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Caso GC, McClain MS, Erwin AL, et al. Functional Properties of Oligomeric and Monomeric Forms of Helicobacter pylori VacA Toxin. Infect Immun. 2021;89(12):e0034821doi: 10.1128/IAI.00348-21.
Caso, G. C., McClain, M. S., Erwin, A. L., Truelock, M. D., Campbell, A. M., Leasure, C. S., Nagel, M., Schey, K. L., Lacy, D. B., Ohi, M. D., & Cover, T. L. (2021). Functional Properties of Oligomeric and Monomeric Forms of Helicobacter pylori VacA Toxin. Infection and immunity, 89(12), e0034821. https://doi.org/10.1128/IAI.00348-21
Caso, Georgia C, et al. "Functional Properties of Oligomeric and Monomeric Forms of Helicobacter pylori VacA Toxin." Infection and immunity vol. 89,12 (2021): e0034821. doi: https://doi.org/10.1128/IAI.00348-21
Caso GC, McClain MS, Erwin AL, Truelock MD, Campbell AM, Leasure CS, Nagel M, Schey KL, Lacy DB, Ohi MD, Cover TL. Functional Properties of Oligomeric and Monomeric Forms of Helicobacter pylori VacA Toxin. Infect Immun. 2021 Nov 16;89(12):e0034821. doi: 10.1128/IAI.00348-21. Epub 2021 Sep 20. PMID: 34543122; PMCID: PMC8594603.
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Infect Immun. 2021 Nov 16;89(12):e0034821. doi: 10.1128/IAI.00348-21. Epub 2021 Sep 20.

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

Infection and immunity

Georgia C Caso, Mark S McClain, Amanda L Erwin, Mandy D Truelock, Anne M Campbell, Catherine S Leasure, Marcus Nagel, Kevin L Schey, D Borden Lacy, Melanie D Ohi, Timothy L Cover

Affiliations

  1. Department of Medicine, Vanderbilt University School of Medicinegrid.471397.f, Nashville, Tennessee, USA.
  2. Life Sciences Institute and Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA.
  3. Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicinegrid.471397.f, Nashville, Tennessee, USA.
  4. Mass Spectrometry Research Center, Department of Biochemistry, Vanderbilt University School of Medicinegrid.471397.f, Nashville, Tennessee, USA.
  5. Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, USA.

PMID: 34543122 PMCID: PMC8594603 DOI: 10.1128/IAI.00348-21

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

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