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.