The Tryptophanase Gene Cluster of Haemophilus influenzae Type b: Evidence for Horizontal Gene Transfer

Among strains of Haemophilus influenzae, the ability to catabolize tryptophan (as detected by indole production) varies and is correlated with pathogenicity. Tryptophan catabolism is widespread (70 to 75%) among harmless respiratory isolates but is nearly universal (94 to 100%) among strains causing serious disease, including meningitis. As a first step in investigating the relationship between tryptophan catabolism and virulence, we have identified genes in pathogenic H. influenzae which are homologous to the tryptophanase (tna) operon of Escherichia coli. The tna genes are located on a 3.1-kb fragment between nlpD and mutS in the H. influenzae type b (Eagan) genome, are flanked by 43-bp direct repeats of an uptake signal sequence downstream from nlpD, and appear to have been inserted as a mobile unit within this sequence. The organization of this insertion is reminiscent of pathogenicity islands. The tna cluster is found at the same map location in all indole-positive strains of H. influenzae surveyed and is absent from reference type d and e genomes. In contrast to H. influenzae, most other Haemophilus species lack tna genes. Phylogenetic comparisons suggest that the tna cluster was acquired by intergeneric lateral transfer, either by H. influenzae or a recent ancestor, and that E. coli may have acquired its tnaA gene from a related source. Genomes of virulent H. influenzae resemble those of pathogenic enterics in having an island of laterally transferred DNA next to mutS. Haemophilus influenzae, a small, gram-negative bacterium, is a causative agent of invasive diseases, such as meningitis and epiglottitis, as well as mucosal infections, including otitis media and chronic bronchitis. The vast majority of H. influenzae strains, however, are harmless commensals of the human upper respiratory tract (25). Invasive disease (meningitis, epiglottitis, and septicemia) is associated with a minority of virulent strains, generally but not exclusively encapsulated and of serotype b (48). With the advent of an effective vaccine against the b capsular antigens, the incidence of H. influenzae type b (Hib)associated meningitis has declined in industrialized countries; however, meningitis associated with serotype a strains and nonencapsulated varieties has occasionally been reported (28, 34). Genetic comparisons between virulent and nonpathogenic strains of H. influenzae are expected to shed light on mechanisms of host invasion (12). In 1995, H. influenzae Rd became the first free-living organism to have its entire genome sequenced (12). The Rd strain, a nonencapsulated derivative of a serotype d isolate, is nonpathogenic; part of the impetus behind sequencing its genome was to provide a baseline comparison with pathogenic isolates. The virulent Eagan strain (serotype b), isolated from a patient with meningitis, has a genome approximately 270 kb larger than that of Rd (9), and comparisons of Hib and Rd genomes have uncovered virulence-associated genes exclusive to Hib (27, 49). The fimbrial gene cluster, which facilitates colonization and adhesion to host cells, is found on an 8-kb fragment of Hib DNA which is missing from Rd (49). In this study, we used genomic comparisons between Rd and Hib (Eagan) to identify