coordinate regulation of virulence factors.

Families of bacterial signal-transducing proteins.

Bacteria can respond to a variety of environmental stimuli by means of systems generally composed of two proteins. The first protein (sensor or transmitter) is usually a transmembrane protein with cytoplasmic and extracytoplasmic domains. The extracytoplasmic domain (sensor) senses the environment and transfers the signal through the transmembrane domain to the cytoplasmic domain (transmitter), which has kinase activity. The second protein is located in the cytoplasm and contains an amino-terminal domain (receiver), which can be phosphorylated by the transmitter, and a carboxy-terminal region (regulator), which regulates gene expression by binding to DNA. The transmitter and receiver modules (the kinase and its target) are conserved in all signal-transducing systems and are the 'core structure' of this two-component system. The sensors and the regulators vary according to the stimuli they respond to and the DNA structure they interact with. On the basis of their sequence homology, the proteins belonging to such two-component systems can be classified into different families, which are summarized in this review.