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.

Complete genome sequence of Neisseria meningitidis serogroup B strain MC58.

The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.

The DNA polymerase-encoding gene of Bacillus subtilis bacteriophage SPO1.

The bacteriophage SPO1 DNA polymerase-encoding gene, which contains a self-splicing intron, has been sequenced and its amino acid (aa) sequence has been deduced. The aa sequence of SPO1 DNA polymerase shows a high degree of similarity with that of DNA polymerase I from Escherichia coli (Po1I). Alignment with the sequences of Po1I, and the phi 29 and SPO1 DNA polymerases indicate that the aa residues that have been implicated in 3'----5' exonuclease activities are conserved.

Transcriptional regulation in the Chlamydia trachomatis pCT plasmid.

We have analyzed transcriptional regulation of the chlamydial plasmid pCT. Transcription of a full-length 2.9-kb ORF1-ORF2 mRNA is likely to be regulated by the sigma 66 transcription factor which recognizes the TATAAT and TNGNCA sequences at the -10 and -35 DNA regions, respectively. RNA synthesis starts 39 nucleotides (nt) upstream from the ATG start codon of ORF1 and terminates within the downstream ORF3 DNA region. A 2.8-kb transcript transverses the ORF3-6 DNA region, while two transcripts of 2.2 and 1.9 kb cover the ORF4-6 DNA region. These mRNAs overlap two abundant transcripts which regulate the expression of the ORF3 and ORF4 genes. The accumulation of transcripts associated with these ORFs is likely to be regulated at the level of RNA synthesis by an unknown sigma factor which could select the RTTTAAA and TTYTTR sequences located at the -10 and -35 DNA regions, respectively. This new promoter consensus sequence could be unique to the gene expression machinery of Chlamydiae.

Sequence of the bacteriophage SP01 gene 30.

The bacteriophage SP01 gene 30, whose function is essential for DNA synthesis, has been analyzed for its primary structural features. Conditionally lethal mutations in the gene 30 locus have been mapped and sequenced, and the wild-type amino acid (aa) sequence has been deduced along with that of a co-transcribed and possibly co-translated upstream unidentified reading frame (URF). The aa sequence deduced for gene 30 shares partial similarity with protein P of bacteriophage lambda, which participated in lambda DNA replication, and also with the exonuclease, gp46, of bacteriophage T4. A lysine-rich region of the hypothetical product of the URF shares similarity with both the T4 DNA topoisomerase and the phi 29 gene 3-encoded protein; the latter codes for a terminal protein which participates in the priming of DNA elongation.

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.

Synthesis, phosphorylation, and nuclear localization of human papillomavirus E7 protein in Schizosaccharomyces pombe.

The complete E7 protein-encoding open reading frame of human papillomavirus type 16 (HPV-16) was expressed in the fission yeast Schizosaccharomyces pombe, under the control of a cloned yeast promoter. The HPV-16 E7 protein synthesized in S. pombe is a 17-kDa phosphoprotein which is recognized by anti-E7 antibodies (raised in rabbits against E7 fusion protein produced in Escherichia coli). The mobility during sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of native E7 phosphoprotein synthesized in S. pombe is identical to that of the E7 phosphoprotein immunoprecipitated from human CaSki cells. Immunofluorescence staining showed that HPV-16 E7 phosphoprotein is localized in the nuclei of transformed S. pombe. These results indicate that E7 protein synthesized by S. pombe is apparently indistinguishable from HPV-16 E7 protein synthesized in higher eukaryotic cells expressing genes of HPV-16, and also that the phosphorylated, nuclear HPV-16 E7 protein is synthesized in S. pombe in a form compatible with its biological activity.

Symmetric transcription of bacteriophage T4 base plate genes.

Dot-blot and Northern-blot experiments, using strand-specific RNA probes, show that part of the bacteriophage T4 DNA that codes for six of the base plate structural genes (gp 51, 27, 28, 29, 48 and 54), is transcribed in vivo from both DNA strands. The r DNA strand transcripts contain sequences which are translated into structural proteins. Antisense l strand RNA is about 100 fold less abundant than RNA molecules transcribed from the r DNA strand.

Characterization of the structural genes for the DNA-binding protein H-NS in Enterobacteriaceae.

The promoter region of Escherichia coli hns, the structural gene for the DNA-binding protein H-NS, has been identified by use of a promoter search vector and the in vivo transcriptional start point by primer extension analysis. The homologous hns genes of two other Enterobacteriaceae, Proteus vulgaris and Serratia marcescens, were identified by heterologous hybridization with a DNA probe derived from E. coli hns, cloned and sequenced. Taking into account only the invariant nucleotides and amino acids, the homology of H-NS among the three organisms was found to be greater than 70% at the DNA level and greater than 75% at the protein level. The three hns genes were also found to have nearly identical transcriptional and translational signals.

Genetic detoxification of bacterial toxins.

Several pathogens, such as Corynebacterium diphtheriae, Clostridium tetani, Bordetella pertussis, Vibrio cholerae, enterotoxigenic Escherichia co1i (1), and even some emerging pathogens, such as Helicobacter pylori (2), produce potent toxins that are responsible for the pathology caused by the bacterium. In most cases the disease, and often even the infection, can be prevented by a vaccine that induces immunity against the toxin. In order to be used in vaccines, the dangerous toxins need to be depleted of their toxic activity in an effective and irreversible manner. The most effective way to inactivate toxins for inclusion in vaccines was developed by Ramon in 1924 by using formaldehyde treatment at 37°C to detoxify diphtheria toxin (3). This method was then used to inactivate other toxins and also viral and bacterial suspensions. Even today, widely used vaccines, such as diphtheria, tetanus, inactivated polio, and whole cell pertussis, and even some of the newly developed acellular pertussis vaccines are produced using formaldehyde or other chemical treatments to inactivate the toxin and/or kill the microorganisms that are present in the vaccine (4, 5).

Bacteriophage T4 gene 28.

The complete nucleotide sequence of bacteriophage T4D gene 28 has been determined. Gene 28 product is a structural component of the viral baseplate for which an enzymatic activity has also been proposed.

Differential response of the bvg virulence regulon of Bordetella pertussis to MgSO4 modulation.

Magnesium sulfate is known to repress the expression of the virulence factors of Bordetella pertussis that are coordinately regulated by the bvg locus. We have tested the time required by MgSO4 to repress the synthesis of several bvg-regulated mRNA species and found that the promoters of the virulence genes (pertussis toxin, adenylate cyclase, and filamentous hemagglutinin) are repressed in 6 min, while the autogenously regulated promoters of the bvg locus (P1, P3, and P4) are repressed only several hours later. These data show a differential behavior between regulated and autoregulated genes of the bvg regulon.

Motility of Helicobacter pylori is coordinately regulated by the transcriptional activator FlgR, an NtrC homolog.

sigma54 is the subunit of bacterial RNA polymerase that transcribes from promoters with enhancer elements bound by enhancer-binding proteins. By computer searches of Helicobacter pylori genomic sequences, chromosomal gene disruption, and RNA analyses, we have identified sigma54-recognized promoters that regulate transcription of flagellar basal body and hook genes, as well as the enhancer-binding protein FlgR (flagellum regulator), a transactivating protein of the NtrC family. We demonstrate that FlgR is required for bacterial motility and transcription of five promoters for seven basal body and hook genes. In addition, FlgR acts as a repressor of transcription of the sigma28-regulated flaA flagellin gene promoter, while changes in DNA topology repress transcription of the sigma54-regulated flaB flagellin gene promoter. Our data indicate that regulation of flagellar gene expression in H. pylori shows similarities with that in enterobacteriaceae and Caulobacter.

The autoregulatory HspR repressor protein governs chaperone gene transcription in Helicobacter pylori.

In the present study, we provide evidence that the groESL, hrcA-grpE-dnaK and cbpA-hspR-orf operons encoding the major chaperones of the human gastric pathogen Helicobacter pylori are transcribed by the vegetative sigma factor sigma80 and are regulated negatively by the transcriptional repressor HspR. In vitro studies with purified recombinant HspR protein established that the protein represses transcription by binding to large DNA regions centred around the transcription initiation site in the case of the Pcbp promoter, and around -85 and -120 in the case of the Pgro and Phrc promoters respectively. All three binding sites contain DNA motifs with some similarity to the HAIR sequence identified as a consensus for the HspR protein of Streptomyces. In contrast to the situation in Streptomyces, in which transcription of HspR-regulated genes is induced in response to heat shock, transcription of the HspR-dependent genes in H. pylori is not inducible by thermal stimuli. Transcription of the groESL and cbpA-hspR-orf operons is induced by osmotic shock, while transcription of the hrcA-grpE-dnaK operon, although HspR dependent, is not affected by salt treatment. The possibility that HspR could constitute a global transcriptional regulator for diverse cellular functions with implications for pathogenesis is discussed.

The pertussis toxin liberation genes of Bordetella pertussis are transcriptionally linked to the pertussis toxin operon.

The DNA sequence of the pertussis toxin operon (ptx) of Bordetella pertussis predicts that transcription of the operon ends downstream from the ptxS3 gene at a possible stem-loop structure. Secretion of the assembled pertussis toxin into the culture medium required the expression of 8 genes arranged in an operon (ptl) and lying 55 bp downstream from the ptx and ptl operons are cotranscribed and coregulated by the P(TOX) promoter. Deletion of the 55-bp DNA region caused an increase in the amount of the ptl transcripts. It is likely that this DNA region is involved in regulation of the ptx-pti expression.

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.

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.

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.

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.

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.