flp-1, the first representative of a new pilin gene subfamily, is requiredfor non-specific adherence of Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans, a Gram-negative bacterium responsible for localized juvenile periodontitis and other infections such as endocard itis, produces long fibrils of bundled pili that are believed to mediate no n-specific, tenacious adherence to surfaces. Previous investigations have i mplicated an abundant, small (approximate to 6.5 kDa), fibril-associated pr otein (Flp/Fap) as the primary fibril subunit. Here, we report studies on f ibril structure and on the function and evolution of Flp. High-resolution e lectron microscopy of adherent clinical strain CU1000N revealed long bundle s of 5- to 7-nm-diameter pili, whose subunits appear to be arranged in a he lical array similar to that observed for type IV pili in other bacteria. Fi brils were found to be associated with the bacterial cell surface and small er structures thought to be membrane vesicles. A modified version of the CU 1000N 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 an tibody to T7-TAG. To determine the importance of Flp1 in fibril formation a nd cell adherence, we used transposon IS903 phi kan to isolate insertion mu tations in the flp-1 gene (formerly designated flp). Mutants with insertion s early in flp-1 fail to produce fibrils and do not adhere to surfaces. Bot h fibril production and adherence were restored by cloned flp-1 in trans, t hus providing the first evidence that flp-1 is required for fibril formatio n and tight, non-specific adherence. One mutant was found to have an insert ion 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-typ e bacteria. These findings and related observations with the Flp1-T7-TAG pr otein indicate that the C-terminus of Flp1 is important for the bundling an d adherence properties of pili. Extensive sequence comparisons and phylogen etic 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. Fur thermore, we found that it is common for bacterial genomes to contain multi ple alleles of flp-like genes, including the open reading frame (flp-2, pre viously designated orfA) immediately downstream of flp-1 in A. actinomycete mcomitans. The duplication and divergence of flp genes in bacteria may be i mportant to the diversification of the colonization properties of these org anisms.