Epitope tagging of a virulence protein in Haemophilus influenzae [abstract]

Abstract

Haemophilus influenzae, a small, gram negative bacterium, is a commensal organism of the human upper respiratory tract. Before the introduction of the Hib vaccine, encapsulated H. influenzae of serotype b was a major cause of childhood meningitis. Nonencapsulated H. influenzae, an important cause of respiratory illnesses, rarely causes invasive disease. However, unusually virulent NTHI strains have occasionally been isolated and raise the concern of vaccine-resistant meningitis. We are studying one such NTHI, strain R2866, isolated from a meningitis patient. R2866 and other unusually virulent NTHI contain a novel, phase variable gene termed lav, which is predicted to encode a member of the AIDA-I/VirG/PerT family of virulence-associated autotransporters. Autotransporters are outer membrane proteins with three domains, passenger, linker and beta-barrel. The "passenger" gives the protein its function, while the beta-barrel imbeds into the outer membrane and forms a pore through which the passenger domain exits from the cell. To understand the function and location of the Lav protein, we engineered an epitope tag within the carboxy terminal beta-domain. Structure of the beta-domain was predicted by homology to the E. coli AIDA-1 protein. We placed a FLAG tag at an external loop near the C-terminus. A tagged construct was used to replace the chromosomal lav gene by natural transformation of R2866. We intend using the FLAG-tagged R2866 to ask whether Lav is cleaved, to determine translational phase variation of lav, and to investigate whether Lav plays a role in adhesion to mammalian tissue.