B-274. Inactivation of mor141 Affects Membrane Morphology and Secretion of Leukotoxin in Aggregatibacter actinomycetemcomitans

C. V. Gallant, E. Chicoine, K. P. Mintz;
Univ. of Vermont, Burlington, VT.

Background: Aggregatibacter actinomycetemcomitans is a gram-negative bacterium that causes localized aggressive periodontitis and is implicated in extra oral infections. Multiple virulence determinants have been described for this pathogen including the secretion of toxins across the outer membrane. The outer membrane acts as a strong permeability barrier against antibiotics and regulates the flow of ions across the membrane. A. actinomycetemcomitans displays an irregular membrane surface and we have isolated a transposon mutant with an outer membrane devoid of any convolutions. The integration site of the transposon was localized to the 5' end of a 3.8 kbp ORF predicted to code for a 141 kDa membrane protein, designated morphogenic protein 141 (Mor141). Homology searches revealed that Mor141 is highly conserved in gram-negative bacteria. The disruption of mor141 is associated with changes in the size and aggregation state of the bacterium. However, the changes in the permeability of the outer membrane and secretion of leukotoxin have not been investigated. Methods: Sensitivity to salts and antibiotics was tested by replica plating of the strains on rich media containing various amounts of the substance. The expression of leukotoxin was examined by immunoblots. The mutant was complemented by transformation with a plasmid containing a 165 bp promoter region located upstream of the mor141 operon. Results: Alteration of the membrane morphology in the mutant strain was associated with an increase in the sensitivity to NaCl, bile salts, as well as chloramphenicol. The level of leukotoxin protein in the mor141 mutant was decreased although leukotoxin transcription was not affected. Complementation of the mutation in trans rescued the phenotypes of the mutation. Conclusion: Disruption of the mor141 gene results in changes in membrane morphology, membrane permeability and leukotoxin secretion. The prevalence of this gene amongst bacterial species suggests a conserved role for this gene product and may be used as a therapeutic target to reduce the pathogenesis of A. actinomycetemcomitans and other pathogens.