Studies On The Function Of Recombinant Subunits A Of A. Actinomycetemcomitans Cytolethal Distending Holotoxin

Cytolethal distending toxin (CDT) is a newly discovered bacterial toxins secreted by a variety of Gram-negative pathogenic bacteria.This toxin can lead to G2 / M phase arrest in epithelial cells, fibroblasts and lymphocytes. Aggregatibacter actinomycetemcomitans (Aa ) is the major pathogens associated with aggressive periodontitis, and is the only oral bacteria that can produce CDT.Now, it is clear that Cdt is encoded by three genes, including cdtA, cdtB, and cdtC. CdtB subunit has been proven to be the toxic one, but the structure and function of CdtA and CdtC are not clear.[Objective] Through cytotoxicity test to sieve out defects or missing mutations to reveal the key amino acid group and the relationship between structure and function of Aa CdtA.[Methods] The toxic CDT subunit encoding wild-type gene cdtA,cdtB,cdtC was amplified by PCR. Through restriction endonuclease digestion,gene cdtA,cdtB,cdtC and vector pET-15b were ligated to form pET-15b-cdtA,pET-15b-cdtB,pET-15b-cdtC. Use wild-type pET-15b-cdtA genomic DNA as a template to construct mutant pET-15b-cdtAY105A,pET-15b-cdtAY181A,pET-15b-cdtAY125A.Protein expression was induced by IPTG and examined by SDS-PAGE and Western blot. The recombinant protein was purified through pre-installed Ni-HisTrap HP column in vitro. Supercoiled plasmid pET-32a DNA was incubated with purified recombinant CdtB protein in vitro to view any changes in the electrophoretic mobility of the plasmid pET-32a DNA band to determine whether the recombinant protein with biological activity. Wild-type,mutant CdtA and wild type CdtB,CdtC was equally mixed in reconstruction buffer, respectively. CHO amd Hela were incubated with these holotoxin. MTT test Hela cell proliferation effect; the number of colony forming(colony-forming units,cfu)calculated CHO living cells; inverted microscope view death and morphological changes of Hela; flow cytometry analysis of Hela cell cycle arrest situation.[Results] The wild-type cdtA,cdtB,cdtC DNA sequence was blast from GenBank and 99%homology was obtained. Mutant constructed prokaryotic expression vector pET-15b-cdtAY105A,pET-15b-cdtAY181A,pET-15b-cdtAY125A is correct.Both of SDS-PAGE and Western blot confirmed that recombinant wild-type and mutant protein was obtained. After incubated with the purified recombinant CdtB in vitro, the supercoiled plasmid pET-32a DNA was observed relaxing by 1% agarose gel electrophoresis test. Separate wild-type CdtA,CdtB and three mutant CdtAY105A,CdtAY181A,CdtAY125A have no inhibitory effect on cell growth proteins. Separate CdtC protein and wild-type CDT holotoxin have cytolethal distending function. Compared with wild-type CDT hotoxin, the biological activity of mutant holotoxin CdtAY105ABC was significantly decreased; biological activity of the mutant holotoxin CdtAY181ABC was slight increase; biological activity of the mutant toxin CdtAY125ABC was no significant changes. [Conclusions] This study has successfully acquired the wild-type and mutant holotoxin.The preliminary findings of Aa CdtA two functional sites CdtAY105A,CdtAY181A is beneficial for further study of the functions of CdtA.