Secretion of RTX leukotoxin by Actinobacillus actinomycetemcomitans

Abstract

Actinobacillus actinomycetemcomitans, the etiologic agent for localized juvenile periodontitis and certain other human infections, such as endocarditis, expresses a leukotoxin that acts on polymorphonuclear leukocytes and macrophages. 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 leukotoxin of A. actinomycetemcomitans is thought to remain associated with the bacterial cell. We have examined leukotoxin production and localization in rough (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, including Y4, JP2 (a strain expressing a high level of toxin), and CU1060N (an isogenic smooth variant of CU1000N), secrete an abundance of leukotoxin into the culture supernatants during early stages of growth. After longer times of incubation, leukotoxin disappears from the supernatants, and its loss is accompanied by the appearance of a number of low-molecular-weight polypeptides. The secreted leukotoxin is active, as evidenced by its ability to kill 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 amounts of leukotoxin. Remarkably, mutations in the tad genes, which are required for tight nonspecific adherence of A. actinomycetemcomitans to surfaces, cause leukotoxin to be released from the bacterial cell. These studies show that A. actinomycetemcomitans has the potential to secrete abundant leukotoxin. It is therefore appropriate to consider a possible role for leukotoxin secretion in the pathogenesis of A. actinomycetemcomitans.

FIG. 1

Localization of leukotoxin from the rough and smooth strains. (A and B) Visualization of cell-free (supernatant) and cell-associated (pellet) leukotoxin from rough strain CU1000N (A) and smooth strain CU1060N (B). Arrows, leukotoxin band (approximately 120 kDa). (C) Leukotoxin in the culture supernatants of the commonly used A. actinomycetemcomitans smooth strains Y4 (left) and JP2, a high-level-leukotoxin producer (right). The times after inoculation at which the samples were taken are noted above each lane. Polypeptides were separated by SDS-PAGE and stained with Coomassie blue. The identity of the leukotoxin band was confirmed by MALDI-MS.

FIG. 2

Activity of secreted leukotoxin on HL-60 cells. (A) HL-60 cells were treated with medium (control) or leukotoxin (LtxA) for various times in the presence of PI and assayed for PI uptake by FACS analysis as described in Materials and Methods. The percentages of cells that were PI positive (dead) are plotted versus the time of incubation. (B) Phase-contrast microscopy of leukotoxin-treated HL-60 cells. Cells were treated with medium (left) or leukotoxin (right) for 30 min at 37°C. Secreted leukotoxin was prepared from culture supernatants as described in Materials and Methods.

FIG. 3

Effect of initial pH on abundance of secreted leukotoxin. CU1060N cells were grown in medium with an initial pH of 7.1 or 8.0. Leukotoxin was prepared from culture supernatants at the times indicated, separated by SDS-PAGE, and visualized by Coomassie blue as described in Materials and Methods.

FIG. 4

Cell-associated leukotoxin in tad mutants. tad mutant strains were grown for 23 h. Cell-associated proteins were prepared and separated by SDS-PAGE and stained with Coomassie blue as described in Materials and Methods. The cell-associated protein is shown for each mutant containing the plasmid vector control (pJAK16) or the appropriate complementing plasmid. The tad mutant is noted below the lanes. Leukotoxin (arrow) was confirmed by MALDI-MS.

FIG. 5

Extracellular proteins from tad mutants. Polypeptides from culture supernatants were prepared, separated by SDS-PAGE, and stained with Coomassie blue as described in Materials and Methods. (Left) Leukotoxin is present in 16-h supernatants of tadB and tadD mutants along with lower-molecular-weight polypeptides. (Right) Leukotoxin is no longer visible in 37-h supernatants of tadB and tadD mutants, which show abundant lower-molecular-weight polypeptides.