Analysis to identify the receptor for cholix toxin from Vibrio cholerae

2021 Fiscal Year Research-status Report

• Project/Area Number: 20K16246
• Research Institution: Osaka Prefecture University
• Principal Investigator: アワスティ シャルダ 大阪府立大学, 生命環境科学研究科, 客員研究員 (30751888)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Keywords: Cholix toxin

Outline of Annual Research Achievements

The objectives for the fiscal year 2021-22 were to evaluate the cytotoxicity of recombinant cholix toxin (rChxA) on eukaryotic cells transformed with plasmid carrying candidate receptor cDNA. Further, cloning, expression and purification of candidate receptor protein (cRP) and binding assays were to be carried out.
The cytotoxicity assay was evaluated using wild-type and transformed cell lines (expressing cRP). The wild-type cells which did not express cRP did not show any cytotoxicity when treated with rChxA up to 20 µg/mL, although growth inhibition was observed at this concentration. On the other hand, transformed cells expressing cRP were susceptible to rChxA and demonstrated cytotoxicity with CD50 of 5 ug/mL rChxA. The results further support the idea that the cRP could be the target of ChxA.
In another objective, cloning and expression of cRP was attempted. Although, the E. coli could be transformed with plasmid carrying cDNA of candidate receptor but no expression was observed. Thus, the cDNA was cloned and the expression was carried out using Bac-to-Bac Baculovirus Expression System and Spodoptera frugiperda (Sf9) cells. The cRP could be expressed in Sf9 cells as confirmed by Western Blotting. As the cloning was carried out without any tag, purification could not be done. Further, cloning and expression of Histidine tagged cRP was carried out. The bacmid DNA carrying gene for His-tagged receptor candidate was prepared. The experiment to transfect the Sf9 cells and isolation of P1 viral stock is ongoing.

Current Status of Research Progress

4: Progress in research has been delayed.

Reason

The research plan is delayed due to corona pandemic. Some of the efforts were diverted to other works and experiments.
As expected for year 2021-22, the cytotoxicity assay of rChxA with transformed cells expressing cRP is completed. The expression of cRP using Baculovirus system was confirmed and further experiments to purify the recombinant cRP are on-going.
The experiment which could not be completed was to carry out in vitro binding assay between ChxA and candidate receptor protein. I expect to progress the work in the current fiscal year.

Strategy for Future Research Activity

In vitro binding assays of rChxA with cRP will be carried out. The expressed and purified recombinant cRP will be used for toxin overlay assay with rChxA. The binding assay will be also carried out by pull-down assay using receptor protein as ‘bait’ and rChxA as ‘prey’ protein. Finally, if the binding between the purified cRP and ChxA will be successful, they will be used for X-ray crystallography.

Causes of Carryover

Some of the experimental objectives could not be completed due to corona pandemic. Those experiments will be carried out along with other objectives in the current fiscal year. The funds will be required to accomplish the incomplete objectives of the research.