Functional Properties of Oligomeric and Monomeric Forms of Helicobacter pylori VacA Toxin

Abstract

Helicobacter pylori VacA is a secreted toxin that assembles into water-soluble oligomeric structures and forms anion-selective membrane channels. Acidification of purified VacA enhances its activity in cell culture assays. Sites of protomer-protomer contact within VacA oligomers have been identified by cryoelectron microscopy, and in the current study, we validated several of these interactions by chemical cross-linking and mass spectrometry. We then mutated amino acids at these contact sites and analyzed the effects of the alterations on VacA oligomerization and activity. VacA proteins with amino acid charge reversals at interprotomer contact sites retained the capacity to assemble into water-soluble oligomers and retained cell-vacuolating activity. Introduction of paired cysteine substitutions at these sites resulted in formation of disulfide bonds between adjacent protomers. Negative-stain electron microscopy and single-particle two-dimensional class analysis revealed that wild-type VacA oligomers disassemble when exposed to acidic pH, whereas the mutant proteins with paired cysteine substitutions retain an oligomeric state at acidic pH. Acid-activated wild-type VacA caused vacuolation of cultured cells, whereas acid-activated mutant proteins with paired cysteine substitutions lacked cell-vacuolating activity. Treatment of these mutant proteins with both low pH and a reducing agent resulted in VacA binding to cells, VacA internalization, and cell vacuolation. Internalization of a nonoligomerizing mutant form of VacA by host cells was detected without a requirement for acid activation. Collectively, these results enhance our understanding of the molecular interactions required for VacA oligomerization and support a model in which toxin activity depends on interactions of monomeric VacA with host cells.

Keywords: bacterial protein toxin; bacterial toxins; gastric cancer; membrane channel proteins; membrane channels; oligomerization; pore-forming proteins; pore-forming toxins.

FIG 1

VacA amino acids targeted for mutagenesis. Three pairs of amino acids predicted to be in close proximity in adjacent protomers within a VacA hexamer (41) were selected based on the results of chemical cross-linking experiments and/or cryo-EM analysis of oligomeric VacA. Sites marked in red correspond to the protomer highlighted in pink, while the sites marked in blue correspond to the protomers highlighted in light blue. The indicated amino acid numbers correspond to K44, K47, K55, E338, and D346. Amino acid substitutions were introduced so that the charges of individual amino acids were reversed, or, alternatively, cysteines were introduced to allow disulfide bond formation between adjacent protomers.

FIG 2

VacA charge-reversal mutants retain an oligomeric structure. Wild-type (WT) VacA and mutant VacA proteins with the indicated amino acid substitutions (corresponding to reversals in amino acid charge) were purified and analyzed by negative-stain EM. The structure of acid-activated WT VacA was compared to that of untreated WT VacA, and the mutant proteins were maintained at neutral pH. The charge change mutants retain an oligomeric structure at neutral pH. Scale bar, 100 nm.

FIG 3

Charge change mutants exhibit increased vacuolating activity in the absence of acid activation. Aliquots of WT VacA and the indicated mutant VacA proteins were acid activated or left untreated. Standardized concentrations of the VacA proteins (5 μg/ml) were then added to HeLa cells in the presence of 5 mM ammonium chloride for 24 h at 37°C. Neutral red uptake assays were performed to assess cell vacuolation (quantified by measuring optical density at 540 nm). WT VacA exhibited minimal vacuolating activity in the absence of acid activation, whereas most of the charge change mutants exhibited detectable activity in the absence of acid activation. Results were analyzed by analysis of variance with a Dunnett’s post hoc test. Asterisks indicate a P value of <0.005 compared to WT (untreated). #, significant differences when comparing untreated VacA proteins with the corresponding acid-activated proteins (P < 0.0001 for WT VacA and P = 0.002 for K44E/K47E/K55D).

FIG 4

VacA mutant proteins harboring paired cysteine substitutions form high-molecular-mass oligomers in solution. WT VacA and the indicated VacA mutant proteins (harboring paired cysteine substitutions) were purified from H. pylori culture supernatants and analyzed by SDS-PAGE and Coomassie blue staining using loading buffer containing BME (A) or loading buffer lacking BME (B). (C) Aliquots of the 47C/338C mutant protein were left untreated, treated with DTT, acid activated, or treated with both DTT and acid. These VacA preparations were diluted in PBS to a concentration of 5 μg/ml, loaded onto SDS-PAGE gels in the absence or presence of BME, and then analyzed by Western blotting. The 47C/338C mutant retains an oligomeric form under either neutral or acidic conditions in the absence of reducing agent but disassembles into monomers under reducing conditions.

FIG 5

Disassembly of VacA oligomers detected by negative-stain EM. WT VacA and the indicated mutants with paired cysteine substitutions were exposed to the indicated conditions (neutral pH with or without 100 mM DTT; acidic pH with or without 100 mM DTT) and then analyzed by negative-stain EM. WT VacA disassembled into monomers at pH 3 in the absence of DTT, whereas the mutant proteins retained an oligomeric structure under these conditions. Both low pH and the reducing agent were required for disassembly of the mutant proteins. Scale bars, 200 Å.

FIG 6

2D class averages of VacA proteins visualized by negative-stain EM. WT VacA and the indicated VacA mutant proteins containing paired cysteine substitutions were left untreated or were acidified and then were analyzed by negative-stain EM. 2D class averages were generated as described in Materials and Methods. Representative class averages of each protein are shown. WT VacA disassembled into monomers at low pH, whereas the mutants retained an oligomeric structure under these conditions. Numbers of particles are shown in the bottom right corner of each class. Box size, 468 Å.

FIG 7

Cell-vacuolating activity of VacA proteins containing paired cysteine substitutions. Aliquots of WT VacA and the indicated mutant proteins containing paired cysteine substitutions were left untreated, acid activated, treated with DTT, or treated with both acid and DTT. The VacA proteins (10 μg/ml final concentration) were then added to HeLa cells in the presence of 5 mM ammonium chloride and incubated for 24 h at 37°C. Neutral red uptake assays were performed to assess cell vacuolation (quantified by measuring optical density at 540 nm). WT VacA exhibited cell-vacuolating activity in response to acid activation, whereas the mutant proteins were only active if treated with both low pH and DTT. Results were analyzed by analysis of variance with a Dunnett’s post hoc test. Asterisks indicate a P value of <0.0001 compared to corresponding preparations treated with acid alone.

FIG 8

Binding and internalization of VacA proteins. WT VacA and the 47C/338C mutant protein were labeled with Alexa Fluor 488 (green) and then were left untreated or were treated with DTT, acid, or acid and DTT. The fluorescently labeled VacA proteins (5 μg/ml final concentration) were incubated with HeLa cells at either 4°C or 37°C, and nuclei were stained with Hoechst 33342 (blue). All images were taken on a Zeiss LSM 880 confocal microscope. (A) Fluorescently labeled VacA proteins were incubated with HeLa cells for 1 h at 4°C. (B) VacA proteins were added to HeLa cells for 5 min at 37°C. The medium containing VacA was replaced with fresh medium supplemented with 5 mM NH₄Cl. The cells were then incubated at 37°C for 4 h. Acid-activated WT VacA bound to cells and was internalized, whereas acid-activated mutants did not. Internalization and binding of the mutant VacA proteins required treatment of the proteins with both acid and a reducing agent.

FIG 9

Internalization of the nonoligomerizing Δ346-347 VacA mutant. The Δ346-347 mutant VacA protein was labeled with Alexa Fluor 488 (green) and was either acidified or left untreated. The labeled protein samples (5 μg/ml final concentration) were then added to HeLa cells for 5 min at 37°C. The medium containing VacA was replaced with fresh medium supplemented with 5 mM NH₄Cl. The cells were then incubated at 37°C for 4 h and subsequently stained to visualize cell nuclei (blue). All images were taken on a Zeiss LSM 880 confocal microscope. Both acid-activated and untreated Δ346-347 mutant VacA proteins were internalized by the cells.