Random Mutagenesis of Helicobacter pylori vacA To Identify Amino Acids Essential for Vacuolating Cytotoxic Activity

Abstract

VacA is a secreted toxin that plays a role in Helicobacter pylori colonization of the stomach and may contribute to the pathogenesis of peptic ulcer disease and gastric cancer. In this study, we analyzed a library of plasmids expressing randomly mutated forms of recombinant VacA and identified 10 mutant VacA proteins that lacked vacuolating cytotoxic activity when added to HeLa cells. The mutations included six single amino acid substitutions within an amino-terminal hydrophobic region and four substitutions outside the aminoterminal hydrophobic region. All 10 mutations mapped within the p33 domain of VacA. By introducing mutations into the H. pylori chromosomal vacA gene, we showed that secreted mutant toxins containing V21L, S25L, G121R, or S246L mutations bound to cells and were internalized but had defects in vacuolating activity. In planar lipid bilayer and membrane depolarization assays, VacA proteins containing V21L and S25L mutations were defective in formation of anion-selective membrane channels, whereas proteins containing G121R or S246L mutations retained channel-forming capacity. These are the first point mutations outside the amino-terminal hydrophobic region that are known to abrogate vacuolating toxin activity. In addition, these are the first examples of mutant VacA proteins that have defects in vacuolating activity despite exhibiting channel activities similar to those of wild-type VacA.

Helicobacter pylori VacA Subdomain Required for Intracellular Toxin Activity and Assembly of Functional Oligomeric Complexes

“…The gel filtration elution properties of VacA ⌬346-347 are similar to those reported for a mutant VacA protein containing a deletion of the p33 domain (30) and similar to several mutant VacA proteins containing large deletions within the p33 domain (43). The gel filtration properties of VacA ⌬346-347 differ markedly from those of several previously described inactive VacA proteins with mutations in the p33 domain, which formed large oligomeric structures similar to wild-type VacA (25,26,43).…”

“…VacA expression plasmids were transformed into the E. coli expression strain ER2566 (New England Biolabs), which encodes an IPTG (isopropyl-␤-D-thiogalactopyranoside)-inducible copy of the RNA polymerase gene from bacteriophage T7. VacA-expressing E. coli strains were cultured in Terrific Broth (Invitrogen) supplemented with 25 g of kanamycin/ml (24,40), and extracts containing soluble proteins were generated as described previously (24,25). Cell culture analysis of VacA proteins expressed in H. pylori or E. coli.…”

“…Thus far, a two-amino-acid deletion mutation (⌬346-347) is the only small alteration within the p55 amino-terminal subdomain that is known to abrogate vacuolating toxin activity (48). Efforts to identify additional small inactivating mutations in the p55 domain using a random mutagenesis approach have not been successful (25). A previous study reported that the VacA ⌬346-347 mutant protein lacked vacuolating activity when expressed intracellularly, but the basis for this lack of activity has not yet been investigated (48).…”

A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain

“…Random mutagenesis experiments revealed only two mutations in this region that abrogated VacA toxin activity (G121R and S246L) (51). Interestingly, comparative sequence analysis of VacA proteins produced by different H. pylori strains reveals heterogeneity in a region (approximately residues 144 to 244, known as the "intermediate region" or the "i-region"), which lies within the putative ␤-helical portion of p33 (23,25).…”

“…This region contains multiple GXXXG motifs, sequences that are predicted to mediate transmembrane dimerization (21,50). Site-directed mutagenesis of several amino acids within the amino-terminal hydrophobic region, including the GXXXG motifs, abolishes membrane channel-forming activity and vacuolating toxin activity (21,49,51). Analysis of crystals formed by a recombinant p55 protein revealed that this domain has a predominantly ␤-helical structure (52,53).…”