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A Helicobacter pylori Vacuolating Cytotoxin A: Mouse DHFR Fusion Protein Triggers Dye Release from Liposomes
Aung Khine Linn1, Nitchakan Samainukul1, Somsri Sakdee1
1Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25 Phutthamonthon 4 Road, Salaya, Nakhon Pathom, 73170, Thailand.
Abstract:
The membrane perturbing action of the VacA toxin from Helicobacter pylori is responsible for vacuole formation in intracellular compartments and the induction of apoptosis. The VacA toxin contains 2 major domains, p33 and p55, which are involved in receptor binding and membrane pore formation, respectively. Improved methodologies for VacA purification and assays are urgently needed for further detailed investigations on the mechanism of action of this significant virulence factor. We found that by fusing mouse DHFR with the N-terminus of the full-length (p88) VacA toxin, expression levels in recombinant E. coli were substantially increased when compared to the conventional (His)6-tagged protein. The DHFR-VacA fusion protein was active in sulforhodamine dye-release assays using liposomes at acidic pH in a concentration-dependent manner. Enzymatic activity of DHFR in the fusion protein was comparable to a commercial reference sample of purified DHFR; however, activity was insensitive to inhibition by methotrexate. Our findings suggest that the VacA p88 toxin with a modified N-terminus still maintains its capability for membrane insertion and that pH-dependent conformational changes occur during interaction of VacA with membranes.
Insights
Researchers enhanced Helicobacter pylori VacA toxin expression using a DHFR fusion. The modified toxin retained membrane insertion and pH-dependent activity, aiding virulence factor studies.
Area of Science:
- Microbiology
- Molecular Biology
- Cell Biology
Background:
- The VacA toxin from Helicobacter pylori causes vacuole formation and apoptosis.
- VacA has p33 (receptor binding) and p55 (pore formation) domains.
- Current VacA purification and assay methods require improvement.
Purpose of the Study:
- To develop improved methods for VacA toxin purification and functional assays.
- To investigate the mechanism of action of the VacA virulence factor.
- To enhance recombinant expression of the full-length VacA toxin.
Main Methods:
- Fusion of mouse dihydrofolate reductase (DHFR) with the N-terminus of full-length VacA (p88).
- Expression of the DHFR-VacA fusion protein in recombinant E. coli.
- Activity assays using liposomes and sulforhodamine dye release at acidic pH.
Main Results:
- DHFR-VacA fusion significantly increased expression levels in E. coli compared to His-tagged VacA.
- The DHFR-VacA fusion protein demonstrated concentration-dependent activity in dye-release assays at acidic pH.
- DHFR enzymatic activity in the fusion was comparable to purified DHFR but insensitive to methotrexate.
Conclusions:
- The modified N-terminus of VacA (p88) does not impede membrane insertion capability.
- pH-dependent conformational changes are crucial for VacA's interaction with membranes.
- The DHFR-VacA fusion strategy offers a promising approach for enhanced VacA studies.
