The Haemophilus influenzae Hia adhesin is an autotransporter protein that remains uncleaved at the C terminus and fully cell associated

Nontypeable Haemophilus influenzae is a gram-negative commensal organism th at is commonly associated with localized respiratory tract disease. The pat hogenesis of disease begins with colonization of the nasopharynx, a process that likely depends on bacterial adherence to respiratory epithelial cells . Hia is the major adhesin expressed by a subset of nontypeable H. influenz ae strains and promotes efficient adherence to a variety of human epithelia l cell lines. Based on previous work, Hia is transported to the surface of Escherichia coli transformants and is capable of mediating E. coli adherenc e without the assistance of other H. influenzae proteins. In the present st udy, we examined the mechanism of Hia secretion. PhoA fusions, deletional m utagenesis, and N-terminal amino acid sequencing established that the signa l for Hia export from the cytoplasm resides in the first 49 amino acids, in cluding a 24-amino-acid stretch with striking similarity to the N terminus of a number of proteins belonging to the autotransporter family. Immunoelec tron microscopy demonstrated that the Hia internal region defined by amino acids 221 to 779 is exposed on the bacterial surface. Secondary-structure a nalysis predicted that the C terminus of Hia forms a beta-barrel with a cen tral hydrophilic channel, and site-specific mutagenesis and fusion protein analysis demonstrated that the C terminus targets Hia to the outer membrane and functions as an outer membrane translocator, analogous to observations with autotransporter proteins. In contrast to typical autotransporter prot eins, Hia undergoes no cleavage between the internal and C-terminal domains and remains fully cell associated. Together, these results suggest that Hi a is the prototype of an important subfamily of autotransporter proteins.