The Tad (tight adherence) macromolecular transport system, which is present in many bacterial and archaeal species, represents an ancient and major new subtype of type II secretion. The tad genes are present on a genomic island named the widespread colonization island (WCI), and encode the machinery that is required for the assembly of adhesive Flp (fimbrial low-molecular-weight protein) pili. The tad genes are essential for biofilm formation, colonization and pathogenesis in the genera Aggregatibacter (Actinobacillus), Haemophilus, Pasteurella, Pseudomonas, Yersinia, Caulobacter and perhaps others. Here we review the structure, function and evolution of the Tad secretion system.

A human antibody facilitates opsonophagocytic killing, inhibits attachment of Pseudomonas aeruginosa, and exerts protective effects in several animal models of P. aeruginosa infection.

Burkholderia cepacia is an important opportunistic human pathogen that affects immunocompromised individuals, particularly cystic fibrosis (CF) patients. Colonization of the lungs of a CF patient by B. cepacia can lead not only to a decline in respiratory function but also to an acute systemic infection, such as bacteremia. We have previously demonstrated that a CF clinical isolate of B. cepacia, strain J2315, can invade and survive within cultured respiratory epithelial cells. In order to further characterize the mechanisms of invasion of B. cepacia, we screened a transposon-generated mutant library of strain J2315 for mutants defective in invasion of A549 respiratory epithelial cells. Here we describe isolation and characterization of a nonmotile mutant of B. cepacia with reduced invasiveness due to disruption of fliG, which encodes a component of the motor-switch complex of the flagellar basal body. We also found that a defined null mutation in fliI, a gene encoding a highly conserved ATPase required for protein translocation via the flagellar type III secretion system, also resulted in loss of motility and a significant reduction in invasion. Both mutants lacked detectable intracellular flagellin and failed to export detectable amounts of flagellin into culture supernatants, suggesting that disruption of fliG and fliI impaired flagellar biogenesis. The reduction in invasion did not appear to be due to defective adherence of the flagellar mutants to A549 cells, suggesting that functional flagella and motility are required for full invasiveness of B. cepacia. Our findings indicate that flagellum-mediated motility may facilitate penetration of host epithelial barriers by B. cepacia, contributing to establishment of infection and systemic spread of the organism.Over the last several decades Burkholderia cepacia has emerged as an important opportunistic human pathogen of the lower respiratory tract that affects immunocompromised individuals, particularly cystic fibrosis (CF) patients (18). Chronic colonization of the lungs of a CF patient by B. cepacia can lead not only to a decline in respiratory function, due to the onset of a necrotizing pneumonia, but also to an acute systemic infection, such as bacteremia or septicemia (11,46). In addition to being an invasive pathogen that is capable of entering deeper tissues and becoming blood borne, B. cepacia can survive in epithelial cells and macrophages (4,35,42,48), which may contribute to the persistence of the organism in the host. The rapid clinical decline due to B. cepacia colonization is known as cepacia syndrome, and this decline leads to mortality in approximately 20 to 35% of chronically colonized individuals (25, 47). Furthermore, colonization by B. cepacia reduces the life expectancy of a CF patient by 50%, from 30 to 15 years (24). The inherent resistance of B. cepacia to multiple antibiotics makes eradication of this pathogen from the lungs of infected individuals especially difficult. Despite the known association of B. cepacia with fatal pulmonary inf...

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...

Type III secretion systems are utilized by a number of gram-negative bacterial pathogens to deliver virulence-associated proteins into host cells. Using a PCR-based approach, we identified homologs of type III secretion genes in the gram-negative bacterium Burkholderia cepacia, an important pulmonary pathogen in immunocompromised patients and patients with cystic fibrosis. One of the genes, designated bscN, encodes a member of a family of ATP-binding proteins believed to generate energy driving virulence protein secretion. Genetic dissection of the regions flanking the bscN gene revealed a locus consisting of at least 10 open reading frames, predicted to encode products with significant homology to known type III secretion proteins in other bacteria. A defined null mutation was generated in the bscN gene, and the null strain and wild-type parent strain were examined by use of a murine model of B. cepacia infection. Quantitative bacteriological analysis of the lungs and spleens of infected C57BL/6 mice revealed that the bscN null strain was attenuated in virulence compared to the parent strain, with significantly lower bacterial recovery from the lungs and spleens at 3 days postinfection. Moreover, histopathological changes, including an inflammatory cell infiltrate, were more pronounced in the lungs of mice infected with the wild-type parent strain than in those of mice infected with the isogenic bscN mutant. These results implicate type III secretion as an important determinant in the pathogenesis of B. cepacia.