Essential Role of a GXXXG Motif for Membrane Channel Formation by Helicobacter pylori Vacuolating Toxin*

Mark S. McClainDagger , Hideki Iwamoto§, Ping CaoDagger , Arlene D. Vinion-Dubiel, Yi Li, Gabor Szabo§, Zhifeng Shao§, and Timothy L. CoverDagger ||**

From the Departments of Dagger  Medicine and  Microbiology and Immunology, Vanderbilt University School of Medicine and the || Veterans Affairs Medical Center, Nashville, Tennessee 37232, and the § Department of Molecular Physiology and Biological Physics and Biophysics Program, University of Virginia School of Medicine, Charlottesville, Virginia 22908

Received for publication, December 10, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Helicobacter pylori secretes a toxin, VacA, that can form anion-selective membrane channels. Within a unique amino-terminal hydrophobic region of VacA, there are three tandem GXXXG motifs (defined by glycines at positions 14, 18, 22, and 26), which are characteristic of transmembrane dimerization sequences. The goals of the current study were to investigate whether these GXXXG motifs are required for membrane channel formation and cytotoxicity and to clarify the role of membrane channel formation in the biological activity of VacA. Six different alanine substitution mutations (P9A, G13A, G14A, G18A, G22A, and G26A) were introduced into the unique hydrophobic region located near the amino terminus of VacA. The effects of these mutations were first analyzed using the TOXCAT system, which permits the study of transmembrane oligomerization of proteins in a natural membrane environment. None of the mutations altered the capacity of ToxR-VacA-maltose-binding protein fusion proteins to insert into a membrane, but G14A and G18A mutations markedly diminished the capacity of the fusion proteins to oligomerize. We then introduced the six alanine substitution mutations into the vacA chromosomal gene of H. pylori and analyzed the properties of purified mutant VacA proteins. VacA-G13A, VacA-G22A, and VacA-G26A induced vacuolation of HeLa cells, whereas VacA-P9A, VacA-G14A, and VacA-G18A did not. Subsequent experiments examined the capacity of each mutant toxin to form membrane channels. In a planar lipid bilayer assay, VacA proteins containing G13A, G22A, and G26A mutations formed anion-selective membrane channels, whereas VacA proteins containing P9A, G14A, and G18A mutations did not. Similarly, VacA-G13A, VacA-G22A, and VacA-G26A induced depolarization of HeLa cells, whereas VacA-P9A, VacA-G14A, and VacA-G18A did not. These data indicate that an intact proline residue and an intact G14XXXG18 motif within the amino-terminal hydrophobic region of VacA are essential for membrane channel formation, and they also provide strong evidence that membrane channel formation is essential for VacA cytotoxicity.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Helicobacter pylori is a Gram-negative bacterium that colonizes the gastric mucosa of humans (1). Colonization of the stomach by H. pylori is a strong risk factor for the development of peptic ulcer disease and distal gastric adenocarcinoma (2-4). Within the gastric mucosa, H. pylori organisms are found predominantly in the gastric mucus layer, but they also adhere to gastric epithelial cells (5).

Adherence of H. pylori to gastric epithelial cells triggers the initiation of processes that ultimately result in alteration of gastric epithelial cell function or gastric epithelial cell death. One of the important effector molecules used by H. pylori to modulate eukaryotic cell function is a secreted toxin known as VacA (6-8). The secreted VacA toxin is an 88-kDa protein, consisting of about 821 amino acids (9). These 88-kDa VacA monomers spontaneously self-assemble into large water-soluble flower-shaped oligomeric complexes, consisting predominantly of 12 or 14 monomeric subunits (10-12). When added to eukaryotic cells in vitro, the oligomeric form of VacA is nearly devoid of cytotoxic activity. However, exposure of oligomeric VacA to either acidic or alkaline pH results in disassembly of the oligomeric complexes into monomers that exhibit potent cytotoxic activity (10, 13-15).

The addition of VacA to eukaryotic cells causes multiple alterations in cell structure and function. When VacA is added to cells in the presence of weak bases, the most apparent effect is the development of cytoplasmic vacuolation (16, 17). VacA also induces permeabilization of epithelial monolayers (18), apoptosis (19-21), depolarization of the resting membrane potential (22, 23), and it interferes with the process of antigen presentation (24). After binding of VacA to the plasma membrane, the toxin is internalized by cells (15, 23, 25, 26). Based on experiments in which cells are transiently transfected with VacA-encoding plasmids (27, 28), it is presumed that VacA cytotoxicity results from toxin action at an intracellular site. Endosomal compartments and mitochondria have both been suggested as possible intracellular sites of VacA action (19, 29, 30).

Water-soluble VacA molecules are able to insert into lipid bilayers and form anion-selective membrane channels (22, 31-33). It has been hypothesized that VacA cytotoxicity results primarily from the formation of such channels. One model proposes that VacA forms anion-selective channels in the membranes of endosomes, which leads to osmotic swelling of these compartments (for review, see Refs. 8 and 34). However, it also has been hypothesized that VacA cytotoxicity may result primarily from an unrecognized enzymatic activity and that the formation of anion-selective membrane channels may be a secondary feature (for review, see Ref. 8). Support for the latter hypothesis is based on the recognition that numerous A-B type bacterial toxins exhibit an enzymatic activity and also exhibit a capacity for membrane channel formation (for review, see Refs. 35 and 36).

In support of the view that VacA cytotoxicity results primarily from its capacity to form membrane channels, it has been reported that an inhibitor of anion-selective channels, NPPB,1 blocks VacA cytotoxicity (22). In addition, we have reported that VacA-Delta (6-27), a VacA mutant containing a 22-amino acid deletion, lacks the capacity to form membrane channels and also lacks cytotoxic activity (37). Unfortunately, neither of these experimental approaches provides conclusive evidence indicating that membrane channel formation is essential for VacA cytotoxicity. Interpretation of experiments using NPPB is limited by the possibility that NPPB might exert multiple effects on cells or on the toxin rather than specifically inhibiting VacA channel activity. Also, conclusions from experiments with VacA-Delta (6-27) are limited by the possibility that a 22-amino acid deletion might alter multiple functional activities of the toxin rather than specifically ablating channel forming activity.

VacA contains a unique hydrophobic region located near its amino terminus, and it seems possible that such a region might be utilized for insertion of VacA into membranes during the process of membrane channel formation. In a previous study, we used a model system known as TOXCAT to analyze the functional role of the VacA hydrophobic region (38, 39). The results indicated that the VacA hydrophobic region (amino acids 1-32) mediated insertion of ToxR-VacA-maltose-binding protein (MBP) fusions into the inner membrane of Escherichia coli and also mediated protein dimerization. In contrast, a fusion protein containing a mutant VacA hydrophobic region (in which glycine 14 was replaced by alanine) inserted into the inner membrane but dimerized less efficiently than the fusion protein containing the wild-type VacA sequence. These experiments demonstrated that the VacA amino-terminal hydrophobic region is capable of inserting into membranes and undergoing dimerization.

Within the VacA hydrophobic region, there are three tandem GXXXG motifs, which are characteristic of transmembrane dimerization sequences (Fig. 1). In the current study, we sought to investigate the functional role of these three GXXXG motifs in membrane channel formation and cytotoxicity and to clarify the role of membrane channel formation in the biological activity of VacA. We report here that alanine substitution mutations at the Gly-14 and Gly-18 positions abolish the capacity of VacA to form membrane channels and also abolish the capacity of VacA to induce cytotoxic effects. We also report that the G14XXXG18 motif is required for dimerization of VacA in a membrane using the TOXCAT system. These results provide further evidence that VacA cytotoxicity occurs via a process that requires membrane channel formation and indicate that the amino-terminal hydrophobic region of VacA plays an essential role in both membrane channel formation and cytotoxicity.


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Fig. 1.   Hydrophobicity plot of the mature secreted VacA toxin from H. pylori 60190 (79). The predicted hydrophobicity of the mature VacA toxin from H. pylori 60190 was analyzed by the method of Kyte and Doolittle (80). Capital letters indicate the amino-terminal sequence of VacA as well as a hydrophilic region of VacA which is susceptible to proteolytic cleavage. The underlined segments represent three tandem GXXXG motifs, characteristic of transmembrane oligomerization regions, located near the amino terminus of VacA.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Analysis of VacA Using the TOXCAT System-- The TOXCAT system (Fig. 2) was originally developed by Russ and Engelman (39) to study transmembrane helix-helix associations in a natural membrane environment. In this system, a putative transmembrane (TM) sequence is cloned between a sequence encoding the transcription activator domain of Vibrio cholerae ToxR and a sequence encoding the periplasmic domain of the E. coli MBP. To determine whether the putative transmembrane sequence mediates transmembrane oligomerization, the ToxR-TM-MBP fusion proteins are expressed in E. coli. Membrane localization of the fusion protein is detected based on complementation of a nonpolar malE mutant E. coli strain. Insertion of the ToxR-TM-MBP fusion protein into the inner membrane, such that the MBP domain localizes to the periplasmic space, is detected by determining whether the bacteria are able to transport maltose and thus grow on maltose-minimal medium. Dimerization of the fusion protein is determined based on expression of the cat gene, which is under the control of the dimerization-dependent transcription activator ToxR. E. coli strains expressing ToxR-TM-MBP fusion proteins that dimerize are resistant to chloramphenicol, whereas strains expressing fusion proteins that lack a dimerization sequence are sensitive to chloramphenicol. Plasmid pccVacA-wt and pccVacA-G14A have been described previously (38). Additional plasmids containing mutations within the vacA amino-terminal sequence were constructed using the method of Perrin and Gilliland (40). As a control, the portion of vacA corresponding to amino acids 295-326 was also cloned into the TOXCAT plasmid, generating pccVacA-295/326. Chloramphenicol acetyltransferase (CAT) enzyme was measured by an antigen-capture enzyme-linked immunosorbent assay (Roche Molecular Biochemicals) according to the manufacturer's instructions.


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Fig. 2.   Schematic illustration of the principles on which the TOXCAT assay is based (39). VacA sequences (depicted as coiled lines) were cloned between a sequence encoding the transcription activator domain of V. cholerae ToxR (squares) and a sequence encoding the periplasmic domain of the E. coli MBP (circles) so that ToxR-VacA-MBP fusion proteins would be expressed. If ToxR-VacA-MBP fusion proteins insert into the E. coli inner membrane such that the MBP domain localizes to the periplasmic space, cells are able to transport maltose and thus can grow in maltose-minimal medium (designated Mal+). Dimerization of the fusion proteins results in the activation of the dimerization-dependent transcription activator ToxR, which leads to expression of the cat gene from the ToxR-activated cholera toxin promoter (pctx). Cells expressing cat are designated Cat+. The figure depicts four possible phenotypes expressed in the TOXCAT assay.

Construction of H. pylori Strains Expressing Mutant VacA Proteins-- Plasmid pAV202 (41), which contains 567 bp of cysS, the cysS-vacA intergenic region, 883 bp of the vacA coding sequence from H. pylori 60190, and a CAT gene inserted at the 3'-terminus of cysS, was used as the template for all site-directed mutagenesis reactions. Mutations encoding single amino acid substitutions were introduced into pAV202 using the method of Perrin and Gilliland (40) or inverse PCR (42). These mutations then were introduced into the H. pylori chromosomal vacA gene by natural transformation and allelic exchange, as described previously (37, 41, 43). Mutants were selected on Brucella agar plates containing 5 µg/ml chloramphenicol. To confirm that the desired mutations had been introduced successfully into the chromosomal vacA gene, fragments of the 5'-end of vacA were PCR amplified and analyzed by restriction endonuclease digestion and nucleotide sequence analysis.

Purification of H. pylori VacA-- H. pylori strains were grown in sulfite-free Brucella broth containing activated charcoal (44). VacA was purified in an oligomeric form from culture supernatants of H. pylori strains, as described previously (10). Purified VacA preparations were routinely acid activated before the addition of VacA to planar lipid bilayer chambers or cell culture wells, as described previously (13, 15).

Planar Lipid Bilayer Methodology-- Planar lipid bilayers, composed of egg phosphatidylcholine/dioleoylphosphatidylserine/cholesterol (55:15:30 mol%) dissolved in n-decane were prepared as described previously (31, 33, 37, 41). Purified acid-activated VacA toxins were added to the lipid bilayers in a buffer consisting of 5 mM citric acid (pH 4.0) and 2 mM EDTA, with the salt composition as described under "Results." Membrane currents were measured as described previously (37, 41). The potential is indicated relative to the cis side, defined as the chamber to which the protein was added. Permeability ratios were determined from the Goldman-Hodgkin-Katz equation (45), after the membrane voltage for zero current (reversal potential) in asymmetric salt concentrations was measured. Statistical significance was analyzed using Student's t test.


                              
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Table I
Analysis of VacA channel forming properties and cytotoxic activity

Analysis of Membrane Potential of HeLa Cells-- Experiments to analyze the membrane potential of HeLa cells were performed as described previously (22, 23). Briefly, HeLa cells were detached with trypsin/EDTA, washed, and then incubated with bis-(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol (oxonol VI) (Molecular Probes) in a final concentration of 2.5 µM for 15 min at 37 °C. A cell suspension (2 ml) was placed in a stirred quartz cuvette at 37 °C in a PerkinElmer Life Sciences LS50B fluorometer. After stabilization of the fluorescence signal (excitation, 585 nm; emission, 645 nm), acid-activated VacA toxins (final concentration 5 µg/ml) were added to the cells, and changes in the fluorescence were monitored. Depolarization causes a potential-dependent change in the cytoplasmic/transmembrane distribution of the fluorophore, which is accompanied by a change in fluorescence (46).

Cell Culture-- HeLa cells were grown in minimal essential medium (modified Eagle's medium containing Earle's salts) containing 10% fetal bovine serum. Acid-activated VacA preparations were incubated with cultured cells in a microtiter format, as described previously (13, 15). After incubation for 24 h, the cells were examined by inverted light microscopy. Samples that induced vacuolation in >50% of cells were scored as positive for the vacuolating cytotoxin phenotype. In some experiments, vacuolation was also quantified by neutral red uptake assay (47).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of VacA Amino Acids Required for Transmembrane Protein Dimerization in a TOXCAT Model System-- The amino-terminal hydrophobic segment of VacA contains three tandem GXXXG motifs, which are characteristic of transmembrane dimerization sequences (Fig. 1). To investigate the role of these GXXXG motifs in greater detail, we used the TOXCAT model system (Fig. 2), which has been used to study transmembrane oligomerization of proteins in a natural membrane environment (38, 39, 48-50). We used a previously described plasmid designated pccVacA-wt (38), which encodes a fusion protein in which the amino-terminal 32 amino acids of VacA are inserted between the transcription activation domain of ToxR and the periplasmic domain of MBP, and which also contains the cat gene under the control of the V. cholerae ctx promoter. To examine the role of the GXXXG motifs, we used four related plasmids that were identical to pccVacA-wt except for the presence of mutations within the vacA sequence which resulted in substitution of the Gly-14, Gly-18, Gly-22, and Gly-26 residues by alanine. We also constructed two related plasmids containing mutations within the vacA sequence which resulted in substitution of the Pro-9 and Gly-13 residues by alanine. Pro-9 was selected for analysis because this residue is reported to be important for VacA cytotoxic activity (51). Gly-13 was selected for analysis as a control because this glycine residue is not located within a GXXXG motif. As an additional control, a 32-amino acid segment (amino acids 295-326) from a predicted hydrophilic region of VacA (Fig. 1) was also cloned into the TOXCAT vector. When analyzed by immunoblot analysis using anti-MBP antiserum, each of these plasmid-containing E. coli strains produced ToxR-TM-MBP fusion proteins (Fig. 3A).


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Fig. 3.   Analysis of mutations in the VacA hydrophobic region using the TOXCAT system (39). A, expression of ToxR-VacA-MBP fusion proteins. E. coli strain MM39 carrying plasmid pccVacA-wt (lane a), pccVacA-P9A (lane b), pccVacA-G13A (lane c), pccVacA-G14A (lane d), pccVacA-G18A (lane e), pccVacA-G22A (lane f), pccVacA-G26A (lane g), pccVacA-295/326 (lane h), and no plasmid (lane i) were cultured in Luria-Bertani medium to an A600 of about 0.3. Equal culture volumes were pelleted, lysed in SDS-sample buffer, separated by SDS-PAGE, and immunoblotted using anti-MBP antiserum (New England Biolabs). B, membrane insertion of ToxR-VacA-MBP fusion proteins determined by growth on M9-maltose. E. coli strain MM39 carrying plasmid pccVacA-wt, pccVacA-P9A, pccVacA-G13A, pccVacA-G14A, pccVacA-G18A, pccVacA-G22A, pccVacA-G26A, and pccVacA-295/326 were grown on M9-glucose plates. Bacteria were resuspended in M9-maltose broth at an A600 of ~0.3. Strains were then spotted onto either M9-glucose or M9-maltose plates. All strains, except for MM39 pccVacA-295/326, grew on M9-maltose, indicating that the fusion proteins inserted into the E. coli inner membrane. C, expression of CAT by E. coli strains. E. coli strains were cultured in Luria-Bertani medium to an A600 of about 0.3. CAT protein levels from each strain was quantified using a CAT enzyme-linked immunosorbent assay. Results represent the mean ± S.D. from triplicate cultures. MM39 pccVacA-G14A, pccVacA-G18A, pccVacA-G26A, and pccVacA-295/326 produced significantly less CAT than did MM39 pccVacA-wt (Student's t test; p < 5 × 10-7).

To determine whether the putative transmembrane sequences encoded by these plasmids promoted membrane localization of the ToxR-TM-MBP fusion proteins, E. coli strains containing these plasmids were cultured on M9-maltose medium. Consistent with previous results (38), E. coli strain MM39 containing pccVacA-wt was able to grow on M9-maltose medium, whereas MM39 with no plasmid was unable to grow on this medium (Fig. 3B, data not shown). As expected, E. coli containing pccVacA-295/326 was unable to grow on this medium. The failure of this strain to grow on M9-maltose medium is consistent with the failure of this VacA hydrophilic segment to mediate insertion of the TOXCAT fusion protein into the inner membrane of E. coli. Each of the plasmids containing mutant vacA sequences grew on M9-maltose medium, similar to E. coli containing the parental wild-type vacA plasmid (Fig. 3B). These results indicate that none of the single amino acid substitutions in the VacA hydrophobic region altered the capacity of this VacA segment to mediate insertion of the ToxR-TM-MBP fusion proteins into a membrane.

We next analyzed whether the mutant VacA segments were able to mediate dimerization of the TOXCAT fusion proteins. As reported previously (38), E. coli MM39 containing pccVacA-wt produced high levels of CAT, whereas E. coli MM39 containing no plasmid produced no CAT enzyme (Fig. 3C and data not shown). The control strain containing pccVacA-295/326 produced barely any detectable CAT. Compared with the E. coli MM39 containing pccVacA-wt, E. coli containing plasmids encoding P9A, G13A, and G22A mutations expressed similar high levels of CAT enzyme. In contrast, E. coli containing plasmids encoding G14A, G18A, or G26A mutations expressed significantly lower levels of CAT than did the strain containing pccVacA-wt. These results indicate that several glycine residues within the VacA hydrophobic region contribute to transmembrane protein dimerization and that the glycines located within the G14XXXG18 motif are particularly important for protein dimerization in this system.

Role of the VacA Amino-terminal Hydrophobic Region in Vacuolating Cytotoxic Activity-- Although the experiments described above using the TOXCAT system provide useful insights into the function of the VacA amino-terminal segment, it seems possible that the conformation and function of this segment might be considerably different in the context of the native VacA protein interacting with eukaryotic cells, rather than the TOXCAT fusion proteins interacting with the inner membrane of E. coli. Therefore, we next undertook studies designed to analyze the function of the amino-terminal hydrophobic region in the context of the intact VacA protein produced by H. pylori.

For these studies, we introduced mutations encoding P9A, G13A, G14A, G18A, G22A, and G26A into the H. pylori chromosomal vacA gene, as described under "Materials and Methods." Each of the mutant strains expressed and secreted VacA proteins of the expected size (Fig. 4A). The level of VacA produced by each of these mutant strains was lower than the level of VacA produced by a wild-type H. pylori strain but similar to the level of VacA produced by a control strain without any vacA mutations in which a chloramphenicol resistance cassette was introduced upstream from the vacA promoter, within the cysS-vacA intergenic region (data not shown). This indicates that the presence of the chloramphenicol cassette in this region results in diminished levels of VacA expression, presumably by altering levels of vacA transcription (52). All of the mutant VacA proteins were detected as large oligomeric structures when H. pylori culture supernatants were fractionated by gel filtration chromatography. Thus, although TOXCAT experiments indicated an apparent role for Gly-14 and Gly-18 in dimerization within a membrane, these residues were not essential for oligomerization of the intact VacA proteins in solution. It seems likely that oligomerization of water-soluble VacA monomers involves interactions of multiple domains rather than only homotypic interactions of the amino-terminal hydrophobic regions of different monomers. In addition, there may be substantial differences between the structure of VacA in solution compared with the structure of VacA in a membrane.


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Fig. 4.   Expression and vacuolating activity of wild-type and mutant VacA proteins secreted by H. pylori. A, expression of VacA proteins. H. pylori mutant strains were grown in broth culture, and broth culture supernatants were analyzed by immunoblot assay using anti-VacA serum. Lane a, H. pylori 60190 expressing wild-type VacA; b, VacA-P9A; c, VacA-G13A; d, VacA-G14A; e, VacA-G18A; f, VacA-G22A; g, VacA-G26A. Each of the strains expressed and secreted a VacA protein. B, vacuolating activity. Equal amounts (20 µg/ml) of purified VacA proteins were added to the medium overlying HeLa cells. Vacuolating activity was measured by neutral red uptake assay (47). Results represent the means ± S.D. from duplicate experiments, each performed in triplicate, and are expressed as the percent of neutral red uptake relative to control cells treated with wild-type VacA purified from H. pylori strain 60190.

Next, we sought to determine the effect of the various mutations described above on the capacity of VacA to induce vacuolating cytotoxic effects on eukaryotic cells. Purified preparations of each of the VacA proteins were standardized by protein concentration, acid-activated, and then added to HeLa cells. As a control, wild-type VacA was purified from the wild-type H. pylori strain 60190. VacA proteins containing G13A, G22A, or G26A mutations each induced vacuolation of HeLa cells (Table I). However, quantification of VacA-induced vacuolation by neutral red uptake assay (47) revealed that VacA-G13A and VacA-G22A were consistently less active than wild-type toxin from H. pylori strain 60190 (Fig. 4B). In contrast to VacA-G13A, VacA-G22A, and VacA-G26A, VacA proteins containing P9A, G14A, or G18A mutations did not induce any detectable vacuolation, even at the highest protein concentration tested (Table I and Fig. 4B). These data indicate that the vacuolating activity of VacA is detectably altered by several of the alanine substitution mutations. However, an intact proline and an intact G14XXXG18 motif are absolutely essential for vacuolating activity.

Role of the VacA Amino-terminal Hydrophobic Region in Formation of Channels in Planar Lipid Bilayers-- To test the hypothesis that the G14XXXG18 motif or perhaps one of the other GXXXG motifs contributes to the capacity of VacA to form anion-selective membrane channels, VacA proteins containing the mutations described above were purified from H. pylori broth culture supernatant. These purified proteins then were standardized according to protein concentration and were tested for the capacity to form membrane channels in a planar lipid bilayer system (37, 41). When tested at a VacA protein concentration of 30 nM, wild-type VacA formed anion-selective membrane channels in the expected manner, as described previously (Table I). VacA proteins containing G13A, G22A, or G26A mutations also formed membrane channels, with an anion selectivity similar to that of channels formed by wild-type VacA. The rate of membrane channel formation was similar among these toxins, except for a slightly slower rate of membrane channel formation by VacA-G22A. In contrast, formation of membrane channels by VacA proteins containing P9A, G14A, or G18A mutations could not be detected under these conditions (Table I). These results indicate that Pro-9, Gly-14, and Gly-18 are essential for channel formation by VacA.

Role of the VacA Amino-terminal Hydrophobic Region in Depolarization of HeLa Cells-- When added to HeLa cells, VacA forms channels in the plasma membrane, which results in depolarization of the resting membrane potential (22, 23). To analyze further the role of the VacA amino-terminal hydrophobic region, we tested the capacity of each of the VacA preparations described above to induce depolarization of HeLa cells. As expected, wild-type VacA induced membrane depolarization in a manner similar to that described previously (Fig. 5). VacA proteins containing G13A, G22A, or G26A mutations also induced depolarization of the resting membrane potential. In contrast, no depolarization was induced by VacA proteins containing P9A, G14A, or G18A mutations. Thus, P9A, G14A, and G18A mutations resulted in loss of the capacity to form membrane channels when analyzed in the planar lipid bilayer system (Table I) as well as loss of the capacity to depolarize HeLa cells (Fig. 5).


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Fig. 5.   Analysis of membrane depolarization induced by mutant VacA proteins. VacA proteins were purified from H. pylori broth culture supernatants as described under "Materials and Methods." HeLa cells were loaded with oxonol VI (a probe used to monitor membrane potential). After the addition of 5 µg/ml acid-activated VacA proteins, changes in fluorescence were monitored. The arrow indicates the time at which toxin was added to the cuvette. Wild-type VacA, VacA-G26A, VacA-G13A, and VacA-G22A induced membrane depolarization, whereas VacA-P9A, VacA-G14A, and VacA-G18A did not.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Several previous studies have shown that amino acid sequences near the amino terminus of VacA play an important role in the process by which VacA exerts cytotoxic effects. The experimental system used in most of these previous studies has involved transiently transfecting HeLa cells with plasmids encoding VacA (27, 28, 37, 51, 53-55). When analyzed in this manner, intracellular expression of wild-type VacA induces cell vacuolation, whereas intracellular expression of truncated forms of VacA which lack as few as 10 amino acids at the amino terminus fails to induce cell vacuolation (28, 53). To identify individual amino acids that might be important for cytotoxic activity, alanine scanning mutagenesis of VacA residues 6-17 has been undertaken (51). When transfected into HeLa cells, plasmids encoding VacA-P9A and VacA-G14A fail to induce intracellular vacuolation, whereas plasmids encoding other single alanine mutations induce cell vacuolation in a manner indistinguishable from wild-type VacA (51). Additional studies indicating the importance of amino acid sequences near the amino terminus of VacA have used allelic exchange mutagenesis of the chromosomally encoded vacA gene in H. pylori to produce mutant toxins (37, 41, 43). When analyzed in this manner, VacA toxins in which a portion of the amino-terminal region was deleted (VacA-(Delta 6-27)) or in which a 12-amino acid extension was added to the amino terminus (s2/m1 VacA) exhibit defects in vacuolating activity (37, 41).

In the current study, we investigated further the functional role of the VacA amino terminus by analyzing a panel of mutant H. pylori toxins. In one or more assays of VacA function, each of the mutant toxins examined exhibited at least a partial diminution in activity compared with wild-type VacA. However, the most dramatic and consistent defects were observed with VacA-P9A, VacA-G14A, and VacA-G18A. Our data confirm results from transient transfection experiments which indicated that P9A and G14A mutations ablate cytotoxic activity (51), and they indicate that a G18A mutation also ablates cytotoxic activity. The essential role played by Pro-9, Gly-14, and Gly-18 in VacA cytotoxic activity thus helps to explain the defect in cytotoxic activity observed in the previously characterized mutant toxin VacA-(Delta 6-27) (37).

It has been proposed that the cytotoxicity of VacA is dependent on the capacity of this toxin to form anion-selective membrane channels (22, 31-33, 37, 41). In the present study, we found that the three nontoxic mutant VacA proteins (VacA-P9A, VacA-G14A, and VacA-G18A) were each defective in membrane channel formation, whereas the other mutant VacA proteins retained the capacity for both cytoplasmic vacuolation and membrane channel formation. The results obtained in a planar lipid bilayer assay were similar to those obtained in a HeLa cell depolarization assay. Thus, the current data provide further support for the hypothesis that VacA channel forming activity is essential for the formation of intracellular vacuoles. However, the current data do not exclude the possibility that VacA may have other biological activities in addition to the capacity for channel formation.

Studies of the mutant toxin VacA-Delta (6-27) indicated that the VacA amino-terminal hydrophobic region does not play a role in toxin binding to HeLa cells or toxin internalization by cells (37), but they suggested that the amino-terminal hydrophobic region plays a role in membrane channel formation. The analysis of mutant toxins containing single alanine substitutions in the current study now provides strong evidence that the amino-terminal hydrophobic region plays an essential role in membrane channel formation. It may be presumed that the VacA amino-terminal hydrophobic region either forms part of the channel or plays an indirect role by stabilizing the structure of the channel. In support of the former possibility, analysis of the predicted hydrophobicity of VacA suggests that the amino-terminal segment is the only contiguous stretch of hydrophobic amino acids long enough to span a membrane, and results obtained using the TOXCAT system indicate that this amino-terminal hydrophobic region is indeed capable of insertion into a membrane (Ref. 38 and the present study). It is not uncommon for the pores of ion channels to be lined with hydrophobic amino acids (56-58). However, it is possible that, in addition to the amino-terminal hydrophobic region, other VacA sequences may also insert into the membrane to form a functional channel. In support of this hypothesis, results of several studies have suggested that multiple regions of the VacA protein are capable of inserting into lipid membranes (59-61).

The appearance of VacA oligomers associated with membranes, as well as the kinetics of membrane channel formation by VacA, suggest that VacA channels are hexameric structures (10, 12, 31, 33). It seems likely that multiple VacA-VacA interactions contribute to the formation of VacA membrane channels. Results obtained using the TOXCAT system indicate that the VacA amino-terminal hydrophobic region is capable of mediating dimerization within a membrane environment (Ref. 38 and the present work). In addition, previous studies have provided evidence indicating the occurrence of interactions between other domains of the toxin (54, 55, 62). The existence of multiple VacA-VacA interaction domains may explain why deletion of the amino-terminal hydrophobic region does not disrupt the formation of VacA oligomeric structures in solution (37).

VacA sequences containing G14A and G18A mutations exhibited defects in the TOXCAT assay of protein dimerization, whereas the P9A mutation did not alter the activity of the VacA sequence in this assay. This suggests that the P9A mutation alters VacA function in a manner different from the effects conferred by the G14A and G18A mutations. Proline residues are known to be associated with turns or bends in secondary structure, and therefore we speculate that the P9A mutation alters the secondary structure of VacA within this region. GXXXG motifs (such as that formed by Gly-14 and Gly-18) are known to be important in the ability of transmembrane alpha -helices to form homo-oligomers within a membrane environment (38, 39, 48-50). The significance of the GXXXG motif has been most widely studied using glycophorin A as a model transmembrane protein (39, 63-66). In addition, the importance of GXXXG motifs has been reported in a variety of other transmembrane proteins (49, 67-69). Recently, the GXXXG motif has been identified as a motif involved in helix-helix interactions in soluble proteins as well (70). Thus, the inactivity of VacA sequences containing G14A and G18A mutations in the TOXCAT system is consistent with the established role of GXXXG motifs in protein-protein interactions.

The structure of the membrane insertion domain is known for only a few bacterial toxins. In the case of toxins such as colicins and the delta -endotoxins of Bacillus thuringiensis, the membrane insertion domain consists of a bundle of hydrophobic and amphipathic alpha -helices (71, 72), and in the case of toxins such as Staphylococcus aureus alpha -toxin and Clostridium perfringens perfringolysin O, the membrane insertion domain consists of a beta -barrel (73, 74). Like VacA, the alpha -toxin from S. aureus also contains three tandem copies of the motif GXXXG (75). However, unlike the VacA amino-terminal hydrophobic region, the glycine-rich region of alpha -toxin is amphipathic. Structural analyses have indicated that this glycine-rich or "hinge" region of alpha -toxin inserts into the membrane of sensitive cells to form a transmembrane beta -barrel (76-78). Thus, although GXXXG motifs are frequently associated with transmembrane alpha -helices (48), these motifs occasionally may be found within transmembrane beta -barrels (76-78). In future investigations, it will be important to correlate the functional studies of the VacA amino-terminal hydrophobic region described in this study with high resolution structural analysis of VacA membrane channels.

    ACKNOWLEDGEMENTS

We thank Beverly Hosse and Wayne Schraw for technical assistance and William Russ and Donald Engelman (who developed TOXCAT with support from the National Institutes of Health) for providing reagents. DNA oligonucleotides were synthesized by the Vanderbilt University DNA Chemistry Core Facility, and DNA sequence analysis was performed by the Vanderbilt University DNA Sequencing Laboratory.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants AI39657 and DK53623 and by the Medical Research Department of the Department of Veterans Affairs.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Division of Infectious Diseases, A3310 Medical Center North, Vanderbilt University School of Medicine, Nashville, TN 37232. Tel.: 615-322-2035; Fax: 615-343-6160; E-mail: timothy.L.cover@vanderbilt.edu.

Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.M212595200

    ABBREVIATIONS

The abbreviations used are: NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid; CAT, chloramphenicol acetyltransferase; MBP, maltose-binding protein; TM, transmembrane; wt, wild-type.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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