Actinobacillus actinomycetemcomitans strains with enhanced levels of production of leukotoxin are characterized by a 530-bp deletion from the promoter region of the leukotoxin gene operon. Previous isolates with this deletion constituted a single clone belonging to serotype b, although they displayed minor differences among each other. We have analyzed the geographic dissemination of this clone by examining 326 A. actinomycetemcomitans isolates from healthy and periodontally diseased individuals as well as from patients with different types of extraoral infections originating from countries worldwide. A total of 38 isolates, all belonging to the same clone, showed the 530-bp deletion. Comparison of a 440-bp sequence from the promoter region of the leukotoxin gene operon from 10 of these strains revealed complete identity, which indicates that the deletion originates from a single mutational event. This particular clone was exclusively associated with localized juvenile periodontitis (LJP). In at least 12 of 28 families from which the clone was isolated, more than one family member had LJP. Notably, all the subjects carrying this clone had a genetic affiliation with the African population. These observations suggest that juvenile periodontitis in some adolescents with an African origin is associated with a disseminating clone of A. actinomycetemcomitans.
Summary
Cytolethal distending toxin (CDT) is a multicomponent bacterial holotoxin that targets most eukarytotic cells causing distension and cell cycle arrest. A number of diverse pathogenic bacterial species associated with diarrhoea, chancroid, chronic hepatitis and periodontal disease produce a CDT. Synthesis of the holotoxin is directed by the expression of three genes, cdtA, cdtB and cdtC. Although
the product of the CdtB gene was previously identified as a type I deoxyribonuclease,
the functions of the cdtA and cdtC products have not been characterized.
Using the periodontal pathogen, Actinobacillus actinomycetemcomitans, we demonstrate that the recombinant product of the CdtA gene binds to the surface of Chinese hamster ovary (CHO) cells. This protein did not induce distension or cytotoxicity when introduced into the cytosol using a lipid‐based protein delivery system. Recombinant CdtB and CdtC proteins failed to bind to CHO cells. However, the delivery of either CdtB or CdtC into the cytosol resulted in the characteristic pattern of distension followed by cell death. Based on these results, it appears that the CdtA protein subunit alone is responsible for anchoring the holotoxin to the cell surface. The CdtC subunit, in concert with CdtB, contributes to the cytotoxic activities of the holotoxin. The specific mechanism of CdtC cytotoxicity is currently unknown.
A leukotoxin from Actinobacillus actinomycetemcomitans was isolated by a procedure that includes polymyxin B extraction, ion-exchange chromatography, and gel filtration chromatography. The procedure resulted in the recovery of 48% of the toxin with a 99-fold increase in specific activity. The isolated toxin has a molecular mass of 180,000 daltons by gel filtration and 115,000 daltons by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. It retains all the major biological characteristics previously documented for crude leukotoxin preparations, including susceptibility to heat and proteolytic enzymes and neutralization by sera from patients with juvenile periodontitis. The isolated leukotoxin destroys human but not rat or guinea pig polyriorphonuclear leukocytes and has no apparent effect on human erythrocytes. The availability of the A. actinomycetemcomitans leukotoxin should facilitate studies on its chemistry and mode of action as well as its role in the pathogenesis of human periodontal disease. Juvenile periodontitis is an inflammatory disease characterized by severe alveolar bone loss in young individuals. Clinical, microbiological, and immunological data implicate Actinobacillus actinomycetemcomitans as a possible etiological agent in juvenile periodontitis. For example, patients harbor relatively high proportions of A. actinomycetemcomitans in diseased sites (13, 19, 20, 22) and have high titers of antibodies against these organisms (10, 12, 15, 24). Furthermore, A. actinomycetemcomitans elaborates several
The periodontal pathogen Actinobacillus actinomycetemcomitans produces a leukotoxin that is considered a primary virulence factor in localized juvenile periodontitis (LJP). Select strains of the bacterium contain a 530‐bp deletion in the promoter region of the leukotoxin gene operon which results in enhanced transcription of the leukotoxin. DNA hybridization and polymerase chain reaction (PCR) were used to examine genetic variants of A. actinomycetemcomitans in 24 LJP‐susceptible children from 21 families having a history of the disease and 34 control children from non‐LJP families. A significant association was found between the detection of variants that had a deletion in the leukotoxin promoter region, indicative of a high level expression leukotoxin genotype, and conversion from a healthy periodontal status to disease. Subjects harboring A. actinomycetemcomitans of this genotype were more likely to convert to LJP than those subjects who had variants containing the full length leukotoxin promoter region (odds ratio = 22.50, 95% C.I.). These findings support the concept that highly virulent strains or clonai types of periodontal pathogens play a major role in the initiation of periodontal disease in susceptible hosts. J Periodontol 1998;69:998–1007.
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