The Arabidopsis hyperpolarization-activated (inward-rectifying) K+ channel KAT1 is structurally more similar to animal depolarization-activated (outward-rectifying) K+ channels than to animal hyperpolarization-activated K+ channels. To gain insight into the structural basis for the opposite voltage dependences of plant inward-rectifying and animal outward-rectifying K+ channels, we constructed recombinant chimeric channels between the hyperpolarization-activated K+ channel KAT1 and a Xenopus depolarization-activated K+ channel. We report here that two of the chimeric constructs, which contain the first third of the KAT1 sequence, including the first four membrane-spanning segments (S1-S4) and the linker sequence between the fourth and fifth membrane-spanning segments, express functional channels that retain activation by hyperpolarization, but not depolarization. These two chimeric channels are no longer selective for K+. The chimeras are selective for cations over anions and are permeable to Ca2+. Therefore, unlike animal hyperpolarization-activated K+ channels, in which the carboxyl terminus is important for inward rectification induced by Mg2+ and polyamine block, the plant KAT1 channel has its major determinants for inward rectification in the amino-terminal region, which ends at the end of the S4-S5 linker.

A high-molecular-mass (120to 128-kDa) Helicobacter pyloni antigen has been associated with peptic ulcer disease. We created a bank of 40,000 random chromosomal fragments of H. pyloni 84-183 by using AZapII. Screening of this bank in Escherichia coli XL1-Blue with absorbed serum from an H. pyloni-infected person permitted the isolation and purification of a clone with a 3.5-kb insert. Subcloning of this insert (pMC3) permitted the expression of a recombinant H. pyloni protein that had a mass of approximately 96 kDa and that was recognized by the human serum. Sera that were obtained from H. pylori-infected persons and that recognized the native 120to 128-kDa H. pylori antigen recognized the recombinant 96-kDa pMC3 protein to a significantly greater extent than did sera that did not recognize the native H. pylori antigen. All 19 H. pyloni isolates producing the 120to 128-kDa antigen hybridized with pMC3; none of 13 nonproducers did so (P < 0.001). Because all 15 isolates producing the vacuolating cytotoxin hybridized with pMC3, we called the gene cagA (cytotoxin-associated gene). Sequence analysis of pMC3 identified an open reading frame of 859 amino acids, without a termination codon. Parallel screening of a Agtll library with human serum revealed positive plaques with identical 0.6-kb inserts and sequences matching the sequence of the downstream region of pMC3. To clone the full-length gene, we used the 0.6-kb fragment as a probe and isolated a clone with a 2.7-kb insert from the AZapII genomic library. Nucleotide sequencing of this insert (pYB2) revealed a 785-bp sequence that overlapped the downstream region of pMC3. Translation of the complete nucleotide sequence ofcagA revealed an open reading frame of 1,181 amino acids yielding a protein of 131,517 daltons. There was no significant homology with any previously reported protein sequence. These findings indicate the cloning and characterization of a high-molecular-mass H. pylori antigen potentially associated with virulence and with cytotoxin production.

Approximately 60% of Helicobacter pylori strains are cagA+ and this genotype is more frequently associated with duodenal ulcer disease. Although most wild-type cagA+ strains are both cytotoxigenic and induce enhanced Interleukin-8 (IL-8) secretion in gastric epithelial cells, isogenic cagA- mutants retain full activity in these assays; thus, cagA appears to be a marker of enhanced virulence. Delineation of the nucleotide sequence of a 4 kb region upstream of cagA allowed the identification of 966 bp (picA) and 2655 bp (picB) open reading frames encoding 36 kDa and 101 kDa polypeptides, respectively. picA and picB constitute an operon in opposite orientation to cagA. The deduced picB product showed significant homology (26% identity and 50% similarity) with the Bordetella pertussis toxin secretion protein (PtlC). Of 55 H. pylori clinical isolates, the picA and picB segment was conserved exclusively in cagA+ strains and present in all isolates from patients with duodenal ulceration, versus 59% of isolates from patients with gastritis alone (P = 0.01). Using gene-replacement techniques, we constructed picA and picB mutant H. pylori strains and demonstrated that the picB gene product is involved in the induction of IL-8 expression in gastric epithelial cells. Further, Northern blot hybridization and RT-PCR data showed that picA and picB are co-transcribed and an insertional mutation in picA ablates picB expression. These studies indicate a role of picA and picB in the induction of an inflammatory response in gastric epithelial cells either directly or by enabling secretion of an unidentified product, and suggest a mechanism for the overrepresentation of strains possessing these genes in patients with peptic ulceration.

Gastric infection with Helicobacter pylori activates a mucosal inflammatory response by mononuclear cells and neutrophils that includes expression of cytokines interleukin-1␤ (IL-1␤), IL-6, tumor necrosis factor alpha, and IL-8. In this study, we analyzed the IL-8 response of human gastric cancer cell lines (Kato III, AGS, and MKN28) to H. pylori infection in vitro. IL-8 mRNA expression was detected by reverse transcription-PCR amplification of RNA extracted from epithelial cells after incubation with different H. pylori wild-type and mutant strains, and IL-8 secretion was measured by an enzyme-linked immunosorbent assay. Exposure to viable H. pylori induced IL-8 mRNA and protein synthesis in all three gastric cell lines but not in nongastric epithelial cell lines. Heat-killed H. pylori and a crude cytotoxin preparation did not induce significant IL-8 secretion. IL-8 mRNA peaked between 2 and 4 h postinfection, and IL-8 protein production was maximal 24 h postinfection. Exposure of gastric carcinoma cells to other gastrointestinal bacteria, such as Pseudomonas aeruginosa, Campylobacter jejuni, and Escherichia coli, but not Campylobacter fetus, induced IL-8 synthesis. Wild-type strains that expressed the vacuolating cytotoxin (Tox ؉) and a cytotoxin-associated gene (cagA) product (CagA ؉) induced significantly more IL-8 than did CagA ؊ Tox ؊ strains. However, there was no decrease in IL-8 induction by isogenic mutants of CagA ؊ , Tox ؊ , or Cag ؊ Tox ؊ strains or by a mutant lacking the urease subunits. These results indicate that exposure to H. pylori and other gram-negative organisms that do not colonize the gastric mucosa induces IL-8 production by gastric carcinoma cells in vitro. Although the CagA ؉ Tox ؉ phenotype of H. pylori is associated with enhanced IL-8 production by gastric cell lines, other bacterial constituents are clearly essential.

Approximately 60% of Helicobacter pylori isolates possess the cagA gene and express its 120-to 140-kDa product (CagA). In this study, the cagA gene was detected in H. pylori isolates from 26 (81.3%) of 32 patients with duodenal ulcers (DU), 17 (68.0%) of 25 patients with gastric ulcers, and 23 (59.0%) of 39 patients with nonulcer dyspepsia (NUD). By Western blotting (immunoblotting) with antiserum to CagA, in vitro CagA expression was demonstrated for 95.5% of cagA ؉ strains compared with 0% of strains lacking cagA. Sera from patients infected with cagA ؉ strains (n ‫؍‬ 66) reacted with recombinant CagA in an enzyme-linked immunosorbent assay to a significantly greater extent than either sera from patients infected with strains lacking cagA (n ‫؍‬ 30) or sera from uninfected persons (n ‫؍‬ 25) (P < 0.001). A strain lacking cagA was isolated from eight patients who had serum immunoglobulin G antibodies to CagA, which suggests that these patients were infected with multiple strains. Serum immunoglobulin G antibodies to CagA were present in 87.5, 76.0, and 56.4% of patients with DU, gastric ulcers, and NUD, respectively (odds ratio, 5.41; 95% confidence interval, 1.44 to 24.72; P ‫؍‬ 0.004 [DU versus NUD]). These data demonstrate an association between infection with cagA ؉ H. pylori and the presence of duodenal ulceration and indicate that serologic testing is a sensitive method for detecting infection with cagA ؉ strains.

analysis has demonstrated that sapA2 was not expressed in the C. fetus strain from which it was cloned. Further Southern analyses revealed increasing sapA diversity as probes increasingly 3 within the ORF were used. Pulsed-field gel electrophoresis and then Southern blotting with the conserved N-terminal region of the sapA homologs as a probe showed that these genes were tightly clustered on the chromosome. Deletion mutagenesis revealed that the S-layer protein bound serospecifically to the C. fetus lipopolysaccharide via its conserved N-terminal region. These data indicated that the S-layer proteins shared functional activity in the conserved N terminus but diverged in a semiconservative manner for the remainder of the molecule. Variation in S-layer protein expression may involve rearrangement of complete gene copies from a single large locus containing multiple sapA homologs.

Wild-type Campylobacterfetus cells possess S-layer proteins (S+ phenotype), whereas after laboratory passage, spontaneous stable mutants that do not express these proteins (S-phenotype) arise. To determine the molecular mechanisms by which C.fetus changes to the S-phenotype, we studied wild-type strain 23D, from which the sapA gene encoding the 97-kDa S-layer protein has been cloned, and strain 23B, a spontaneous S-mutant. We compared these strains with another pair of strains, LP (S+) and HP (S5 Northern RNA blot analysis showed no sapA mRNA in strain 23B. These data indicate that the lack of S-layer protein expression in spontaneous mutant strains is caused by the deletion of promoter sequences.Many microorganisms possess regular surface layers (Slayers) that are noncovalently attached to membrane structures (35). The superficial location of these proteins suggests that these layers are important mediators of interactions with hosts (4,5,21,24). Although the morphological properties of S-layers have been extensively studied for a wide range of microorganisms (22,34,35), relatively little is known about the regulatory mechanisms involved in the biosynthesis, transport, and assembly of S-layer proteins.Campylobacterfetus is an important veterinary pathogen that causes infertility and infectious abortion in sheep and cattle (36) and extraintestinal infections (16) and a thrombotic diathesis (8) in humans. The ability of C. fetus to produce these diseases is associated with the presence of surface array proteins (4,5,11,21,24). The C. fetus S-layer is composed of high-molecular-mass protein subunits that migrate at approximately 97, 127, and 149 kDa; usually a subunit of a single size predominates for a given strain (10, 25). C. fetus strains may contain either type A or type B lipopolysaccharide, and their S-layer proteins are lipopolysaccharide type specific (38). The type A S-layer proteins are antigenically related and have identical N-terminal amino acid sequences (25) but differ from the type B proteins (10, 38).S-layer protein expression in C. fetus is subject to antigenic variation (10,25,(30)(31)(32). It is possible to isolate C. fetus variants that express from zero to up to three different S-layer proteins (10, 25), but the determinants of which protein is predominantly expressed are not known. carrying S-layer proteins (S+ phenotype), but not spontaneous mutants lacking them (S-phenotype), resist complement-mediated killing by normal or immune serum and resist phagocytosis (4,5,21). Strain 23B is an S-spontaneous mutant of the wild-type S+ strain 23D (21).The mechanisms by which pathogens alter their surface antigens are varied but often involve programmed DNA rearrangements (7). As a first step in elucidating the molecular mechanisms involved in the antigenic variation of the C. fetus S-layer proteins, we recently cloned and sequenced the strain 23D sapA gene, which encodes a polypeptide of 97 kDa (3). The aim of our present studies is to assess the molecular principles of C. fetus S-layer protein pro...

The Campylobacter fetus surface-layer (Slayer) proteins mediate both complement resistance and antigenic variation in mammalian hosts. Wild-type strain 23D possesses the sapA gene, which encodes a 97-kDa S-layer protein, and several sapA homologs are present in both wildype and mutant strains. Here we report that a cloned silent gene (sapAI) in C. fetus can express a functional full-length S-layer protein in Escherichia coli. Analysis of sapA and sapAl and partial analysis of sapA2 indicate that a block of -600 bp b nning upstream and continuing into the open reading frames is completely conserved, and then the sequences diverge completely, but immediately downstream of each gene is another conserved 50-bp sequence. Conservation of sapAl among stains, the presence of a putative Chi (RecBCD recognition) site upstream of sapA, sapAl, and sapA2, and the sequence identities of the sapA genes suggest a system for homologous recombination. Comparison of the wild-type strain (23D) with a phenotypic variant (23D-11) indicates that variation is associated with removal of the divergent region of sapA from the expression locus and exchange with a corresponding region from a sapA homolog. We propose that site-specific reciprocal recombination between sapA homologs leads to expression of divergent S-layer proteins as one of the mechanisms that C. fetus uses for antigenic variation.