[논문]Co-expression of CdtA and CdtC subunits of cytolethal distending toxin from Aggregatibacter actinomycetemcomitans

Lee, Seung-Jae; Lee, Kyung-Yeol; Kim, Hyung-Seop

doi:10.5051/jkape.2009.39.s.231

Co-expression of CdtA and CdtC subunits of cytolethal distending toxin from Aggregatibacter actinomycetemcomitans 원문보기

대한치주과학회지 = The journal of Korean academy of periodontology, v.39 no.2 suppl., 2009년, pp.231 - 237

Lee, Seung-Jae (Department of Periodontology, College of Dentistry, Chonbuk National University) , Lee, Kyung-Yeol (Department of Oral Microbiology and Institute of Oral Bioscience, Brain Korea 21 Project, Chonbuk National University) , Kim, Hyung-Seop (Department of Periodontology, College of Dentistry, Chonbuk National University)

Abstract ▼ AI-Helper

Purpose: Cytolethal distending toxin (CDT) is a family of heat-labile cytotoxins produced by several gram-negative mucosa-associated pathogens, including Aggregatibacter actinomycetemcomitans. CDT is well known to be capable of inducing growth arrest, morphological alterations, and eventually death in various cells. CDT belongs to a tripartite $AB_2$ toxin (CdtB: the enzymatic A subunit; CdtA and CdtC: the heterodimeric B subunit). Previous studies proposed that CdtA and CdtC together bind to a cell surface receptor and glycolipids act as a receptor for A. actinomycetemcomitans CDT (AaCDT). In this study, recombinant CdtA and CdtC proteins of AaCDT were co-expressed in a bacterial expression system and tested for their affinity for $GM_1$ ganglioside. Methods: The genes for CdtA and CdtC from A. actinomycetemcomitans Y4 were utilized to construct the expression vectors, pRSET-cdtA and pET28a-cdtC. Both CdtA and CdtC proteins were expressed in Escherichia coli BL21(DE3) and then purified using hexahistidine (His6) tag. The identity of purified protein was confirmed by anti-His6 antibody and monoclonal anti-CdtA antibody. Furthermore, the affinity of recombinant protein to $GM_1$ ganglioside was checked through ELISA. Results: Recombinant CdtA and CdtC proteins were expressed as soluble proteins and reacted to anti-His6 and monoclonal anti-CdtA antibodies. ELISA revealed that purified soluble CdtA-CdtC protein bound to $GM_1$ ganglioside, while CdtA alone did not. Conclusions: Co-expression of CdtA and CdtC proteins enhanced the solubility of the proteins in E. coli, leading to convenient preparation of active CdtA-CdtC, a critical material for the study of AaCDT pathogenesis.

주제어

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가설 설정

A CDT groove mutant exhibited diminished binding to cells²⁵⁾. This data support the hypothesis that the groove formed between CdtA and CdtC are involved in the binding of the toxin to target cells. Also, many studies reported that CdtA and CdtC together bind to the cellular membrane^14,17,18).

제안 방법

In this study, CdtA and CdtC subunits were expressed as soluble proteins from E. coli transformed with both cdtA- and cdtC-expressing vectors, while each of CDT subunit expressed as an insoluble protein. These expressed CDT subunits were purified with Ni-NTA column and also responded to anti-His6 and anti-CdtA antibodies (Fig.
The goal of this study was to co-express CdtA and CdtC subunits of CDT from A. actinomycetemcomitans and to find whether purified CdtA-CdtC protein binds to GM₁ ganglioside.

성능/효과

In conclusion, CdtA and CdtC proteins were co-expressed as soluble protein and CdtA-CdtC protein bound to GM₁ ganglioside. Further studies will be necessary to characterize precisely CdtA-CdtC protein.

후속연구

ganglioside. Further studies will be necessary to characterize precisely CdtA-CdtC protein.
The exact role of CdtA/CdtC heterodimer in CDT toxicity is not yet clearly understood, and further work is required to fully identify the role of CdtA/CdtC heterodimer, especially in its mechanism of host cell binding. Therefore, the soluble CdtA-CdtC protein will serve to investigate the pathological process of A.

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DOI : 10.5051/jkape.2009.39.S.231 [무료]
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