Identification of a Haemophilus influenzae 5 '-nucleotidase protein: Cloning of the nucA gene and immunogenicity and characterization of the NucA protein

We report on the identification of a surface-exposed, highly conserved, imm unogenic nontypeable Haemophilus influenzae (NTHi) protein, which elicits c ross-reactive bactericidal antibodies against NTHi, The protein was extract ed from NTHi strain P860295 with KSCN and purified; it migrated as a single band on a sodium dodecyl sulfate-polyacrylamide gel with an apparent molec ular mass of 63 kDa. Mouse antiserum generated against the purified protein was reactive on whole-cell enzyme-linked immunosorbent assay (ELISA) with seven NTHi strains and type b Eagan and Whittier strains and exhibited bact ericidal activity to homologous and heterologous NTHi strains. However, the protein is made in small amounts in NTHi as corroborated by immunoelectron microscopy. To further study this protein, we cloned, sequenced, and expre ssed it recombinantly in Escherichia coli. The recombinant protein is local ized in the periplasm of E. coli and has been purified to homogeneity. Both the recombinant and native proteins possess 5'-nucleotidase activity; henc e, the protein has been called NucA, Mouse antiserum directed against the r ecombinant NucA protein was reactive on Western immunoblots and whole-cell ELISA with all H. influenzae strains tested including Eagan and was bacteri cidal for two heterologous strains tested. The antiserum also resulted in a log reduction in bacteremia, in an infant-rat protection study with H. inf luenzae type b as the challenge strain. These features suggest that NucA is a potential subunit vaccine candidate against NTHi disease.