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.

Helicobacter pylori infection is strongly associated with histologic gastritis and peptic ulcer disease. Broth culture supernatants from a subset of H. pyloni strains induce vacuolization in cultured cells, a phenomenon that has been attributed to cytotoxin activity. Concentrated culture supernatants from 15 of 28 (53.6%) H. pylori strains tested induced vacuolization in HeLa cells in titers ranging from 1:10 to 1:180. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of supernatants from these 28 strains and 2 control strains demonstrated an 82-kilodalton (kDa) protein band in 3 of 16 supernatants with vacuolizing activity, but in none of 14 supernatants without vacuolizing activity. By immunoblotting with human sera, a 128-kDa band was recognized in all 16 supernatants with vacuolizing activity, compared with 9 of 14 (64%) supernatants without vacuolizing activity (P = 0.014). Serologic recognition of the 128-kDa band in H. pyloni culture supernatants was more prevalent among persons infected with vacuolizing H. pylori strains than among persons infected with nonvacuolizing strains, but the difference was not statistically significant (80 versus 45%; P = 0.079); human serologic recognition of the 82-kDa band was less common. The 128-kDa band was recognized by 100% of 31 serum samples from H. pyloni-infected patients with duodenal ulcer disease, compared with 60.8% of 74 serum samples from H. pyloni-infected persons without peptic ulcer disease (P = 0.0001). These data indicate that antigenic 128-and 82-kDa proteins are present in H. pyloni broth culture supernatants with vacuolizing activity and that serologic responses to the 128-kDa protein are more prevalent among H. pyloni-infected persons with duodenal ulceration than among infected persons without peptic ulceration.

Concentrated broth culture supernatants from 50 to 60% of Helicobacterpylori strains induce eukaryotic cell vacuolation in vitro. A quantitative assay for cell vacuolation was developed on the basis of the rapid uptake of neutral red dye into intracellular vacuoles. The neutral red dye uptake (NRU) of visibly vacuolated HeLa cells was significantly greater than that of nonvacuolated cells. By using the rapid NRU assay, we sought to determine the roles of H. pylori cytotoxin, urease, and ammonia in the vacuolation of HeLa cells. The NRU of HeLa cells incubated in medium containing ammonium chloride or ammonium sulfate was significantly greater than that of cells incubated in medium alone. In addition, ammonium salts augmented the NRU induced by H. pylori supernatants. The NRU induced by jack bean urease was augmented by the addition of urea to cell culture medium; this suggests that urease-mediated NRU occurs via the generation of ammonia. Acetohydroxamic acid blocked the NRU induced by jack bean urease and urea but failed to block the uptake induced by H. pyloni supernatants. Supernatant from a non-urease-producing H. pylori mutant strain induced NRU identical to that of the urease-positive parental strain. These observations indicate that the vacuolating activity in H. pyloni supernatants is not mediated solely by urease activity but that it may be potentiated by urease-mediated ammonia production. Helicobacter pylori infection has been strongly associated with type B gastritis and peptic ulcer disease, but the mechanisms whereby infection may lead to tissue inflammation and damage are poorly understood (3). Concentrated broth culture supernatants from 50 to 60% of H. pylori strains induce vacuolation of cells in vitro (5, 12), a phenomenon that has been attributed to cytotoxin activity. The vacuolating activity in supernatants is abolished by heating to 70°C or by incubation with proteases (12), and thus it is probable that vacuolation is mediated at least in part by a protein. Recently, the vacuolating activity in H. pylori culture supernatants has been attributed to urease activity rather than to a specific cytotoxin (25). The degradation of urea by urease liberates ammonia, which is known to induce cell injury (2, 22, 25); in addition, ammonia has long been known to induce cell vacuolation (6, 18, 20). To study further the vacuolation of cells induced by H. pylori supernatants, we developed a quantitative assay for cell vacuolation on the basis of the rapid uptake of neutral red dye by cell vacuoles. By using this assay, we sought to clarify the roles of H. pylori cytotoxin, urease, and ammonia in the phenomenon of cell vacuolation. MATERIALS AND METHODS Bacterial strains. H. pylori 60190 and Tx3Oa, Tox+ and Tox-, respectively (5, 12), were used for most studies. These strains were cultured in brucella broth (BBL Microbiology Systems, Cockeysville, Md.) containing 5% fetal bovine serum for 48 h on a rotary shaker at 37°C in ambient air supplemented with 5% CO2. H. pylori 88-3887 (urease positive) and 26U1 (a...

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.

We performed polymerase chain reaction analysis, for Mycobacterium species 16S rRNA, rpoB , and IS 6110 sequences, on 25 tissue specimens from patients with sarcoidosis and on 25 control tissue specimens consisting of mediastinal or cervical lymph nodes and lung biopsies. Mycobacterium species 16S rRNA sequences were amplified from 12 (48%) rpoB sequences from 6 (24%) of the sarcoidosis specimens. In total, 16S rRNA or rpoB sequences were amplified from 15 sarcoidosis specimens (60%) but were not detected in any of the control tissues (p=0.00002, Chi square). In three specimens, the sequences resembled Mycobacterium species other than M. tuberculosis . All specimens with sequences consistent with M. tuberculosis were negative for IS 6110 . We provide evidence that one of a variety of Mycobacterium species , especially organisms resembling M. tuberculosis, is found in most patients with sarcoidosis.

Alleles of the vacuolating cytotoxin gene (vacA) ofHelicobacter pylori vary between strains, particularly in the region encoding the signal sequence (which may be type s1 or s2) and the midregion (which may be type m1 or m2). Using a PCR-based typing system developed in the United States, we showed that 36 strains from Asia and South America were all vacA signal sequence type s1; 3 were midregion type m1 and 11 were m2, but 22 could not be typed for the vacA midregion. All strains possessedcagA (cytotoxin-associated gene A), another virulence marker. vacA nucleotide sequence analysis showed that midregion typing failure was due to base substitutions at the primer annealing sites. Using the new sequence data, we developed two new PCR-based vacA midregion typing systems, both of which correctly typed 41 U.S. strains previously typed by the old system and successfully typed all 36 of the non-U.S. strains. All previously untypeable strains were vacA m1, other than one m1/m2 hybrid. In summary, we describe and validate a simple PCR-based system for typing vacuolating cytotoxin (vacA) alleles of H. pylori and show that this system correctly identifies the signal and midregion types of vacA in 77 strains from Asia and North and South America.

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 ؉ ]). The pathological effects on histological sections of gastric mucosae were assessed and were compared with the effects of treatments with extracts from noncytotoxic strains (G21 and G50 [CagA ؊ /Tox ؊ ]) and from strains that express either CagA alone (D931 [CagA ؉ / Tox ؊ ]) or the cytotoxin alone (G104 [CagA ؊ /Tox ؉ ]). The treatment with extracts from cytotoxic strains induced various epithelial lesions (vacuolation, erosions, and ulcerations), recruitment of inflammatory cells in the lamina propria, and a marked reduction of the mucin layer. Extracts of noncytotoxic strains induced mucin depletion but no other significant pathology. Crude extracts of strain D931, expressing CagA alone, caused only mild infiltration of inflammatory cells, whereas extracts of strain G104, expressing cytotoxin alone, induced extensive epithelial damage but little inflammatory reaction. Loss of the mucin layer was not associated with a cytotoxic phenotype, since this loss was observed in mice treated with crude extracts of all strains. The pathogenic roles of CagA, cytotoxin, and urease were further assessed by using extracts of mutant strains of H. pylori defective in the expression of each of these virulence factors. The results obtained suggest that (i) urease activity does not play a significant role in inducing the observed gastric damage, (ii) cytotoxin has an important role in the induction of gastric epithelial cell lesions but not in eliciting inflammation, and (iii) other components present in strains which carry the cagA gene, but distinct from CagA itself, are involved in eliciting the inflammatory response.

LRRC8 family proteins on the plasma membrane play a critical role in cellular osmoregulation by forming volume-regulated anion channels (VRACs) necessary to prevent necrotic cell death. We demonstrate that intracellular LRRC8 proteins acting within lysosomes also play an essential role in cellular osmoregulation. LRRC8 proteins on lysosome membranes generate large lysosomal volume-regulated anion channel (Lyso-VRAC) currents in response to low cytoplasmic ionic strength conditions. When a double-leucine L706L707 motif at the C terminus of LRRC8A was mutated to alanines, normal plasma membrane VRAC currents were still observed, but Lyso-VRAC currents were absent. We used this targeting mutant, as well as pharmacological tools, to demonstrate that Lyso-VRAC currents are necessary for the formation of large lysosome-derived vacuoles, which store and then expel excess water to maintain cytosolic water homeostasis. Thus, Lyso-VRACs allow lysosomes of mammalian cells to act as the cell`s “bladder.” When Lyso-VRAC current was selectively eliminated, the extent of necrotic cell death to sustained stress was greatly increased, not only in response to hypoosmotic stress, but also to hypoxic and hypothermic stresses. Thus Lyso-VRACs play an essential role in enabling cells to mount successful homeostatic responses to multiple stressors.