The Distribution Of Actinobacillus Actinomycetemcomitans In Subgingival Plaque And Reconstitution Of Cytolethal Distending Toxin

Periodontitis is a kind of chronic bacterial infection. Periodontal bacteria can locate at susceptible sites, invade the periodontal epithelium barrier and escape the host immune response. At the same time these microorganisms cause periodontal connective tissue destruction followed by tooth mobility and eventually tooth loss. It is acknowledged that Porphyromonas gingivalis, Tannerella forsythensis and Actinobacillus actinomycetemcomitans are the major pathogens involved in periodontitis. A. actinomycetemcomitans is a gram-negative non-spore-forming, non-motile, facultatively anaerobic coccobacillus, which is most probably located in subgingival plaque.A. actinomycetemcomitans can produce a large number of virulence factors, which can modulate inflammation, induce tissue distruction and inhibit tissue repair. Substantial evidences have proved that the pathogen is closely associated with localized aggressive periodontitis and possesses the ability to induce rapid loss of alveolar bone of certain teeth. A. actinomycetemcomitans is also detected in subgingival plaque of chronic periodontitis patients and periodontal healthy individuals. However, data vary a lot in different research about the prevalence of A.actinomycetemcomitans in chronic periodontitis. In our research, we utilized the 16S ribosomal polymerase chain reaction to determine the prevalence of A. actinomycetemcomitans and tried to analyze the distribution characteristic in chronic periodontitis patients.116 chronic periodontitis patients and 111 periodontally healthy adults were included. In each patient, subgingival plaque samples were collected from two sites of different molars with the greatest probing depth (PD) and one periodontally healthy site (PD<3mm). The samples of periodontally healthy adults were obtained from the mesio-buccal site of one first upper molar. Bacteria DNA were extracted for detection of A. actinomycetemcomitans by 16S rRNA PCR. The prevalence for A. actinomycetemcomitans of diseased sites (33.62%) was significantly higher than that of healthy sites from patients (0.9%) and the periodontally sites (0.9%) (P<0.01). No significant difference was observed between male and female chronic periodontitis patients (P>0.05). A. actinomycetemcomitans was more observed in young patients than older ones. A.actinomycetemcomitans was detected in advance periodontal destruction sites with highest prevalence, and moderate higher than mild involved sites. It is showed that the pocket depth and clinical attachment loss was associated with the occurrence of A. actinomycetemcomitans in a positive mode. And A.actinomycetemcomitans was more often detected in the sites of bleeding on probing.Among various virulence factors of A. actinomycetemcomitans, cytolethal distending toxin is a newly found one. It's consisted of three subunits and coded by successive operons, cdtA, cdtB and cdtC. CDT can induce intoxicated cells distending, arrest cell cycle at G₂/M phase, cause lymphocytes apoptosis and stimulate pro-inflammetion cytokine secretion.We applied polymerase chain reaction to clone the CDT-coding gene cdtABC. Following the purification of the amplicon, the reconstitution apparatus pQE60-cdtABC was constituted through TA clone, endonuclease digestion and ligation of target gene and vector. The sequence we cloned into the plasmid was compared with the original sequence in GenBank and the homology is 99%. The competent E. coli cells were transfected with our reconstitution plasmid pQE60-cdtABC and induced by IPTG. The expressed recombinant protein CDT was detected in the cells by both SDS-PAGE and Western blot. Purification of rCDT was performed by Ni2+ chelated magnetic beads, which can recognize and combine with the six His-tag located on N-terminus of recombinant protein. Recombinant subunit CdtC was detected by SDS-PAGE, which suggested that the expression plasmid is successfully constituted and can be induced to express rCDT.We concluded that A. actinomycetemcomitans was more frequently detected in periodontitis sites than periodontally healthy sites. For chronic periodontitis patients, a higher prevalence was associated with the serious involved sites than those moderate and mild implicated sites. A. actinomycetemcomitans is considered to be the one of the periopathogens involved in the etiology of chronic periodontitis. Furthermore, we constituted the expression plasmid of cytolethal distending toxin and successfully induced rCDT expression. The work we finished in the research laid a foundation for further research for cytolethal distending toxin activity and mechanism is chronic periodontitis.