flp‐1, the first representative of a new pilin gene subfamily, is required for non‐specific adherence of Actinobacillus actinomycetemcomitans

Abstract

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.

Tight-adherence genes of Actinobacillus actinomycetemcomitans are required for virulence in a rat model

“…The genes appear to be arranged in an operon. Our results show that the tad locus encodes a secretion system responsible for the secretion and assembly of bundled pili (18,21). Remarkably, tad-related loci are present in diverse Bacteria and Archaea, and we have proposed that the role of tad-like loci in other microbes is to mediate adherence in different environments (16).…”

“…Using a rat model, we compared the wild-type strain to mutant strains defective in flp-1 or tadA. Flp1 is the major pilus subunit (18,21), and TadA is an ATP hydrolase that is likely to energize or serve as a chaperone in the secretion and assembly of Flp1 (22). Our results show that this model system can be used to study disease resembling LAP of humans.…”

“…Cells adhere avidly to solid surfaces, including plastic, glass, and hydroxyapatite (14)(15)(16). Clinical isolates also autoaggregate and produce bundled pili that mediate nonspecific adherence (16)(17)(18)(19). The tight-adherence phenotype is thought to be important for colonization of host surfaces, and recently we reported that an adherent clinical isolate was able to colonize, survive, and persist in the oral cavity of rats (2).…”

tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans

“…The bacteria are known to produce several virulence factors, including a leukotoxin and a cytolethal distending toxin, and they adhere to solid surfaces and cells (Taichman and Wilton, 1981;Kolodrubetz et al, 1989;Lally et al, 1989;Eastcott et al, 1990;Mangan et al, 1991;Fives-Taylor et al, 1999;van Winkelhoff and Slots, 1999;Kachlany et al, 2001a;Narayanan et al, 2002;Henderson et al, 2003;Rose et al, 2003;Paturel et al, 2004;Rhodes et al, 2005;Diaz et al, 2006;Fine et al, 2005Fine et al, , 2006Smith and Bayles, 2006). Clinical isolates autoaggregate and secrete long, bundled pili (Flp fibrils) (Holt et al, 1980;Scannapieco et al, 1987;Rosan et al, 1988;Inoue et al, 1998;Kachlany et al, 2001b;Henderson et al, 2003;Fine et al, 2006). In vitro A. actinomycetemcomitans can produce rare, stable, nonadherent, and nonaggregative genetic variants that form smooth, large colonies and fail to produce Flp fibrils (Wyss, 1989;Inouye et al, 1990;Fine et al, 1999Fine et al, , 2006Kachlany et al, 2000Kachlany et al, , 2001aHenderson et al, 2003).…”

“…The three natural methods for gene transfer (transformation, conjugation, and transduction) have all been observed in A. actinomycetemcomitans (Tϕnjum et al, 1990;Goncharoff et al, 1993;Willi et al, 1997;Galli et al, 2001). We previously developed an efficient method for conjugationbased transposon-insertion mutagenesis (Thomson et al, 1999) that allowed us to isolate three independent mutations in the catalase gene (katA) of A. actinomycetemcomitans (Thomson et al, 1999) and in genes involved in non-specific adherence (Kachlany et al, , 2001bPlanet et al, 2003). For the latter, we identified the tad (tight adherence) locus, which encodes a novel secretion system responsible for the synthesis, assembly, and secretion of bundled type IVb pili, termed Flp fibrils (Kachlany et al, 2001a).…”