A HEMOGLOBIN-BINDING OUTER-MEMBRANE PROTEIN IS INVOLVED IN VIRULENCE EXPRESSION BY HAEMOPHILUS-DUCREYI IN AN ANIMAL-MODEL

Haemophilus ducreyi exhibits a requirement for exogenously supplied he me for aerobic growth in vitro, Nine of ten wild-type isolates of H. d ucreyi were shown to contain a readily detectable hemoglobin-binding a ctivity. Spontaneous hemoglobin-binding-negative mutants of two of the se wild-type isolates lost the ability to express an outer membrane pr otein with an apparent molecular mass of approximately 100 kDa, Simila rly, the single wild-type isolate that lacked the ability to bind hemo globin also appeared to lack expression of this same 100-kDa protein, A monoclonal antibody (5A9) to this 100-kDa protein was used to identi fy a recombinant clone which possessed an H. ducreyi chromosomal fragm ent containing the gene encoding the 100-kDa protein; this protein was designated hemoglobin utilization protein A (HupA). Nucleotide sequen ce analysis of the hupA gene revealed that the predicted protein, with a calculated molecular mass of 108 kDa, was similar to TonB-dependent outer membrane proteins of other bacteria, Increasing the concentrati on of heme in the growth medium resulted in decreased expression of th e HupA protein. Mutant analysis was used to prove that the HupA protei n was essential for the utilization by H. ducreyi of both hemoglobin a nd hemoglobin-haptoglobin as sources of heme in vitro. In addition, it was found that an isogenic hupA mutant was less virulent than the wil d-type parent strain in the temperature-dependent rabbit model for der mal lesion production by H. ducreyi.