2022 Fiscal Year Research-status Report
Elucidation of receptor binding mechanism of Enterotoxigenic Escherichia coli colonization factor CS6
| Project/Area Number |
22K15466
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| Research Institution | National Center for Global Health and Medicine |
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Principal Investigator |
アイビエケ アラファテ 国立研究開発法人国立国際医療研究センター, 研究所, 感染症制御研究部 細菌感染研究室 研究員 (80933647)
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| Project Period (FY) |
2022-04-01 – 2025-03-31
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| Keywords | Enterotoxigenic E. coli / Colonization factor / CS6 / Receptor |
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| Outline of Annual Research Achievements |
Prior research has proposed mucin as a potential receptor for the ETEC colonization factor CS6. We performed RNA sequencing and subsequent differential gene expression analysis on INT407 and Caco-2 cell lines, which display high and low CS6 binding affinity, respectively. Based on gene expression profiles and previous findings, Mucin 1 and Mucin 16, two trans-membrane glycoproteins, were selected as potential receptor candidates. Using shRNA interference through lentiviral delivery, we created knockdown strains of these candidates in the INT407 cell line. We assessed knockdown efficiencies at both RNA and protein levels employing RT-qPCR and ELISA. We analyzed the adhesion of ETEC CS6 to the knockdown cells and compared it with control and non-target knockdown cells, finding no significant decrease in the number of adhesive bacteria. Additionally, we conducted immuno-fluorescent assays on the adhesion of EYFP-expressing ETEC bacteria to INT407 cells to observe co-localization of Mucin 1 and Mucin 16 with the CS6-expressing bacteria. The results demonstrated that Mucin 1 did not co-localize with ETEC 4266, and various antibodies were unable to distinctly label Mucin 16 in INT407 cells. These findings suggest that the transmembrane proteins Mucin 1 and Mucin 16 may not be potential receptors for CS6.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
While our selected receptor candidates, Mucin 1 and Mucin 16, were determined not to be the receptors for CS6, we have nevertheless obtained valuable data. This data strongly suggests that these transmembrane mucins may not serve as receptors for CS6. Additionally, we have gathered insightful information on the differential gene expression in the INT407 and Caco-2 cell lines, which demonstrate high and low CS6 binding affinity respectively. This information is instrumental in guiding our ongoing efforts to select and confirm further receptor candidates for CS6 within these cell lines.
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| Strategy for Future Research Activity |
Our upcoming research activities aim to employ two distinct proteomic methods to select new receptor candidates.
1.Pull-down Assay: Our first approach involves conducting a pull-down assay to identify potential CS6 receptor proteins. We will use a heat-saline extract of Top10pCS6, which contains CS6, as a bait protein. This will be mixed with whole cell lysates or membrane protein extracts from INT407 and Caco-2 to form a CS6-receptor ligand. This ligand will then be captured using a CS6 antibody and protein G. Subsequent SDS-PAGE will be conducted to examine protein candidates that are either specific to or are present in higher concentrations in INT407. The candidate proteins will be identified using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS).
2.Far-Western Blotting Assay: Our second strategy involves conducting a far-western blotting assay to identify potential receptor candidates. We will use membrane protein extracts from INT407 and Caco-2 to conduct SDS-PAGE, after which the proteins will be transferred to a PVDF membrane. The membrane will then be probed with CS6 to select protein bands that are either specific to INT407 or are present in higher concentrations in INT407 compared to Caco-2. Similar to the first strategy, LC-MS/MS will be used to identify the candidate proteins.
If successful, these two strategies will enable us to pinpoint potential receptor candidates for CS6, therefore to achieve better understanding the binding mechanism.
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| Causes of Carryover |
The cost of reagents used in this fiscal year was slightly lower than expected, and no travel expenses were incurred this year. The remaining amount will be used for the purchase of reagents and consumables to be used next year.
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