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A Helicobacter pylori Vacuolating Cytotoxin A: Mouse DHFR Fusion Protein Triggers Dye Release from Liposomes

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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.

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Acknowledgements

We would like to thank Anchalee Nirachanon for excellent secretarial assistance. This work was supported by grant RSA5580047 (to GK) from the Thailand Research Fund (TRF). A Royal Golden Jubilee PhD Scholarship (to AKL) by the Thailand Research Fund is gratefully acknowledged.

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  1. Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25 Phutthamonthon 4 Road, Salaya, Nakhon Pathom, 73170, Thailand

    Aung Khine Linn, Nitchakan Samainukul, Somsri Sakdee, Chanan Angsuthanasombat & Gerd Katzenmeier

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  1. Aung Khine Linn
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  2. Nitchakan Samainukul
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  3. Somsri Sakdee
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Correspondence to Gerd Katzenmeier.

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Linn, A.K., Samainukul, N., Sakdee, S. et al. A Helicobacter pylori Vacuolating Cytotoxin A: Mouse DHFR Fusion Protein Triggers Dye Release from Liposomes. Curr Microbiol 75, 223–230 (2018). https://doi.org/10.1007/s00284-017-1369-9

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  • DOI: https://doi.org/10.1007/s00284-017-1369-9

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