Nonspecific Adherence and Fibril Biogenesis by Actinobacillus actinomycetemcomitans : TadA Protein Is an ATPase

Abstract

Cells of Actinobacillus actinomycetemcomitans, a gram-negative pathogen responsible for an aggressive form of juvenile periodontitis, form tenaciously adherent biofilms on solid surfaces. The bacteria produce long fibrils of bundled pili, which are required for adherence. Mutations in flp-1, which encodes the major subunit of the pili, or any of seven downstream tad genes (tadABCDEFG) cause defects in fibril production, autoaggregation, and tenacious adherence. We proposed that the tad genes specify part of a novel secretion system for the assembly and transport of Flp pili. The predicted amino acid sequence of TadA (426 amino acids, 47,140 Da) contains motifs for nucleotide binding and hydrolysis common among secretion NTP hydrolase (NTPase) proteins. In addition, the tadA gene is the first representative of a distinct subfamily of potential type IV secretion NTPase genes. Here we report studies on the function of TadA. The tadA gene was altered to express a modified version of TadA that has the 11-residue epitope (T7-TAG) fused to its C terminus. The TadA-T7 protein was indistinguishable from the wild type in its ability to complement the fibril and adherence defects of A. actinomycetemcomitans tadA mutants. Although TadA is not predicted to have a transmembrane domain, the protein was localized to the inner membrane and cytoplasmic fractions of A. actinomycetemcomitans cells, indicating a possible peripheral association with the inner membrane. TadA-T7 was purified and found to hydrolyze ATP in vitro. The ATPase activity is stimulated by Triton X-100, with maximal stimulation at the critical micellar concentration. TadA-T7 forms multimers that are stable during sodium dodecyl sulfatepolyacrylamide gel electrophoresis in nonreducing conditions, and electron microscopy revealed that TadA-T7 can form structures closely resembling the hexameric rings of other type IV secretion NTPases. Site-directed mutagenesis was used to substitute Ala and Gln residues for the conserved Lys residue of the Walker A box for nucleotide binding. Both mutants were found to be defective in their ability to complement tadA mutants. We suggest that the ATPase activity of TadA is required to energize the assembly or secretion of Flp pili for tight adherence of A. actinomycetemcomitans.

Type IV Pilin Proteins: Versatile Molecular Modules

“…O'Toole and Kolter (304) first reported that P. aeruginosa mutants lacking T4P are deficient in biofilm formation. Since then, similar findings have been reported for a number of bacteria and archaea, including (but not limited to) V. cholerae, A. actinomycetemcomitans, Aeromonas caviae, Shewanella oneidensis, Legionella pneumophila, Caulobacter crescentus, Deinococcus geothermalis, C. perfringens, and Sulfolobus acidocaldarius (40,44,102,133,178,342,389,398,408,431).…”

Functional role of conserved residues in the characteristic secretion NTPase motifs of the Pseudomonas aeruginosa type IV pilus motor proteins PilB, PilT and PilU

“…Shown are the average twitching zone areas (12 zones each, four technical replicates from three independent plates) for unmodified and mutant proteins. and DotB of Legionella pneumophila (T4S) have been shown to be dominant-negative, whereas VirB11 (T4S) WA mutants have not (Bhattacharjee et al, 2001;Sakai et al, 2001;Stephens et al, 1995). Turner et al (1993) have shown that a WA mutant of XcpR (P. aeruginosa T2S ATPase) causes dominant-negative effects on secretion in the wild-type, but that the identical mutation in PilB does not affect the presence of external T4P in a wild-type strain, as assessed by electron microscopy.…”

Haemophilus ducreyi Requires the flp Gene Cluster for Microcolony Formation In Vitro

“…Moreover, it was recently proposed that tadA of A. actinomycetemcomitans is the first representative of a distinct subfamily of potential type IV secretion NTPase genes and that the flp-1-tadG gene cluster of this periodontal pathogen may encode a secretion system that is distinct from type II and type IV secretion systems (10). As it is found in both pathogenic and nonpathogenic species of bacteria, it is possible that this gene cluster encodes products that are not directly involved in pathogenesis.…”