Helicobacter pylori and its secreted immunomodulator VacA protect against anaphylaxis in experimental models of food allergy.

BACKGROUND: Food allergy is an increasingly common health problem in Western populations. Epidemiological studies have suggested both positive and negative associations between food allergy and infection with the gastric bacterium Helicobacter pylori. OBJECTIVE: The objective of this work was to investigate whether experimental infection with H. pylori, or prophylactic treatment with H. pylori-derived immunomodulatory molecules, affects the onset and severity of food allergy, either positively or negatively. METHODS: We infected neonatal C57BL/6 or C3H mice with H. pylori or treated animals with H. pylori components (bacterial lysate or the immunomodulator VacA) and subsequently subjected them to four different protocols for food allergy induction, using either ovalbumin or peanut extract as allergens for sensitization and challenge. Readouts included anaphylaxis scoring, quantification of allergen-specific serum IgE and IgG1 and of the mast cell protease MCPT1, as well as splenic T-helper-2 cell-derived cytokine production. Mesenteric lymph node CD4+ FoxP3+ regulatory T cells were subjected to flow cytometric quantification and sorting followed by qRT-PCR, and to DNA methylation analyses of the Treg-specific demethylated region (TSDR) within the FOXP3 locus. RESULTS: Mice that had been infected with H. pylori or treated with H. pylori-derived immunomodulators showed reduced anaphylaxis upon allergen sensitization and challenge, irrespective of the allergen used. Most of the immunologic assays confirmed a protective effect of H. pylori. CD4+ FoxP3+ T cells were more abundant in protected mice and exhibited a stable Treg phenotype characterized by FOXP3 TSDR demethylation. CONCLUSIONS AND CLINICAL RELEVANCE: Helicobacter pylori confers protection against the anaphylaxis associated with ovalbumin and peanut allergy and affects the epigenome of T cells, thereby promoting stable Treg differentiation and functionality. Prophylactic treatment with H. pylori-derived immunomodulators appears to be a promising strategy for food allergy prevention.

A 12-amino-acid segment, present in type s2 but not type s1 Helicobacter pylori VacA proteins, abolishes cytotoxin activity and alters membrane channel formation.

Helicobacter pylori, a gram-negative bacterium associated with gastritis, peptic ulceration, and gastric adenocarcinoma in humans, secretes a protein toxin, VacA, that causes vacuolar degeneration of epithelial cells. Several different families of H. pylori vacA alleles can be distinguished based on sequence diversity in the "middle" region (i.e., m1 and m2) and in the 5' end of the gene (i.e., s1 and s2). Type s2 VacA toxins contain a 12-amino-acid amino-terminal hydrophilic segment, which is absent from type s1 toxins. To examine the functional properties of VacA toxins containing this 12-amino-acid segment, we analyzed a wild-type s1/m1 VacA and a chimeric s2/m1 VacA protein. Purified s1/m1 VacA from H. pylori strain 60190 induced vacuolation in HeLa and Vero cells, whereas the chimeric s2/m1 toxin (in which the s1 sequence of VacA from strain 60190 was replaced with the s2 sequence from strain Tx30a) lacked detectable cytotoxic activity. Type s1/m1 VacA from strain 60190 formed membrane channels in a planar lipid bilayer assay at a significantly higher rate than did s2/m1 VacA. However, membrane channels formed by type s1 VacA and type s2 VacA proteins exhibited similar anion selectivities (permeability ratio, P(Cl)/P(Na) = 5). When an equimolar mixture of the chimeric s2/m1 toxin and the wild-type s1/m1 toxin was added to HeLa cells, the chimeric toxin completely inhibited the activity of the s1/m1 toxin. Thus, the s2/m1 toxin exhibited a dominant-negative phenotype similar to that of a previously described mutant toxin, VacA-(Delta6-27). Immunoprecipitation experiments indicated that both s2/m1 VacA and VacA-(Delta6-27) could physically interact with a c-myc epitope-tagged s1/m1 VacA, which suggests that the dominant-negative phenotype results from the formation of heterooligomeric VacA complexes with defective functional activity. Despite detectable differences in the channel-forming activities and cytotoxic properties of type s1 and type s2 VacA proteins, the conservation of type s2 sequences in many H. pylori isolates suggests that type s2 VacA proteins retain an important biological activity.

In search of the Helicobacter pylori VacA mechanism of action.

Helicobacter pylori secretes an approximately 88 kDa VacA toxin that is considered to be an important virulence factor in the pathogenesis of peptic ulcer disease. Over the past decade, research on the molecular mechanisms and biological functions of VacA has generated a complex and often puzzling scenario. VacA is secreted into the extracellular space and also is partially retained on the bacterial cell surface, exists in monomeric and oligomeric forms, and binds to multiple eukaryotic cell-surface receptors. The cellular effects induced by VacA include vacuolation, alteration of endo-lysosomal function, pore formation in the plasma membrane, apoptosis, and epithelial monolayer permeabilisation. VacA has been reported to target several different cell components, including endocytic vesicles, mitochondria, the cytoskeleton, and epithelial cell-cell junctions. It remains unclear whether VacA should be classified as an A/B type toxin, a channel-forming toxin, or both. This review is intended to summarise our current knowledge about VacA, and to orient the reader to this fascinating and challenging research area.

Antigenic diversity among Helicobacter pylori vacuolating toxins.

Helicobacter pylori vacuolating cytotoxin (VacA) is a secreted protein that induces vacuolation of epithelial cells. To study VacA structure and function, we immunized mice with purified type s1-m1 VacA from H. pylori strain 60190 and generated a panel of 10 immunoglobulin G1kappa anti-VacA monoclonal antibodies. All of the antibodies reacted with purified native VacA but not with denatured VacA, suggesting that these antibodies react with conformational epitopes. Seven of the antibodies reacted with both native and acid-treated VacA, which suggests that epitopes present on both oligomeric and monomeric forms of the toxin were recognized. Two monoclonal antibodies, both reactive with epitopes formed by amino acids in the carboxy-terminal portion of VacA (amino acids 685 to 821), neutralized the cytotoxic activity of type s1-m1 VacA when toxin and antibody were mixed prior to cell contact but failed to neutralize the cytotoxic activity of type s1-m2 VacA. Only 3 of the 10 antibodies consistently recognized type s1-m1 VacA toxins from multiple H. pylori strains, and none of the antibodies recognized type s2-m2 VacA toxins. These results indicate that there is considerable antigenic diversity among VacA toxins produced by different H. pylori strains.

Helicobacter pylori genotypes, host factors, and gastric mucosal histopathology in peptic ulcer disease.

From 183 patients undergoing upper gastrointestinal endoscopy, we used antral and corpus gastric biopsies for bacterial culture and histopathologic examination, blood samples to detect immunoglobulin G antibodies against Helicobacter pylori, and H pylori genomic DNA to analyze cytotoxin-associated gene A (cagA) and vacuolating cytotoxin (vacA) genotypes. As expected, among H pylori biopsy-positive patients, those with duodenal ulcer (DU) (n = 34) had significantly more severe chronic and acute inflammation (P <.001) and epithelial degeneration (P =.004) in the gastric antrum than in the gastric corpus. Each of those 3 parameters and H pylori density were significantly higher in the antrum of patients with DU than in patients with gastric ulcer (GU) or no ulcer. Colonization with vacA s1/cagA-positive strains of H pylori was associated with inflammation and epithelial degeneration in gastric mucosa and increased risk for peptic ulcer disease (PUD), whereas colonization with vacA s2m2/cagA-negative strains was associated with mild gastric histopathology and was not associated with any significant risk for PUD. The predominant H pylori strains in African Americans were vacA s1bm1/cagA-positive, whereas all genotypes were well represented in non-Hispanic-Caucasians. By multivariate analysis, H pylori colonization was significantly associated with DU (Adjusted odds ratio [AdjOR] = 3.2 [1.4-7.2]) and nonsteroidal anti-inflammatory drugs (NSAID) use was inversely associated (AdjOR = 0.3 [0.2-0.7]). NSAID use (AdjOR = 4.3 [1.02-18.5]) and African-American ethnicity (AdjOR = 10.9 [2.6-50]) were significantly associated with GU. Smoking and age were not significantly associated with either DU or GU. These data indicate that DU is associated with an antral-dominant gastritis, and H pylori genotype and NSAID use independently contribute to the pathogenesis of PUD. HUM PATHOL 32:264-273. This is a US Government work. There are no restrictions on its use.

Amino-terminal hydrophobic region of Helicobacter pylori vacuolating cytotoxin (VacA) mediates transmembrane protein dimerization.

Helicobacter pylori VacA is a secreted protein toxin that forms channels in lipid bilayers and induces multiple structural and functional alterations in eukaryotic cells. A unique hydrophobic segment at the amino terminus of VacA contains three tandem repeats of a GxxxG motif that is characteristic of transmembrane dimerization sequences. To examine functional properties of this region, we expressed and analyzed ToxR-VacA-maltose binding protein fusions using the TOXCAT system, which was recently developed by W. P. Russ and D. M. Engelman (Proc. Natl. Acad. Sci. USA 96:863-868, 1999) to study transmembrane helix-helix associations in a natural membrane environment. A wild-type VacA hydrophobic region mediated insertion of the fusion protein into the inner membrane of Escherichia coli and mediated protein dimerization. A fusion protein containing a mutant VacA hydrophobic region (in which glycine 14 of VacA was replaced by alanine) also inserted into the inner membrane but dimerized significantly less efficiently than the fusion protein containing the wild-type VacA sequence. Based on these results, we speculate that the wild-type VacA amino-terminal hydrophobic region contributes to oligomerization of the toxin within membranes of eukaryotic cells.

Carboxy-terminal proteolytic processing of Helicobacter pylori vacuolating toxin.

The vacA gene of Helicobacter pylori strain 60190 encodes a 1, 287-amino-acid protoxin, which undergoes cleavage of a 33-amino-acid amino-terminal signal sequence and carboxy-terminal proteolytic processing to yield a mature secreted toxin. Several features of VacA suggest that it belongs to the autotransporter family of gram-negative bacterial secreted proteins. Based on matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis, we calculate that the mature toxin has a mass of 88.2+/-0.2 kDa and consists of approximately 821 amino acids.

Acid activation of Helicobacter pylori vacuolating cytotoxin (VacA) results in toxin internalization by eukaryotic cells.

Helicobacter pylori VacA is a secreted toxin that induces multiple structural and functional alterations in eukaryotic cells. Exposure of VacA to either acidic or alkaline pH ('activation') results in structural changes in the protein and a marked enhancement of its cell-vacuolating activity. However, the mechanism by which activation leads to increased cytotoxicity is not well understood. In this study, we analysed the binding and internalization of [125I]-VacA by HeLa cells. We detected no difference in the binding of untreated and activated [125I]-VacA to cells. Binding of acid-activated [125I]-VacA to cells at 4 degrees C was not saturable, and was only partially inhibited by excess unlabelled toxin. These results suggest that VacA binds either non-specifically or to an abundant, low-affinity receptor on HeLa cells. To study internalization of VacA, we used a protease protection assay. Analysis by SDS-PAGE and autoradiography indicated that the intact 87 kDa toxin was internalized in a time-dependent process at 37 degrees C but not at 4 degrees C. Furthermore, internalization of the intact toxin was detected only if VacA was acid or alkaline activated before being added to cells. The internalization of activated [125I]-VacA was not substantially inhibited by the presence of excess unlabelled toxin, but was blocked if cells were depleted of cellular ATP by the addition of sodium azide and 2-deoxy-D-glucose. These results indicate that acid or alkaline pH-induced structural changes in VacA are required for VacA entry into cells, and that internalization of the intact 87 kDa toxin is required for VacA cytotoxicity.

Intercellular communication in Helicobacter pylori: luxS is essential for the production of an extracellular signaling molecule.

Individual bacteria of numerous species can communicate and coordinate their actions via the production, release, and detection of extracellular signaling molecules. In this study, we used the Vibrio harveyi luminescence bioassay to determine whether Helicobacter pylori produces such a factor. Cell-free conditioned media from H. pylori strains 60190 and 26695 each induced >100-fold-greater luminescence in V. harveyi than did sterile culture medium. The H. pylori signaling molecule had a molecular mass of <10 kDa, and its activity was unaffected by heating to 80 degrees C for 5 min or protease treatment. The genome sequence of H. pylori 26695 does not contain any gene predicted to encode an acyl homoserine lactone synthase but does contain an orthologue of luxS, which is required for production of autoinducer-2 (AI-2) in V. harveyi. To evaluate the role of luxS in H. pylori, we constructed luxS null mutants derived from H. pylori 60190 and 26695. Conditioned media from the wild-type H. pylori strains induced >100-fold-greater luminescence in the V. harveyi bioassay than did conditioned medium from either mutant strain. Production of the signaling molecule was restored in an H. pylori luxS null mutant strain by complementation with a single intact copy of luxS placed in a heterologous site on the chromosome. In addition, Escherichia coli DH5alpha produced autoinducer activity following the introduction of an intact copy of luxS from H. pylori. Production of the signaling molecule by H. pylori was growth phase dependent, with maximal production occurring in the mid-exponential phase of growth. Transcription of H. pylori vacA also was growth phase dependent, but this phenomenon was not dependent on luxS activity. These data indicate that H. pylori produces an extracellular signaling molecule related to AI-2 from V. harveyi. We speculate that this signaling molecule may play a role in regulating H. pylori gene expression.

Cell vacuolation induced by the VacA cytotoxin of Helicobacter pylori is regulated by the Rac1 GTPase.

Chronic gastric infection with the Gram-negative bacterium Helicobacter pylori is a major contributing factor in the development of duodenal ulcers and is believed to be a significant risk factor in the development of gastric tumors. The VacA cytotoxin of H. pylori is a 90-kDa secreted protein that forms trans-membrane ion channels. In epithelial cells, VacA activity is associated with the rapid formation of acidic vacuoles enriched for late endosomal and lysosomal markers. Rac1 is a member of the Rho family of small GTP-binding proteins that regulate reorganization of the actin cytoskeleton and intracellular signal transduction and are being shown increasingly to play a role in membrane trafficking events. In this study we report that: (i) green fluorescent-tagged Rac1 localizes around the perimeter of the vacuoles induced by VacA; (ii) expression of dominant negative Rac1 in epithelial cells inhibits vacuole formation; (iii) expression of constitutively active Rac1 potentiates the activity of VacA. Taken together, these data demonstrate a role for Rac1 in the regulation of VacA activity.

A dominant negative mutant of Helicobacter pylori vacuolating toxin (VacA) inhibits VacA-induced cell vacuolation.

Most Helicobacter pylori strains secrete a toxin (VacA) that causes structural and functional alterations in epithelial cells and is thought to play an important role in the pathogenesis of H. pylori-associated gastroduodenal diseases. The amino acid sequence, ultrastructural morphology, and cellular effects of VacA are unrelated to those of any other known bacterial protein toxin, and the VacA mechanism of action remains poorly understood. To analyze the functional role of a unique strongly hydrophobic region near the VacA amino terminus, we constructed an H. pylori strain that produced a mutant VacA protein (VacA-(Delta6-27)) in which this hydrophobic segment was deleted. VacA-(Delta6-27) was secreted by H. pylori, oligomerized properly, and formed two-dimensional lipid-bound crystals with structural features that were indistinguishable from those of wild-type VacA. However, VacA-(Delta6-27) formed ion-conductive channels in planar lipid bilayers significantly more slowly than did wild-type VacA, and the mutant channels were less anion-selective. Mixtures of wild-type VacA and VacA-(Delta6-27) formed membrane channels with properties intermediate between those formed by either isolated species. VacA-(Delta6-27) did not exhibit any detectable defects in binding or uptake by HeLa cells, but this mutant toxin failed to induce cell vacuolation. Moreover, when an equimolar mixture of purified VacA-(Delta6-27) and purified wild-type VacA were added simultaneously to HeLa cells, the mutant toxin exhibited a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA. A dominant negative effect also was observed when HeLa cells were co-transfected with plasmids encoding wild-type and mutant toxins. We propose a model in which the dominant negative effects of VacA-(Delta6-27) result from protein-protein interactions between the mutant and wild-type VacA proteins, thereby resulting in the formation of mixed oligomers with defective functional activity.

Kinetics and mechanisms of extracellular protein release by Helicobacter pylori.

To investigate the kinetics and mechanisms of extracellular protein release by Helicobacter pylori, we analyzed the entry of metabolically radiolabeled bacterial proteins into broth culture supernatant. At early time points, vacuolating cytotoxin (VacA) constituted a major extracellular protein. Subsequently, culture supernatants accumulated many proteins that were components of intact bacterial cells. This nonselective release of proteins was associated with a decreasing turbidity of cultures and loss of bacterial viability, indicative of an autolytic process. The rates of VacA secretion and autolysis were each influenced by medium composition, and therefore these may be regulated phenomena. Extracellular release of proteins by H. pylori may be an important adaptation that facilitates the persistence of H. pylori in the human gastric mucus layer. Moreover, entry of proinflammatory proteins into the gastric mucosa may contribute to the induction of a mucosal inflammatory response.

Vacuolating cytotoxin (vacA) alleles of Helicobacter pylori comprise two geographically widespread types, m1 and m2, and have evolved through limited recombination.

Vacuolating cytotoxin (vacA) alleles of Helicobacter pylori vary, particularly in their mid region (which may be type m1 or m2) and their signal peptide coding region (type s1 or s2). We investigated nucleotide diversity among vacA alleles in strains from several locales in Asia, South America, and the USA. Phylogenetic analysis of vacA mid region sequences from 18 strains validated the division into two main groups (m1 and m2) and showed further significant divisions within these groups. Informative site analysis demonstrated one example of recombination between m1 and m2 alleles, and several examples of recombination among alleles within these groups. Recombination was not sufficiently extensive to destroy phylogenetic structure entirely. Synonymous nucleotide substitution rates were markedly different between regions of vacA, suggesting different evolutionary divergence times and implying horizontal transfer of genetic elements within vacA. Non-synonymous/synonymous rate ratios were greater between m1 and m2 sequences than among m1 sequences, consistent with m1 and m2 alleles encoding functions fitting strains for slightly different ecological niches.

Helicobacter pylori vacuolating cytotoxin (VacA) disorganizes the cytoskeletal architecture of gastric epithelial cells.

Helicobacter pylori colonization of the gastric mucosa induces peptic ulcer disease and interferes with ulcer healing. Re-epithelialization is an essential component of ulcer healing. It requires cell migration and proliferation which are dependent on the cell cytoskeleton. Most H. pylori strains produce a toxin (VacA) that induces multiple structural and functional changes in epithelial cells. In this study, we investigated the effects of VacA on the gastric epithelial cell cytoskeletal architecture. Exposure of rat gastric epithelial cells to purified VacA from H. pylori 60190 significantly inhibited actin stress fiber formation (83 +/- 5% reduction; p < 0.0001) and disorganized microtubule pattern (90 +/- 8%; p < 0.001). Furthermore, VacA treatment significantly reduced tyrosine phosphorylation of focal adhesion kinase (FAK) (by 45 +/- 6%; p < 0.002) and its expression in focal adhesions (73 +/- 8%; p < 0.0001). These findings suggest that H. pylori VacA interferes with cytoskeleton-dependent cell functions and with the transmission of signals related to cell spreading and growth.

Simple and accurate PCR-based system for typing vacuolating cytotoxin alleles of Helicobacter pylori.

Alleles of the vacuolating cytotoxin gene (vacA) of Helicobacter 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 possessed cagA (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.

Release of Helicobacter pylori vacuolating cytotoxin by both a specific secretion pathway and budding of outer membrane vesicles. Uptake of released toxin and vesicles by gastric epithelium.

The mechanisms by which Helicobacter pylori releases its virulence factors are poorly known. Active secretion has been proposed for some products, including a vacuolating toxin (VacA). Outer membrane vesicles represent another mechanism by which some Gram-negative bacteria may release virulence factors. This study sought to localize VacA by immunocytochemistry in H. pylori cells, to determine whether H. pylori produces outer membrane vesicles, and to investigate whether such vesicles might constitute a vehicle for the delivery of bacterial virulence factors to the gastric mucosa. Small (50-300 nm) membrane vesicles were found in H. pylori culture media from both H. pylori strain 60190 and strain CCUG 17874. These vesicles appeared to originate from blebs arising on the bacterial outer membrane. VacA was immunolocalized in the periplasm and outer membrane of intact bacteria and also in outer membrane blebs and vesicles. Both soluble secreted VacA and VacA-containing vesicles bound to, and were internalized by, MKN28 cells and were detectable in the gastric mucosa from H. pylori-infected humans. The release of outer membrane vesicles by H. pylori may represent a mechanism, additional to secretory pathways, for the delivery of bacterial toxins and antigens to the gastric mucosa.

Detection of anti-VacA antibody responses in serum and gastric juice samples using type s1/m1 and s2/m2 Helicobacter pylori VacA antigens.

Several different families of vacuolating toxin (vacA) alleles are present in Helicobacter pylori, and they encode products with differing functional activities. H. pylori strains containing certain types of vacA alleles have been associated with an increased risk for peptic ulcer disease. In this study, we tested serum samples and gastric juice from 19 H. pylori-negative and 39 H. pylori-positive patients for enzyme-linked immunosorbent assay reactivity with two different types of VacA antigens (types s1/m1 and s2/m2), which were purified from H. pylori 60190 and 86-338, respectively. Both antigens were recognized better by serum immunoglobulin G (IgG) from H. pylori-positive persons than by serum IgG from H. pylori-negative persons (P < 0.01). The s1/m1 VacA antigen was better recognized by sera from patients carrying vacA type s1/m1 strains than by sera from patients carrying vacA type s2/m2 or s1/m2 strains (P < 0.01). Conversely, the s2/m2 VacA antigen was better recognized by sera from patients carrying type s2/m2 or s1/m2 strains (P = 0.03). Serum IgG anti-VacA antibodies were present more frequently in patients carrying type s1/m1 strains than in other H. pylori-positive patients (P = 0.0002). In addition, the highest levels of IgA anti-VacA antibodies were detected in the gastric juice of patients carrying type s1/m1 strains. These data indicate that different VacA isoforms have distinct antigenic properties and that multiple forms of VacA elicit antibody responses in H. pylori-positive humans.

VacA from Helicobacter pylori: a hexameric chloride channel.

VacA is a unique protein toxin secreted by the human pathogen Helicobacter pylori. At a neutral pH, the cytotoxin self-associates into predominantly dodecameric complexes. In this report, we show that at an acidic pH, VacA forms anion selective channels in planar phospholipid bilayers. Similar to several other chloride channels, the VacA channel exhibits a moderate selectivity for anions over cations (P(Cl):P(Na) = 4.2:1), inhibition by the blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and a permeability sequence, SCN- >> I- > Br- > Cl- > F, consistent with a 'weak field strength' binding site for the permeant anion. Single channel recordings reveal rapid transitions (486 s(-1)) between the closed state and a single open state of 24 pS (+60 mV, 1.5 M NaCl). Evaluation of the rate of increase in macroscopic current as well as atomic force microscopy suggest that this VacA channel is a hexamer, formed by the assembly of membrane-bound monomers. Not only are these VacA channels likely to play an important role in the pathological activity of this toxin, but they may also serve as a model system to further investigate the mechanism of anion selectivity in general.

Mutational analysis of the vacA promoter provides insight into gene transcription in Helicobacter pylori.

Analysis of 12 Helicobacter pylori promoters indicates the existence of a consensus -10 hexamer (TAtaaT) but little conservation of -35 sequences. In this study, mutations in either the H. pylori vacA -10 region or the -35 region resulted in decreased vacA transcription and suggested that an extended -10 motif is utilized. Thus, despite the lack of a -35 consensus sequence for H. pylori promoters, the -35 region plays a functional role in vacA transcription.