MUTATIONAL ANALYSIS OF THE PUTATIVE LEUKOTOXIN TRANSPORT GENES IN ACTINOBACILLUS-ACTINOMYCETEMCOMITANS

The periodontal pathogen, Actinobacillus actinomycetemcomitans, produces leukotoxin, a protein that specifically lyses host defense cells. The leukotoxin is similar in sequence and operon organization to the Escherichia coli alpha-hemolysin and other members of the RTX family of toxins. However, unlike the other RTX toxins, the A. actinomycetemcomitans leukotoxin is not secreted from the cell and instead remains associated with the outer membrane. Nonetheless, the A. actinomycetemcomitans lkt operon contains two genes, lktB and lktD, that appear analagous to the toxin localization genes found In the other Gram-negative bacteria. Thus, to determine the roles of these putative transport genes in A. actinomycetemcomitans, we have used insertional mutagenesis to generate mutant strains lacking functional LktB and/or LktD. When either lktD or both lktB and lktD were inactivated, the level of detectable leukotoxin protein in the cell decreased significantly. However, the lktB and lktD mutations had no effect on the levels of leukotoxin RNA. Thus, the lack of LktB and LktD proteins must affect LktA synthesis post-transcriptionally. It is proposed that this is an indirect effect of leukotoxin mislocalization in lkfB(-) and lktD(-) mutants. Finally, analysis of the mutants revealed that LktB and LktD are not essential for the formation of extracellular membrane vesicles in A. actinomycetemcomitans.