Identification and Molecular Analysis of Rough-Colony-Specific Outer Membrane Proteins of Actinobacillus actinomycetemcomitans

Abstract

Actinobacillus actinomycetemcomitans, a gram-negative bacterium isolated from the human mouth, has been implicated in the pathogenesis of early-onset periodontitis. Primary isolates cultured from subgingival plaque exhibit an adherent, rough colony phenotype which spontaneously converts to a nonadherent, smooth phenotype upon in vitro subculture. The rough colony variant produces abundant fimbriae and autoaggregates, while the smooth colony variant is planktonic and produces scant fimbriae. To begin to understand the significance of colony variation in biofilm formation by A. actinomycetemcomitans, outer membrane protein profiles of four isogenic rough and smooth colony variants were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two proteins with relative molecular masses of 43 and 20 kDa were expressed by the rough colony variants exclusively. Expression of these proteins was not found to be dependent on growth phase, oxygen tension, or type of complex medium. N-terminal amino acid sequences of these proteins obtained by Edman degradation were compared with sequences from the University of Oklahoma A. actinomycetemcomitans genome database. Two contiguous open reading frames (ORFs) encoding proteins having sequence homology with these proteins were identified. The 43-kDa protein (RcpA [rough colony protein A]) was similar to precursor protein D of the general secretion pathway of gram-negative bacilli, while the 20-kDa protein (RcpB [rough colony protein B]) appeared to be unique. The genes encoding these proteins have been cloned from A. actinomycetemcomitans 283 and sequenced. A BLASTX (gapped BLAST) search of the surrounding ORFs revealed homology with other fimbria-related proteins. These data suggest that the genes encoding the 43-kDa (rcpA) and 20-kDa (rcpB) proteins may be functionally related to each other and to genes that may encode fimbria-associated proteins.

Aggregatibacter, a Low Abundance Pathobiont That Influences Biogeography, Microbial Dysbiosis, and Host Defense Capabilities in Periodontitis: The History of a Bug, and Localization of Disease

“…Ultimately, several studies including work by Inoue et al [24], Haase et al [25], and Planet et al [26] established that adherence was dependent on a 14 gene operon, the tad operon, that was widespread in nature [27]. Pieces of this important operon were found in most microbes that had pathogenic potential as well as all archae sequenced to date, suggesting the biological significance of this operon in nature [26].…”

Aggregatibacter Actinomycetemcomitans: Clinical Significance of a Pathobiont Subjected to Ample Changes in Classification and Nomenclature

“…Fimbriae are assembled as bundles of 5-to 7-nm-diameter pili composed of a 6.5 kDa protein designated Flp (fimbrial low-molecular-weight protein) [80,81]. The RcpA/B (rough colony proteins) were the first outer membrane proteins identified that were associated with rough colony variants [82], and they are encoded by a 14-gene locus designated the tad locus. The Tad (tight adherence) macromolecular transport system is a subtype of type II secretion.…”

The highly leukotoxic JP2 clone of Aggregatibacter actinomycetemcomitans: evolutionary aspects, epidemiology and etiological role in aggressive periodontitis

“…The fimbriae are composed of a glycosylated protein, Flp‐1 ( Inoue et al ., 1998, 2000). The genetic basis for the conversion of rough to smooth morphology has been explained by mutations in the genes involved in fimbriae biogenesis ( Haase et al . 1999, 2003; Kachlany et al ., 2000A, 2001A; Planet et al ., 2003; Wang et al ., 2005).…”