The gram-negative coccobacillus, Actinobacillus actinomycetemcomitans, is the putative agent for localized juvenile periodontitis, a particularly destructive form of periodontal disease in adolescents. This bacterium has also been isolated from a variety of other infections, notably endocarditis. Fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms, a property likely to be critical for colonization of teeth and other surfaces. Here we report the identification of a locus of seven genes required for nonspecific adherence of A. actinomycetemcomitans to surfaces. The recently developed transposon IS903kan was used to isolate mutants of the rough clinical isolate CU1000 that are defective in tight adherence to surfaces (Tad ؊ ). Unlike wild-type cells, Tad ؊ mutant cells adhere poorly to surfaces, fail to form large autoaggregates, and lack long, bundled fibrils. Nucleotide sequencing and genetic complementation analysis revealed a 6.7-kb region of the genome with seven adjacent genes (tadABCDEFG) required for tight adherence. The predicted TadA polypeptide is similar to VirB11, an ATPase involved in macromolecular transport. The predicted amino acid sequences of the other Tad polypeptides indicate membrane localization but no obvious functions. We suggest that the tad genes are involved in secretion of factors required for tight adherence of A. actinomycetemcomitans. Remarkably, complete and highly conserved tad gene clusters are present in the genomes of the bubonic plague bacillus Yersinia pestis and the human and animal pathogen Pasteurella multocida. Partial tad loci also occur in strikingly diverse Bacteria and Archaea. Our results show that the tad genes are required for tight adherence of A. actinomycetemcomitans to surfaces and are therefore likely to be essential for colonization and pathogenesis. The occurrence of similar genes in a wide array of microorganisms indicates that they have important functions. We propose that tad-like genes have a significant role in microbial colonization.

Actinobacillus actinomycetemcomitans, a Gram‐negative bacterium responsible for localized juvenile periodontitis and other infections such as endocarditis, produces long fibrils of bundled pili that are believed to mediate non‐specific, tenacious adherence to surfaces. Previous investigations have implicated an abundant, small (≈ 6.5 kDa), fibril‐associated protein (Flp/Fap) as the primary fibril subunit. Here, we report studies on fibril structure and on the function and evolution of Flp. High‐resolution electron microscopy of adherent clinical strain CU1000N revealed long bundles of 5‐ to 7‐nm‐diameter pili, whose subunits appear to be arranged in a helical array similar to that observed for type IV pili in other bacteria. Fibrils were found to be associated with the bacterial cell surface and smaller structures thought to be membrane vesicles. A modified version of the CU1000N Flp1 polypeptide with the T7‐TAG epitope fused to its C‐terminus was expressed in the wild‐type strain, and the presence of the modified Flp1 in fibrils was confirmed by immunogold electron microscopy with monoclonal antibody to T7‐TAG. To determine the importance of Flp1 in fibril formation and cell adherence, we used transposon IS903φkan to isolate insertion mutations in the flp‐1 gene (formerly designated flp). Mutants with insertions early in flp‐1 fail to produce fibrils and do not adhere to surfaces. Both fibril production and adherence were restored by cloned flp‐1 in trans, thus providing the first evidence that flp‐1 is required for fibril formation and tight, non‐specific adherence. One mutant was found to have an insertion near the 3′ end of flp‐1 that results in the expression of a truncated and altered C‐terminus of Flp1. This mutant produced short, unbundled pili, and its adherence to surfaces was significantly less than that of wild‐type bacteria. These findings and related observations with the Flp1‐T7‐TAG protein indicate that the C‐terminus of Flp1 is important for the bundling and adherence properties of pili. Extensive sequence comparisons and phylogenetic analysis of 61 predicted prepilin genes of bacteria revealed flp‐1 to be a member of a novel and widespread subfamily of type IV prepilin genes. Thus, Flp pili are likely to be expressed by diverse bacterial species. Furthermore, we found that it is common for bacterial genomes to contain multiple alleles of flp‐like genes, including the open reading frame (flp‐2, previously designated orfA) immediately downstream of flp‐1 in A. actinomycetemcomitans. The duplication and divergence of flp genes in bacteria may be important to the diversification of the colonization properties of these organisms.

Genomic islands, such as pathogenicity islands, contribute to the evolution and diversification of microbial life. Here we report on the Widespread Colonization Island, which encompasses the tad (tight adherence) locus for colonization of surfaces and biofilm formation by the human pathogen Actinobacillus actinomycetemcomitans. At least 12 of the 14 genes at the tad locus are required for tenacious biofilm formation and synthesis of bundled Flp pili (fibrils) that mediate adherence. The pilin subunit, Flp1, remains inside the cell in tad-locus mutants, indicating that these genes encode a secretion system for export and assembly of fibrils. We found tad-related regions in a wide variety of Bacterial and Archaeal species, and their sequence characteristics indicate possible horizontal transfer. To test the hypothesis of horizontal transfer, we compared the phylogeny of the tad locus to a robust organismal phylogeny using statistical tests of congruence and tree reconciliation techniques. Our analysis strongly supports a complex history of gene shuffling by recombination and multiple horizontal transfers, duplications and losses. We present evidence for a specific horizontal transfer event leading to the establishment of this region as a determinant of disease.

Macromolecular transport systems in bacteria currently are classified by function and sequence comparisons into five basic types. In this classification system, type II and type IV secretion systems both possess members of a superfamily of genes for putative NTP hydrolase (NTPase) proteins that are strikingly similar in structure, function, and sequence. These include VirB11, TrbB, TraG, GspE, PilB, PilT, and ComG1. The predicted protein product of tadA, a recently discovered gene required for tenacious adherence of Actinobacillus actinomycetemcomitans, also has significant sequence similarity to members of this superfamily and to several unclassified and uncharacterized gene products of both Archaea and Bacteria. To understand the relationship of tadA and tadA-like genes to those encoding the putative NTPases of type II͞IV secretion, we used a phylogenetic approach to obtain a genealogy of 148 NTPase genes and reconstruct a scenario of gene superfamily evolution. In this phylogeny, clear distinctions can be made between type II and type IV families and their constituent subfamilies. In addition, the subgroup containing tadA constitutes a novel and extremely widespread subfamily of the family encompassing all putative NTPases of type IV secretion systems. We report diagnostic amino acid residue positions for each major monophyletic family and subfamily in the phylogenetic tree, and we propose an easy method for precisely classifying and naming putative NTPase genes based on phylogeny. This molecular key-based method can be applied to other gene superfamilies and represents a valuable tool for genome analysis.conjugation ͉ ATPase ͉ Archaea ͉ molecular key ͉ Walker box

Actinobacillus actinomycetemcomitans is a Gram-negative coccobacillus that has been associated with localized aggressive periodontitis and infections of the heart, brain, and urinary tract. Wild-type clinical isolates have the remarkable ability to adhere tenaciously and nonspecifically to solid surfaces such as glass, plastic, and hydroxyapatite. Adherence by A. actinomycetemcomitans is mediated by the tight-adherence (tad) gene locus, which consists of 14 genes (flp-1-flp-2-tadV-rcpCAB-tadZABCDEFG). All but 2 of the genes have been shown to be required for the secretion and assembly of long, bundled Flp1 fibrils. To test whether the tad locus is required for colonization and disease, we developed a rat model for periodontal disease. To mimic the natural route of infection, Sprague-Dawley rats were inoculated orally by adding bacteria directly to their food for 8 days. After inoculation with wild-type or mutant strains defective in adherence (flp-1 and tadA), the rats were assessed for colonization of the oral cavity and pathogenesis. Wild-type A. actinomycetemcomitans was able to colonize and persist for at least 12 weeks in the oral cavity, elicit a humoral immune response, and cause significant bone loss in rats. In contrast, rats fed flp-1 or tadA mutant strains showed no bone loss and their immune responses were indistinguishable from those of the uninoculated controls. These results demonstrate the critical importance of the tad locus in the colonization and pathogenesis of A. actinomycetemcomitans. nonspecific adherence ͉ tad genes ͉ pathogenesis ͉ localized juvenile periodontitis

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Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium that colonizes the human oral cavity and is the causative agent for localized aggressive periodontitis (LAP), an aggressive form of periodontal disease that occurs in adolescents. A. actinomycetemcomitans secretes a protein toxin, leukotoxin (LtxA), which helps the bacterium evade the host immune response during infection. LtxA is a membrane-active toxin that specifically targets white blood cells (WBCs). In this review, we discuss recent developments in this field, including the identification and characterization of genes and proteins involved in secretion, regulation of LtxA, biosynthesis, newly described activities of LtxA, and how LtxA may be used as a therapy for the treatment of diseases.