Secretion of RTX leukotoxin by Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans, the etiologic agent for localized juv enile periodontitis and certain other human infections, such as endocarditi s, expresses a leukotoxin that acts on polymorphonuclear leukocytes and mac rophages. Leukotoxin is a member of the highly conserved repeat toxin (RTX) family of bacterial toxins expressed by a variety of pathogenic bacteria. While the RTX toxins of other bacterial species are secreted, the leukotoxi n of A. actinomycetemcomitans is thought to remain associated with the bact erial cell. We have examined leukotoxin production and localization in roug h (adherent) and smooth (nonadherent) strains of A. actinomycetemcomitans. We found that leukotoxin expressed by the rough, adherent, clinical isolate CU1000N is indeed cell associated, as expected. However, we were surprised to find that smooth, nonadherent strains of A. actinomycetemcomitans, incl uding Y4, JP2 (a strain expressing a high level of toxin), and CU1060N tan isogenic smooth variant of CU1000N), secrete an abundance of leukotoxin int o the culture supernatants during early stages of growth. After longer time s of incubation, leukotoxin disappears from the supernatants, and its Loss is accompanied by the appearance of a number of low-molecular-weight polype ptides. The secreted leukotoxin is active, as evidenced by its ability to k ill HL-60 cells in vitro. We found that the growth phase and initial pH of the growth medium significantly affect the abundance of secreted leukotoxin , and we have developed a rapid (<2 h) method to partially purify large amo unts of leukotoxin. Remarkably, mutations in the tad genes, which are requi red for tight nonspecific adherence of A. actinomycetemcomitans to surfaces , cause leukotoxin to be released from the bacterial cell. These studies sh ow that A. actinomycetemcomitans has the potential to secrete abundant leuk otoxin. It is therefore appropriate to consider a possible role for leukoto xin secretion in the pathogenesis of A. actinomycetemcomitans.