Adherence to ciliated respiratory epithelial cells is considered a critical early step in Bordetella pathogenesis. ForBordetella pertussis, the etiologic agent of whooping cough, several factors have been shown to mediate adherence to cells and cell lines in vitro. These putative adhesins include filamentous hemagglutinin (FHA), fimbriae, pertactin, and pertussis toxin. Determining the precise roles of each of these factors in vivo, however, has been difficult, due in part to the lack of natural-host animal models for use with B. pertussis. Using the closely related species Bordetella bronchiseptica, and by constructing both deletion mutation and ectopic expression mutants, we have shown that FHA is both necessary and sufficient for mediating adherence to a rat lung epithelial (L2) cell line. Using a rat model of respiratory infection, we have shown that FHA is absolutely required, but not sufficient, for tracheal colonization in healthy, unanesthetized animals. FHA was not required for initial tracheal colonization in anesthetized animals, however, suggesting that its role in establishment may be dedicated to overcoming the clearance action of the mucociliary escalator.

Streptococcus equi (Streptococcus equi subsp. equi), a Lancefield group C streptococcus, causes strangles, a highly contagious purulent lymphadenitis and pharyngitis of members of the family Equidae. The antiphagocytic 58-kDa M-like protein SeM is a major virulence factor and protective antigen. The amino acid sequence and structure of SeM has been determined and compared to that of a second, 40-kDa M-like protein (SzPSe) of S. equi and to those of other streptococcal proteins. Both SeM and SzPSe are mainly alpha-helical fibrillar molecules with no homology other than that between their signal and membrane anchor sequences and are only distantly related to other streptococcal M and M-like proteins. The sequence of SzPSe indicates that it is an allele of SzP that encodes the variable protective M-like and typing antigens of S. zooepidemicus (S. equi subsp. zooepidemicus). SeM is opsonogenic for S. equi but not for the closely related S. zooepidemicus, whereas SzPSe is strongly opsonogenic for S. zooepidemicus but not for S. equi. Both proteins bind equine fibrinogen. SeM and SzPSe proteins from temporally and geographically separated isolates of S. equi are identical in size. The results taken together support previous evidence that S. equi is a clonal pathogen originating from an ancestral strain of S. zooepidemicus. We postulate that acquisition of SeM synthesis was a key element in the success of the clone because of its effect in enhancing resistance to phagocytosis and because protective immunity entails a requirement for SeM-specific antibody.

Human monocytes and macrophages bind Bordetella pertussis through multiple specific receptor-ligand interactions; however, the effect of these interactions on monocyte and macrophage function is not well understood. In an in vitro system, B. pertussis infection of human monocytes significantly impaired T cell proliferation to exogenous antigen at MOIs as low as 1.0. B. pertussis isogenic mutant strains deficient in filamentous hemagglutinin or adenylate cyclase toxin were incapable of proliferation inhibition, suggesting that these virulence-associated factors are essential for this activity. B. pertussis-induced monocyte death alone did not explain these results, nor did differences in intracellular survival. In addition, B. pertussis infection did not significantly alter monocyte phagocytosis of complement-opsonized latex particles, indicating that B. pertussis infection does not globally impair monocyte functions in this system. These results suggest that B. pertussis may be capable of subverting cellular immune defenses in an infected host.

The differential host species specificities of Bordetella pertussis, B. parapertussis, and B. bronchiseptica might be explained by polymorphisms in adherence factor genes. We have found that B. parapertussis and B. bronchiseptica, unlike B. pertussis, contain a full-length gene for the fimbrial subunit FimA. B. bronchiseptica expresses fimA in a BvgAS-dependent fashion.The genetic basis for fimbrial expression in Bordetella pertussis has been well characterized. A fimbrial operon located downstream of the filamentous hemagglutinin structural gene fhaB is regulated by the BvgAS two-component system; it contains genes encoding accessory proteins (FimB and FimC) and the fimbrial minor subunit (FimD) (18). The genes for the major fimbrial subunits, Fim2 and Fim3, are expressed elsewhere on the Bordetella chromosome (8, 10). Fimbrial phase variation can be observed in vivo and is controlled by small insertions or deletions in a C-rich region upstream of both fim2 and fim3 (17). The B. pertussis fimbrial operon also contains a pseudogene designated fimA, located at the 5Ј end of the fimbrial gene cluster (Fig. 1) (18). This gene contains a DNA sequence homologous to those of fim2 and fim3 but lacks sequences predicted to encode the N-terminal third of the fimbrial subunit (18).The organization of the Bordetella bronchiseptica and Bordetella parapertussis fimbrial genes and their function are less well characterized than those of the B. pertussis fimbrial genes (5, 6). B. bronchiseptica expresses proteins that are recognized by polyclonal and monoclonal antisera generated against B. pertussis Fim2 and Fim3 (11, 16). Coding sequences for Fim2 and Fim3 are 74 and 94% similar at the nucleotide level, respectively, between B. pertussis and B. bronchiseptica (16).Comparison of 5Ј sequences upstream of fim2 and fim3 in the two species suggests similar mechanisms of transcriptional control. Accessory fimbrial proteins in B. bronchiseptica have not been examined in any detail, although the minor fimbrial subunit in this species, FimD, is predicted to differ from that of B. pertussis by only one amino acid (19). Differences in the host species specificities of these three Bordetella species might reflect polymorphisms at the genetic loci that encode fimbriae and other adherence factors.Structure of B. bronchiseptica and B. parapertussis genomic regions encompassing fimA. Using PCR primers derived from previously published B. pertussis fhaB (positions 10756 to 10781; GenBank no. X52156) and fimA (746 to 729; GenBank no. X64876) sequences (4, 18), we detected a 400-bp amplicon size polymorphism between B. pertussis BP536 (13) and B. bronchiseptica GP1, RB50, 110H, B133, and VPI-FE1 (2, 3) that corresponded to the region just downstream of fhaB. (Sequence analysis at the 3Ј end of GP1 fhaB revealed a substitution, G10781T, at the 3Ј end of the forward priming site.) The 1,105-bp amplicon from B. bronchiseptica guinea pig isolate GP1 (1) was cloned in pBluescript II KS (Stratagene, La Jolla, Calif.), and its sequence was determined...