J Korean Acad Periodontol.  2009 Aug;39(Suppl):231-237.

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

• Lee SJ
• Lee KY
• Kim HS

Abstract

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 AB2 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 GM1 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 GM1 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 GM1 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.

Keyword

Aggregatibacter actinomycetemcomitans; toxin

MeSH Terms

Antibodies
Bacterial Toxins
Cytotoxins
Edetic Acid
Enzyme-Linked Immunosorbent Assay
Escherichia coli
Glycolipids
Histidine
Humans
Oligopeptides
Proteins
Solubility
Antibodies
Bacterial Toxins
Cytotoxins
Edetic Acid
Glycolipids
Histidine
Oligopeptides
Proteins

Figure

• 

  Figure 1 SDS-PAGE analysis of purified CdtA and CdtC protein. After expression of recombinant proteins from pRSET-cdtA and pET28a-cdtC, the transformed bacteria were sonicated and centrifuged. The supernatant was loaded onto Ni-NTA agarose column and Flow-through (FT) was saved. The sample-loaded column was washed three times (W1~W3) and eluted (E1~E3) as described in Materials and Methods. rCdtA indicated purified recombinant CdtA protein from pET-cdtA and served as control.

• 

  Figure 2 Western blot analysis of purified CdtA and CdtC protein using anti-His6 antibody. rCdtA-CdtC protein was purified from E. coli transformed with both pRSET-cdtA and pET28a-cdtC, while rCdtA from E. coli transformed with pET-cdtA. Protein samples were electrophoresed in SDS-PAGE gel, transferred to nitrocellulose membrane, and detected by anti-His6 antibody.

• 

  Figure 3 Western blot analysis of purified CdtA and CdtC protein using monoclonal anti-CdtA antibody. Protein samples were purified by Ni-NTA column from bacterial lysates from E. coli transformed with both pRSET-cdtA and pET28a-cdtC (rCdtA-CdtC), and E. coli transformed with pET-cdtA (rCdtA). Samples were separated in SDS-PAGE gel and then transferred to nitrocellulose membrane, and detected by anti-CdtA antibody. Bands around 25 kDa was clearly shown but band at 20 kDa not shown.

• 

  Figure 4 GM1 ELISA of purified CdtA-CdtC protein using anti-His6 antibody. ELISA plate was coated with GM1 ganglioside and further incubated with diverse concentration of CdtA and CdtA-CdtC protein. After washing, the remaining amount of CdtA and CdtA-CdtC protein was quantified using anti-His6 antibody at OD405.

• 

  Figure 5 GM1 ELISA of purified CdtA-CdtC protein using monoclonal anti-CdtA antibody. ELISA plate was coated with GM1 ganglioside and further incubated with diverse concentration of CdtA and CdtA-CdtC protein. After washing, the remaining amount of CdtA and CdtA-CdtC protein was quantified using monoclonal anti-CdtA antibody at OD405.

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