TdeA, a TolC-like protein required for toxin and drug export in Aggregatibacter (Actinobacillus) actinomycetemcomitans

Abstract

Aggregatibacter actinomycetemcomitansW is an oral bacterium that causes localized aggressive periodontitis (LAP) and extra-oral infections such as sub-acute infective endocarditis. As part of its array of virulence factors, A. actinomycetemcomitans produces leukotoxin (LtxA), a member of the RTX family of toxins. LtxA kills human leukocytes and we have recently shown that the toxin is required for β -hemolysis by A. actinomycetemcomitans on solid medium. In other RTX toxinproducing bacteria, an outer membrane channel-forming protein, TolC, is required for toxin secretion and drug export. We have identified an ORF in A. actinomycetemcomitans that encodes a putative protein having predicted structural properties similar to TolC. Inactivation of this ORF resulted in a mutant that was no longer β -hemolytic and did not secrete LtxA. This mutant was significantly more sensitive to antimicrobial agents compared to the wild type strain and was unable to export the antimicrobial agent berberine. Thus, this ORF was named tdeA for "toxin and drug export". Examination of the DNA sequence surrounding tdeA revealed two upstream ORFs that encode proteins similar to the drug efflux proteins, MacA and MacB. Inactivation of macB in A. actinomycetemcomitans did not alter the drug sensitivity profile or the hemolytic activity of the mutant. The genes macA, macB and tdeA are organized as an operon and are constitutively expressed as a single transcript. These results show that A. actinomycetemcomitans indeed requires a TolC-like protein for LtxA secretion and that this protein, TdeA, also functions as part of a drug efflux system.

Aggregatibacter actinomycetemcomitans Leukotoxin

“…Like E. coli mutants of tolC (Wandersman and Delepelaire, 1990;Nishino et al, 2003Nishino et al, , 2006Tamura et al, 2005), A. actinomycetemcomitans tdeA mutants are significantly more sensitive to antimicrobial agents than are wild-type strains (Crosby and Kachlany, 2007). The reason for this is that TdeA is also part of one or more drug efflux pumps, similar to other TolC proteins.…”

“…Thus, the data are consistent with the hypothesis that LtxA also plays a role in iron acquisition during infection. The mechanism by which iron regulates secretion is not known, but iron may interact with TdeA to cause a decrease in the pore size to prevent the passage of LtxA out of the cell (Crosby and Kachlany, 2007).…”

Aggregatibacter actinomycetemcomitans Leukotoxin Utilizes a Cholesterol Recognition/Amino Acid Consensus Site for Membrane Association

“…Four of the genes, ltxCABD (in transcriptional order) are located in the leukotoxin operon. A fifth gene, tdeA (6), is not part of the ltx operon and is located 572 kb downstream of this site. Prior to secretion, Gram-negative bacterial protein toxins are translated as protoxins and must be posttranslationally modified to achieve biological activity.…”

Membrane Morphology and Leukotoxin Secretion Are Associated with a Novel Membrane Protein of Aggregatibacter actinomycetemcomitans

“…The secretion of leukotoxin is mediated by a type I secretion system, and in order for the toxin to reach the extracellular milieu, the toxin must be transported across two membranes separated by a periplasmic space containing the peptidoglycan cell wall (7,19). Current understanding of the secretion of leukotoxin in A. actinomycetemcomitans is based on its homology with the E. coli HlyA secretion system.…”