Protection against Helicobacter pylori infection in mice by intragastric vaccination with H. pylori antigens is achieved using a non-toxic mutant of E. coli heat-labile enterotoxin (LT) as adjuvant.

We have previously shown that infection of mice with H. pylori can be prevented by oral immunization with H. pylori antigens given together with E.

Transcriptional analysis of the divergent cagAB genes encoded by the pathogenicity island of Helicobacter pylori.

Helicobacter pylori strains isolated from most patients with peptic ulcer disease and adenocarcinoma express the vacuolating toxin VacA and contain a pathogenicity island named cag. The cag pathogenicity island codes for more than 40 putative proteins with features similar to bacterial secretion systems. One of these proteins, CagA, is an immunodominant antigen with unknown function encoded by the cagA gene.

Therapeutic intragastric vaccination against Helicobacter pylori in mice eradicates an otherwise chronic infection and confers protection against reinfection.

Chronic infection of the gastroduodenal mucosae by the gram-negative spiral bacterium Helicobacter pylori is responsible for chronic active gastritis, peptic ulcers, and gastric cancers such as adenocarcinoma and low-grade gastric B-cell lymphoma. The success of eradication by antibiotic therapy is being rapidly hampered by the increasing occurrence of antibiotic-resistant strains. An attractive alternative approach to combat this infection is represented by the therapeutic use of vaccines.

Identification and characterization of an operon of Helicobacter pylori that is involved in motility and stress adaptation.

We identified a novel stress-responsive operon (sro) of Helicobacter pylori that contains seven genes which are likely to be involved in cellular functions as diverse as chemotaxis, heat shock response, ion transport, and posttranslational protein modification. The products of three of these genes show amino acid homologies to known proteins, such as the flagellar motor switch protein CheY, a class of heat shock proteins, and the ribosomal protein L11 methyltransferase, and to a phosphatidyltransferase. In addition to containing an open reading frame of unknown function, the product of which is predicted to be membrane associated, the sro locus contains three open reading frames that have previously been described as constituting two separate loci, the ftsH gene and the copAP operon of H.

Efficient production of heat-labile enterotoxin mutant proteins by overexpression of dsbA in a degP-deficient Escherichia coli strain.

Escherichia coli heat-labile enterotoxin (LT) mutants containing Val60-->Gly or Ser114-->Lys substitutions in the A subunit do not produce the A subunit efficiently in E. coli. These mutants accumulate mostly the B pentamer devoid of the A subunit in the periplasmic space.

Mutations in the A subunit affect yield, stability, and protease sensitivity of nontoxic derivatives of heat-labile enterotoxin.

Heat-labile toxin (LT) is a protein related to cholera toxin, produced by enterotoxigenic Escherichia coli strains, that is organized as an AB5 complex. A number of nontoxic derivatives of LT, useful for new or improved vaccines against diarrheal diseases or as mucosal adjuvants, have been constructed by site-directed mutagenesis. Here we have studied the biochemical properties of the nontoxic mutants LT-K7 (Arg-7-->Lys), LT-D53 (Val-53-->Asp), LT-K63 (Ser-63-->Lys), LT-K97 (Val-97-->Lys), LT-K104 (Tyr-104-->Lys), LT-K114 (Ser-114-->Lys), and LT-K7/K97 (Arg-7-->Lys and Val-97-->Lys).

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.

Novel molecular biology approaches to acellular vaccines.

Bacterial toxins are commonly detoxified by chemical treatment in order to use them in human vaccines. We have used site-directed mutagenesis of toxin genes to obtain bacteria that produce naturally nontoxic mutants of bacterial toxins, such as pertussis toxin (PT), cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT). Genetically detoxified PT showed a superior safety and immunogenicity in animal models, phase I and phase II clinical trials, and a superior protective efficacy in the early and late stage of a phase III efficacy trial, proving in a definitive and extensive way that genetic detoxification of bacterial toxins can, and should, replace chemical treatment.

Acellular pertussis vaccines: a turning point in infant and adolescent vaccination.

Whooping cough, an infectious disease caused by the gram-negative bacterium Bordetella pertussis, is a life-threatening disease that cannot be controlled by antibiotic treatment or other procedures of modern medicine. Immunization, using a vaccine made of heat-killed bacteria, has been the only way to prevent the disease and keep the infection under control. However, the high reactogenicity of the whole-cell vaccine available so far has made vaccination very controversial, and vaccine use has been restricted to the minimum doses strictly necessary to protect infants during the first few years of life, when the disease is most dangerous.

Genetic detoxification of bacterial toxins: a new approach to vaccine development.

Chemically detoxified bacterial toxins (toxoids) have been successfully used as vaccines for the prevention of many bacterial infectious diseases. Today, nontoxic derivatives of bacterial toxins can be obtained by mutagenesis of the toxin genes. These genetically inactivated toxins are superior to the classical toxoids both in safety and in immunogenicity and therefore they should replace the old toxoids in the existing vaccines.

Helicobacter pylori cytotoxin: importance of native conformation for induction of neutralizing antibodies.

We have attempted to express the Helicobacter pylori vacuolating cytotoxin in Escherichia coli. Although the 95-kDa VacA polypeptide was expressed abundantly, it completely lacked any biological activity. In addition, this material failed to induce neutralizing antibodies after immunization of rabbits.

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.

Role of the Helicobacter pylori virulence factors vacuolating cytotoxin, CagA, and urease in a mouse model of disease.

The pathogenic role of Helicobacter pylori virulence factors has been studied with a mouse model of gastric disease. BALB/c mice were treated orally with different amounts of sonic extracts of cytotoxic H. pylori strains (NCTC 11637, 60190, 84-183, and 87A300 [CagA+/Tox+]).

Cyclodextrins for growth of Helicobacter pylori and production of vacuolating cytotoxin.

Growth of Helicobacter pylori in liquid culture requires the addition of media supplements that often interfere with subsequent purification of bacterial antigens. In order to determine whether cyclodextrins can substitute for conventional H. pylori growth supplements, we cultured H.

Construction of nontoxic derivatives of cholera toxin and characterization of the immunological response against the A subunit.

Using computer modelling, we have identified some of the residues of the A subunit of cholera toxin (CT) and heat-labile toxin that are involved in NAD binding, catalysis, and toxicity. Here we describe the site-directed mutagenesis of the CT gene and the construction of CT mutants. Nine mutations of the A subunit gene were generated.

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 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.

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.

Analysis of expression of CagA and VacA virulence factors in 43 strains of Helicobacter pylori reveals that clinical isolates can be divided into two major types and that CagA is not necessary for expression of the vacuolating cytotoxin.

Colonization of the mucosa of the stomach and the duodenum by Helicobacter pylori is the major cause of acute and chronic gastroduodenal pathologies in humans. Duodenal ulcer formation strongly correlates with the expression of an antigen (CagA) that is usually coeexpressed with the vacuolating cytotoxin (VacA), a protein that causes ulceration in the stomach of mice. However, the relationship between these two virulence factors is unknown.

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.

A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit.

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies.

Humoral immune response to plasmid protein pgp3 in patients with Chlamydia trachomatis infection.

We identified, by two-dimensional electrophoretic analysis and microsequencing, a protein of Chlamydia trachomatis elementary bodies which corresponds to the polypeptide (pgp3) encoded by open reading frame 3 (ORF3). Amino acid analysis showed that the first residue (Gly) found in the native protein is the one encoded by the second ORF3 codon, implying a typical bacterial removal of the first Met residue. Relatively large amounts of recombinant pgp3 (r-pgp3) in a stable, water-soluble form were obtained by overexpressing ORF3 in Escherichia coli and purifying the product from periplasmic extracts under nondenaturing conditions.

Optimized conditions for the fermentation of Helicobacter pylori and production of vacuolating cytotoxin.

We report here improvements to the growth media and fermentation conditions which result in a substantial increase of Helicobacter pylori growth and in the enhanced production of vacuolating cytotoxin. Addition of glucose to the medium resulted in the increase of cell yield, cell viability and a significant improvement in the production of vacuolating cytotoxin.

Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis.

Computer analysis of the crystallographic structure of the A subunit of Escherichia coli heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53-->Glu or Asp, Ser-63-->Lys, Val-97-->Lys, Tyr-104-->Lys or Asp, and Ser-114-->Lys or Glu.

Common features of the NAD-binding and catalytic site of ADP-ribosylating toxins.

Computer analysis of the three-dimensional structure of ADP-ribosylating toxins showed that in all toxins the NAD-binding site is located in a cavity. This cavity consists of 18 contiguous amino acids that form an alpha-helix bent over a beta-strand. The tertiary folding of this structure is strictly conserved despite the differences in the amino acid sequence.