IDENTIFICATION AND CHARACTERIZATION OF GENES ENCODING THE HUMAN TRANSFERRIN-BINDING PROTEINS FROM HAEMOPHILUS-INFLUENZAE

Haemophilus influenzae, a strict human pathogen, acquires iron in vivo through the direct binding and removal of iron from human transferrin by an as yet uncharacterized process at the bacterial cell surface, I n this study, the tbpA and tbpB genes of H. influenzae, encoding the t ransferrin-binding proteins Tbp1 and Tbp2, respectively, were cloned a nd sequenced. Alignments of the H, infuenzae Tbp1 acid Tbp2 protein se quences with those of related proteins from heterologous species were analyzed, On the basis of similarities between these and previously ch aracterized proteins, Tbp1 appears to be a member of the TonB-dependen t family of outer membrane proteins while Tbp2 is lipid modified by si gnal peptidase II. Isogenic mutants deficient in expression of Tbp1 or Tbp2 or both proteins were prepared by insertion of the Tn903 kanamyc in resistance cassette into cloned sequences and reintroduction of the interrupted sequences into the wild-type chromosome, Binding assays w ith the mutants showed that a significant reduction in transferrin-bin ding ability resulted from the loss of either of the Tbps and a comple te loss of binding was evident when neither protein was expressed, Los s of either Tbp2 or both proteins correlated with an inability to grow on media supplemented with transferrin-bound iron as the sole source of iron, whereas the Tbp1(+) Tbp2(-) mutant was able to grow only at h igh transferrin concentrations.