The TadV Protein ofActinobacillus actinomycetemcomitansIs a Novel Aspartic Acid Prepilin Peptidase Required for Maturation of the Flp1 Pilin and TadE and TadF Pseudopilins

Abstract

The tad locus of Actinobacillus actinomycetemcomitans encodes genes for the biogenesis of Flp pili, which allow the bacterium to adhere tenaciously to surfaces and form strong biofilms. Although tad (tight adherence) loci are widespread among bacterial and archaeal species, very little is known about the functions of the individual components of the Tad secretion apparatus. Here we characterize the mechanism by which the pre-Flp1 prepilin is processed to the mature pilus subunit. We demonstrate that the tadV gene encodes a prepilin peptidase that is both necessary and sufficient for proteolytic maturation of Flp1. TadV was also found to be required for maturation of the TadE and TadF pilin-like proteins, which we term pseudopilins. Using sitedirected mutagenesis, we show that processing of pre-Flp1, pre-TadE, and pre-TadF is required for biofilm formation. Mutation of a highly conserved glutamic acid residue at position ؉5 of Flp1, relative to the cleavage site, resulted in a processed pilin that was blocked in assembly. In contrast, identical mutations in TadE or TadF had no effect on biofilm formation, indicating that the mechanisms by which Flp1 pilin and the pseudopilins function are distinct. We also determined that two conserved aspartic acid residues in TadV are critical for function of the prepilin peptidase. Together, our results indicate that the A. actinomycetemcomitans TadV protein is a member of a novel subclass of nonmethylating aspartic acid prepilin peptidases.Actinobacillus actinomycetemcomitans is a gram-negative, facultatively anaerobic coccobacillus that inhabits the oral cavities of humans and other mammals (4, 30, 52). A. actinomycetemcomitans is an opportunistic pathogen, primarily known as the etiologic agent of localized aggressive periodontitis, a particularly severe form of periodontal disease (3, 30). A. actinomycetemcomitans has also been associated with nonoral infections, including endocarditis, septicemia, and abscesses (77). We have identified a locus of 14 genes , designated the tad (tight adherence) locus, which is essential for the ability of the organism to adhere tenaciously to surfaces and form biofilms (40,42,59,62). The A. actinomycetemcomitans tad locus is required for the biogenesis of long and bundled pili, termed Flp fibrils, which confer the tight adherence phenotype (35,38,40,42). A functional tad locus is essential for colonization, persistence, and bone loss in a rat model of localized aggressive periodontitis (68), indicating that adherence is a critical component of the virulence repertoire of A. actinomycetemcomitans.At least 12, and probably 13, of the genes in the tad locus are essential for Flp pilus biogenesis (40,42,59,62). Certain components of the A. actinomycetemcomitans Flp pilus biogenesis system, including the RcpA predicted outer membrane secretin (29), the TadA ATPase (7), and the PilC-like TadB and TadC proteins (57), share similarity to those of bacterial type II secretion (T2S) and type IV pilus (T4P) systems, while TadA also has homology to the...

Ralstonia solanacearum Needs Flp Pili for Virulence on Potato

“…Interestingly, the NB336-1085 mutant strain overproduced biofilms compared to the wild-type NB336 strain. The result was somewhat unexpected, since tad mutants of A. actinomycetemcomitans and P. aeruginosa are impaired in biofilm formation on abiotic surfaces (Kachlany et al 2000;Perez et al 2006;Tomich et al 2006;de Bentzmann et al 2006 by the fact that T4a pili mutants of R. solanacearum AWI (Kang et al 2002) and A. avenae subsp. citrulli (Bahar et al 2009) are severely impaired in biofilm formation on abiotic surfaces.…”

“…Due to its unique features, it has been described as the Flp subfamily of T4b pili . In A. actinomycetemcomitans, the Flp pilin requires for its assembly a subset of components, called Tad and Rcp, which includes the RcpA secretin (Clock et al 2008), the TadD pilotin or docking protein (Clock et al 2008), the TadA trafficking NTPase (Bhattacharjee et al 2001), the PilC-like proteins TadB and TadC (Wang and Chen 2005), Charles K. Wairuri 4 MPMI the TadE and TadF pseudopilins (Kachlany et al 2000), and the TadV prepilin peptidase (Perez et al 2006;Tomich et al 2006). The genes of other Tad system components (RcpB, RcpC, TadG and TadZ) do not show extensive homologies to any genes and have not been assigned predicted functions.…”

Ocorrência de genes tad associados à formação de biofilme em isolados de Pasteurella multocida de pulmões de suínos com pneumonia

“…Alguns autores demonstraram que mutações nos genes tadE e tadF em A. actinomycetecomitans ocasionam uma diminuição na produção de pili e incapacidade de adesão em superfície (Kachlany et al 2000, Wang et al 2003, Tomich et al 2006. Nos isolados do nosso estudo apenas dois não apresentaram amplificação para esses genes (17A e 18A), sendo ambos de pulmões sem lesão, indicando que possa ter ocorrido uma baixa capacidade de fixação no epitélio pulmonar por parte destes.…”

Organization and PprB-Dependent Control of the Pseudomonas aeruginosa tad Locus, Involved in Flp Pilus Biology

“…Similar tad loci have been identified in archaea, actinobacteria, and gram-negative bacteria, usually as a single copy, but in up to four copies in some species (19,31). Several conserved elements have been identified in Tad machines to date: the TadA trafficking NTPase (17,24), the RcpA secretin, one or two inner membrane PilC homologues, TadB and TadC, the TadV/FppA prepilin peptidase (4,30), the Flp pilin, and the TadE and TadF pseudopilins (30). These components are also shared by type IVa pilus machines (T4P) and type II secretion systems (T2S).…”