Nonspecific Adherence by Actinobacillus actinomycetemcomitans Requires Genes Widespread in Bacteria and Archaea

Actinobacillus actinomycetemcomitans is a gram-negative bacterium associated with several human diseases (8, 36, 47). The most predominant of these is known as localized juvenile periodontitis, a severe disease of adolescents that is characterized by bone and tissue destruction and ultimately loss of teeth if untreated. A. actinomycetemcomitans is also a member of a clinically important group of bacteria implicated in infective endocarditis (39). These bacteria are referred to as the HACEK group (Haemophilus aphrophilus, A. actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) (9). Vegetative growths and inflammation of the heart valves caused by the HACEK bacteria result in serious complications owing to the formation of bacterial masses on the valves. A. actinomycetemcomitans expresses several potential virulence factors (8), but only the RTX-type leukotoxin has been studied in detail at the molecular and genetic levels (14, 24). Other possible factors include a cytolethal distending toxin, iron and hemin binding proteins, a trypsin-like protease, an OmpA family member, capsular polysaccharide biosynthetic proteins, catalase, and a GroEL-like protein (5, 12, 13, 23, 26, 28, 35, 41, 43, 46). However, their potential roles in pathogenesis are unknown. A. actinomycetemcomitans is also able to invade epithelial cells, and the bacteria can be transferred between cells (8, 27). A striking and characteristic property of fresh clinical isolates of A. actinomycetemcomitans is their ability to form tenacious biofilms on solid surfaces, including glass, plastics, and hydroxyapatite (6, 21, 30). This property is very likely required for pathogenesis by allowing for colonization of teeth in an environment of continuous salivary flow. Clinical isolates form rough-appearing colonies, autoaggregate, and express bundles of fimbria-like structures that may be important for adherence and colonization (18, 30, 34). Genetic analysis of rough, adherent strains has proven to be difficult. The distinctive adherence property of clinical strains is easily lost, as nonadherent, smooth-colony variants readily emerge during subculture (7, 45). In addition, while DNA can be introduced into A. actinomycetemcomitans by transformation (37) or conjugation (11), the efficiency of DNA transfer into rough, adherent strains is too low for standard transposon mutagenesis protocols involving suicide vectors. Recently we reported the development of transposon IS903φkan, which carries a cryptic kanamycin resistance gene that can be activated upon insertion of the transposon into an expressed gene, and we have demonstrated its utility in the direct selection of random insertions in genetically recalcitrant bacteria, such as A. actinomycetemcomitans (38). Here we report the use of IS903φkan to obtain adherence-defective mutants of A. actinomycetemcomitans CU1000, a well-characterized rough clinical isolate (7, 20, 30). Genetic and nucleotide sequence analyses of these mutants have allowed us to identify a locus of seven novel genes that are required for tenacious adherence, autoaggregation, and the production of bundled fibers. Examination of genome sequences of Bacteria and Archaea revealed that surprisingly diverse microorganisms are predicted to carry tad-related genes. We discuss the likely function of these genes and the significance of their widespread occurrence.