Increased zinc finger protein zFOC1 transcripts in gastric cancer compared with normal gastric tissue.

BACKGROUND: Screening of cDNA arrays of the IMAGE library identified human zFOC1 as a differentially expressed cDNA that was upregulated in KATO III gastric cancer cells following stimulation with the gastric pathogen Helicobacter pylori. AIMS: To determine the expression of zFOC1 in gastric mucosa with and without H pylori infection and in patients with gastric cancer. RESULTS: zFOC1 is localised on chromosome 12q24.3 and encodes a zinc finger protein. Expression studies in human H pylori infected and uninfected gastric biopsies, gastric tumours, and gastric cancer cell lines revealed that zFOCI gene transcripts are significantly higher in gastric cancer than in non-cancerous gastric tissues. CONCLUSIONS: The zFOC1 gene appears to be a tumour marker associated with gastric cancer.

cDNA array analysis of cag pathogenicity island-associated Helicobacter pylori epithelial cell response genes.

Helicobacter pylori strains containing the cag pathogenicity island (PAI) induce NF-kappaB activation and interleukin-8 secretion in gastric epithelial cells. The aim of this study was to investigate changes in epithelial gene expression induced by cag PAI-positive and -negative strains of H. pylori using high-density cDNA array hybridization technology. Radio-labeled cDNA prepared from H. pylori-infected Kato 3 gastric epithelial cells was hybridized to high-density cDNA arrays to identify changes in epithelial gene expression compared to noninfected controls. In vivo expression of selected, differentially expressed genes was examined by reverse transcription-PCR analysis of H. pylori-positive and -negative gastric mucosa. Screening of ca. 57,800 cDNAs identified 208 known genes and 48 novel genes and/or expressed sequence tags of unknown function to be differentially expressed in Kato 3 cells following H. pylori infection. Marked differences in gene expression profiles were observed following cag PAI-positive and cag PAI-negative infection with 15 novel cDNAs and 92 known genes being differentially expressed. H. pylori was found to change the expression of genes encoding growth factors and cytokine/chemokines and their receptors, apoptosis proteins, transcription factors and metalloprotease-disintegrin proteins (ADAMs), and tissue inhibitors of metalloproteinases. Gastric differential expression of selected known genes (amphiregulin and ADAM 10) and a novel gene (HPYR1) was confirmed in vivo in patients with H. pylori infection. Confirmation of the in vivo expression of selected genes demonstrates the usefulness of this approach for investigating pathogen-induced changes in host gene expression.

Iron acquisition and virulence in Helicobacter pylori: a major role for FeoB, a high-affinity ferrous iron transporter.

The genome sequence of Helicobacter pylori suggests that this bacterium possesses several Fe acquisition systems, including both Fe2+- and Fe3+-citrate transporters. The role of these transporters was investigated by generating insertion mutants in feoB, tonB, fecA1 and fecDE. Fe transport in the feoB mutant was approximately 10-fold lower than in the wild type (with 0.5 microM Fe), irrespective of whether Fe was supplied in the Fe2+ or Fe3+ form. In contrast, transport rates were unaffected by the other mutations. Complementation of the feoB mutation fully restored both Fe2+ and Fe3+ transport. The growth inhibition exhibited by the feoB mutant in Fe-deficient media was relieved by human holo-transferrin, holo-lactoferrin and Fe3+-dicitrate, but not by FeSO4. The feoB mutant had less cellular Fe and was more sensitive to growth inhibition by transition metals in comparison with the wild type. Biphasic kinetics of Fe2+ transport in the wild type suggested the presence of high- and low-affinity uptake systems. The high-affinity system (apparent Ks = 0.54 microM) is absent in the feoB mutant. Transport via FeoB is highly specific for Fe2+ and was inhibited by FCCP, DCCD and vanadate, indicating an active process energized by ATP. Ferrozine inhibition of Fe2+ and Fe3+ uptake implied the concerted involvement of both an Fe3+ reductase and FeoB in the uptake of Fe supplied as Fe3+. Taken together, the results are consistent with FeoB-mediated Fe2+ uptake being a major pathway for H. pylori Fe acquisition. feoB mutants were unable to colonize the gastric mucosa of mice, indicating that FeoB makes an important contribution to Fe acquisition by H. pylori in the low-pH, low-O2 environment of the stomach.

Identification of two novel diurnal genes by screening of a rat brain cDNA library.

While the hypothalamus is fundamental for sleep and circadian regulation, the molecular mechanism involved are poorly understood. We have used a differential gene expression technique to identify hypothalamic genes which have altered expression in rat sleep periods. Complex cDNA probes from rat hypothalami removed at Zeitgeber times 4 and 15 were hybridised to rat brain cDNA library girds. From 30 differentially expressed clones, six were further analysed and two were confirmed to exhibit increased expression at Zeitgeber time 4. A Northern blot hybridization of brain, heart, kidney, lung, testis and skin mRNA showed that both clones were brain specific. Therefore, we have identified two novel brain specific diurnally expressed hypothalamic genes. Both genes may have roles in sleep or circadian regulation.

Helicobacter pylori porCDAB and oorDABC genes encode distinct pyruvate:flavodoxin and 2-oxoglutarate:acceptor oxidoreductases which mediate electron transport to NADP.

Helicobacter pylori, a major cause of human gastric disease, is a microaerophilic bacterium that contains neither pyruvate nor 2-oxoglutarate dehydrogenase activity. Previous studies (N. J. Hughes, P. A. Chalk, C. L. Clayton, and D. J. Kelly, J. Bacteriol. 177:3953-3959, 1995) have indicated that the major routes for the generation of acetyl coenzyme A (acetyl-CoA) and succinyl-CoA are via pyruvate:flavodoxin oxidoreductase (POR) and 2-oxoglutarate:acceptor oxidoreductase (OOR), respectively. The purified POR is a heterotetrameric protein, with subunits of 48 (PorA), 36 (PorB), 24 (PorC), and 14 (PorD) kDa. In this study OOR has been purified, and it is similarly composed of polypeptides of 43 (OorA), 33 (OorB), 24 (OorC), and 10 (OorD) kDa. Both POR and OOR are oxygen labile and are likely to be major contributors to the microaerophilic phenotype of H. pylori. Unlike POR, OOR was unable to use a previously identified flavodoxin (FldA) as an electron acceptor. Although the purified enzymes were unable to reduce NAD(P), electrons from both pyruvate and 2-oxoglutarate could reduce NADP in cell extracts, consistent with a role for these oxidoreductases in the provision of NADPH as a respiratory electron donor. The H. pylori por, oor, and fldA genes were cloned and sequenced. The deduced por gene products showed significant sequence similarity to archaeal four-subunit 2-oxoacid:acceptor oxidoreductases. However, the amino acid sequences of OorA and -B were more closely related to that of the two-subunit POR of the aerobic halophile Halobacterium halobium. Both porD and oorD encode integral ferredoxin-like subunits. POR and OOR are probably essential enzymes in H. pylori, as insertion inactivation of porB and oorA appeared to be lethal.

Identification of two novel diurnal genes by screening of a rat brain cDNA library.

While the hypothalamus is fundamental for sleep and circadian regulation, the molecular mechanisms involved are poorly understood. We have used a differential gene expression technique to identify hypothalamic genes which have altered expression in rat sleep periods. Complex cDNA probes from rat hypothalami removed at Zeitgeber times 4 and 15 were hybridised to rat brain cDNA library girds. From 30 differentially expressed clones, six were further analysed and two were confirmed to exhibit increased expression at Zeitgeber time 4. A Northern blot hybridization of brain, heart, kidney, lung, testis and skin mRNA showed that both clones were brain specific. Therefore, we have identified two novel brain specific diurnally expressed hypothalamic genes. Both genes may have roles in sleep or circadian regulation.

Cloning, sequencing, expression, purification and preliminary characterization of a type II dehydroquinase from Helicobacter pylori.

A heat-stable dehydroquinase was purified to near homogeneity from a plate-grown suspension of the Gram-negative stomach pathogen Helicobacter pylori, and shown from both its subunit and native molecular masses to be a member of the type II family of dehydroquinases. This was confirmed by N-terminal amino acid sequence data. The gene encoding this activity was isolated following initial identification, by random sequencing of the H. pylori genome, of a 96 bp fragment, the translated sequence of which showed strong identity to a C-terminal region of other type II enzymes. Southern blot analysis of a cosmid library identified several potential clones, one of which complemented an Escherichia coli aroD point mutant strain deficient in host dehydroquinase. The gene encoding the H. pylori type II dehydroquinase (designated aroQ) was sequenced. The translated sequence was identical to the N-terminal sequence obtained directly from the purified protein, and showed strong identity to other members of the type II family of dehydroquinases. The enzyme was readily expressed in E. coli from a plasmid construct from which several milligrams of protein could be isolated, and the molecular mass of the protein was confirmed by electrospray MS. The aroQ gene in H. pylori may function in the central biosynthetic shikimate pathway of this bacterium, thus opening the way for the construction of attenuated strains as potential vaccines as well as offering a new target for selective enzyme inhibition.

Identification of carboxylation enzymes and characterization of a novel four-subunit pyruvate:flavodoxin oxidoreductase from Helicobacter pylori.

The enzyme activities responsible for carboxylation reactions in cell extracts of the gastric pathogen Helicobacter pylori have been studied by H14CO3- fixation and spectrophotometric assays. Acetyl coenzyme A carboxylase (EC 6.4.1.2) and malic enzyme (EC 1.1.1.40) activities were detected, whereas pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxylase (EC 4.1.3.1) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) activities were absent. However, a pyruvate-dependent, ATP-independent, and avidin-insensitive H14CO3- fixation activity, which was shown to be due to the isotope exchange reaction of pyruvate:flavodoxin oxidoreductase (EC 1.2.7.1), was present. The purified enzyme is composed of four subunits of 47, 36, 24, and 14 kDa. N-terminal sequence analysis showed that this enzyme is related to a recently recognized group of four-subunit pyruvate:ferredoxin oxidoreductases previously known only from hyperthermophiles. This enzyme from H. pylori was found to mediate the reduction of a number of artificial electron acceptors in addition to a flavodoxin isolated from H. pylori extracts, which is likely to be the in vivo electron acceptor. Indirect evidence that the enzyme is capable of in vitro reduction of the anti-H. pylori drug metronidazole was also obtained.

Genes encoding homologues of three consecutive enzymes in the butyrate/butanol-producing pathway of Clostridium acetobutylicum are clustered on the Clostridium difficile chromosome.

Screening of a Clostridium difficile lambda EMBL3 gene library with antisera raised against C. difficile culture supernatant identified several clones expressing a 31-kDa protein. A 1.8-kb HindIII fragment subcloned from one of the clones was sufficient for expression of the 31-kDa polypeptide. Southern blot analysis showed a region homologous to this fragment to be present in all of 13 different C. difficile strains tested. Sequence analysis of the 1.8-kb fragment revealed three adjacent open reading frames. A database search showed that these three open reading frames appeared to encode homologues of three consecutive enzymes in the butanol/butyrate-producing pathway of Clostridium acetobutylicum (crotonase, beta-hydroxybutyryl coenzyme A dehydrogenase and thiolase).

Rapid fingerprinting of Helicobacter pylori by polymerase chain reaction and restriction fragment length polymorphism analysis.

A simple and reliable technique was developed for differentiating Helicobacter pylori strains by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified DNAs. Oligonucleotide primer pairs developed to the urease, 48-kDa stress protein (htrA), and 26-kDa antigen-encoding genes were used to amplify fragments of the appropriate size from crude boiled cell preparations. The PCR-amplified products were digested with Sau3A, HaeIII, MspI, AluI, MluI, HinfI, and XbaI restriction endonucleases. Restriction fragment length polymorphisms were particularly evident within the urease and htrA genes and were easily detected by Sau3A, HaeIII, MspI, and AluI restriction endonuclease analysis. Double digestion of these separately amplified products or restriction analysis of multiple PCR-amplified fragments was found to discriminate 17 of 17 (100%) H. pylori strains which had unique genomic DNA fingerprints. Results of an investigation of multiple isolate sets obtained from patients before and after therapy was consistent with the hypothesis that treatment failures were due to the persistence of the same strain but did not discount the possibility that the patients were reinfected with a strain shared by family members or close contacts. The results indicate that the PCR-restriction endonuclease analysis method can be applied directly to biopsy samples, has the potential to fingerprint H. pylori isolates rapidly, and may permit detailed epidemiological investigations on the transmission of this important pathogen.

Sensitive detection of Helicobacter pylori by using polymerase chain reaction.

A polymerase chain reaction (PCR) for the specific detection of Helicobacter pylori was developed with a single primer pair derived from the nucleotide sequence of the urease A gene of H. pylori. We achieved specific amplification of a 411-bp DNA fragment in H. pylori. After 35 cycles of amplification, the product could be detected by agarose gel electrophoresis and contained conserved single HinfI and AluI restriction sites. This fragment was amplified in all 50 strains of H. pylori tested, but it was not detected in other bacterial species, showing the PCR assay to be 100% specific. PCR DNA amplification was able to detect as few as 10 H. pylori cells. PCR detected H. pylori in 15 of 23 clinical human gastric biopsy samples, whereas culturing and microscopy detected H. pylori in only 7 of the samples found to be positive by PCR. Additional primer pairs based on the urease genes enabled the detection of H. pylori in paraffin-embedded human gastric biopsy samples. The detection of H. pylori by PCR will enable both retrospective and prospective analyses of clinical samples, elucidating the role of this organism in gastroduodenal disease.

Evaluation of fingerprinting methods for identification of Helicobacter pylori strains.

Variation amongst strains of Helicobacter pylori was examined by 35S-methionine-labelled protein SDS-PAGE (Radio-PAGE), immunoblot and DNA fingerprinting techniques. These methods allowed discrimination amongst isolates and showed total correlation. Pig and baboon gastric Helicobacter pylori-like organisms were found to be very similar to Helicobacter pylori by both Radio-PAGE and immunoblotting, whereas a Helicobacter mustelae isolate was markedly different. The HindIII restriction endonuclease digest patterns of Helicobacter pylori DNA illustrated the considerable genomic variation of this organism. The Radio-PAGE and immunoblot typing methods both gave precise identification of Helicobacter pylori strains, but Radio-PAGE was found to give higher resolution and represents a standardised universally applicable fingerprinting method for Helicobacter pylori.

Genomic investigation of phenotypic variation in Campylobacter jejuni flagellin.

Western blots of whole-cell sonicates of 10 different clones of a faecal isolate of Campylobacter jejuni 533 detected the expression of flagella antigens of either 59 or 62 kDa. Other antigenic proteins appeared identical both in the parent and all the clones. The mechanism for this phenotypic variation was studied using Southern blotting with a flagellin-specific gene probe and products of a polymerase chain reaction (PCR) using flagellin-gene primers. Restriction-enzyme digestion and Southern blotting did not detect any genomic rearrangements in the flagellin genes of the different phenotypes nor did restriction-enzyme analysis of the PCR products.

Characterization of a plasmid from Helicobacter pylori encoding a replication protein common to plasmids in gram-positive bacteria.

A 1.5 kb cryptic plasmid was isolated from Helicobacter pylori. Low-stringency hybridization analysis using this plasmid as a DNA probe revealed base sequence homology with other plasmids in this species. Nucleotide sequence analysis identified an open reading frame encoding a putative polypeptide of 25 kDa. This protein showed marked amino acid sequence similarity to replication-initiation proteins commonly found in small plasmids endogenous to Gram-positive bacteria which replicate by the 'rolling-circle' mechanism. Sequence motifs corresponding to the origins-of-replication consensus sequences were found on this cryptic plasmid. DNA and oligonucleotide probes to these plasmid replication sequences were used in hybridization analysis to identify similar sequences in other H. pylori plasmids. We believe this is the first plasmid isolated from a Gram-negative bacterium to show replication determinants characteristic of the 'rolling-circle' group of plasmids from Gram-positive bacteria. The cloned plasmid will be used to develop a shuttle-vector for H. pylori.

Identification of toxigenic Clostridium difficile strains by using a toxin A gene-specific probe.

A 4.5-kilobase PstI fragment encoding part of the toxin A gene was isolated and used as a DNA probe in colony hybridization studies with 58 toxigenic and 17 nontoxigenic Clostridium difficile strains. All 58 toxigenic strains showed positive hybridization, in contrast to the 17 nontoxigenic strains. Southern blot analysis with the toxin A gene probe showed hybridization to a single fragment of equal intensities for HindIII-digested genomic DNAs isolated from C. difficile strains of wide-ranging toxin production. The positive hybridization signals were due to fragments of heterogeneous lengths (9 to 13 kilobases) for toxigenic strains of different types but were absent for the nontoxigenic strains. These results suggest the presence of a single copy of the toxin A gene on the genome of C. difficile strains, and the wide variation of toxin expression is not a reflection of gene copy number. The lack of toxin activity for nontoxigenic strains can be explained by the absence of at least part of the toxin A gene. The toxin A gene probe was tested against clostridial strains from 18 other species, of which only toxigenic C. sordellii strains showed positive hybridization. The specificity of the toxin A gene probe for toxigenic strains may lead to improved methods for the specific identification of toxigenic C. difficile strains from clinical specimens.

Nucleotide sequence of Clostridium difficile toxin A gene fragment and detection of toxigenic strains by polymerase chain reaction.

A 1947 base pair (bp) fragment of the toxin A gene of Clostridium difficile was sequenced. A continuous open reading frame was found, which contained 4 distinct groups of repeat nucleotide sequence with 88 to 100% identity within each group. The arrangement of the groups (A, 81 bp, B, C and D, 63 bp) was ABCCCDABCDDABCCCDABCCDABCDABC. Based on nucleotide sequence data from the C repeat group, a pair of oligonucleotide primers were synthesised and used in the polymerase chain reaction (PCR) to amplify fragments from the toxin A gene. Several products of multiples of 63 bp length were amplified for all 33 toxigenic C. difficile strains tested in contrast to the 12 non-toxigenic strains tested which failed to amplify any product. This rapid technique is of potential use in the specific identification of toxigenic C. difficile strains in mixed culture and from clinical specimens.

Molecular cloning and expression of Campylobacter pylori species-specific antigens in Escherichia coli K-12.

A gene bank of Campylobacter pylori DNA in Escherichia coli was constructed by cloning Sau3A-cleaved DNA fragments into the bacteriophage vector lambda EMBL3. The expression of C. pylori antigens was determined by screening the gene library with adsorbed C. pylori whole-cell rabbit antisera. One recombinant clone which reacted positively (lambda CP2) was studied further. Immunoblot analysis with lambda CP2 showed a polypeptide band of 66 kilodaltons (kDa) reacting antigenically with the adsorbed antiserum. Extraction of DNA from lambda CP2 and digestion with SalI revealed a DNA insert of 17 kilobases (kb). Subcloning with SalI and the E. coli vector pUC18 showed that the DNA also encoded a 31-kDa antigen. The cloned antigens were shown by immunoblotting to have the same molecular weight in E. coli as in C. pylori and to be present in all C. pylori strains. Antiserum was raised against the cloned polypeptides and found to react only with C. pylori when analyzed by dot blotting and indirect immunofluorescence. The cloned antigens were determined to be expressed from the pUC18 lac promoter. The DNA encoding these antigens was radiolabeled with 32P and found to hybridize only to C. pylori strains. Immunoblotting with affinity-purified polyclonal antibody to the urease enzyme of C. pylori revealed that the cloned antigens may be part of the urease enzyme.

Identification and molecular cloning of a 70 kDa species-specific antigen common to Clostridium difficile.

Three common antigens (CB 1, 2 and 3), characteristic of Clostridium difficile species were identified by immunoblot analysis using homologous and heterologous rabbit antisera, raised against whole cells from 9 distinct strains of C. difficile. A gene library of C. difficile genomic DNA was constructed in Escherichia coli by cloning in Sau 3A-cleaved clostridial DNA fragments into the bacteriophage vector lambda EMBL3. OUt of 3000 plaques screened using the whole cell antisera, 27 clones were positively identified. One of these clones, designated gamma Cd21, expressed high levels of an antigen which could be immunologically identified using whole cell antisera against the 9 C. difficile strains. Antiserum raised against the clone gamma Cd21 identified a 70 kDa antigen (previously named CB1) as demonstrated by immunoblot analysis. Monospecific antiserum against gamma Cd21 recognises the 70 kDa antigen in all 97 strains of C. difficile derived from worldwide sources and does not cross-react with 17 strains from 13 other clostridial species.