Lipoprotein e(P4) is essential for hemin uptake by Haemophilus influenzae

Heme uptake is a common means of iron and porphyrin acquisition by many pathogenic bacteria. The genus Haemophilus includes several important pathogenic bacterial species that characteristically require hemin-, protoporphyrin-, or heme-substituted proteins as essential growth factors under aerobic conditions. However, the mechanism of heme transport is not understood for Haemophilus. We have cloned a DNA fragment from H. influenzae that allows an Escherichia coli hemA mutant to employ exogenous hemin or protoporphyrin IX as sole sources of porphyrin. DNA sequencing of the cloned DNA fragment suggested that a previously characterized gene (hel) encoding an antigenic, outer membrane lipoprotein e(P4) was responsible for the complementation activity. Construction of hel insertion mutations in strain H. influenzae Rd demonstrated that hel is essential for growth under aerobic conditions but not under anaerobic conditions. The aerobic growth defect of hel mutants could be reversed by providing exogenous hemin in the presence of outer membrane perturbants suggesting that these hel mutants are defective in transport of hemin through the outer membrane. The analysis of hybrids between e(P4) and beta-lactamase demonstrated that a domain of e(P4) near its NH2' terminus was required for its function in hemin use. Within this domain is a short amino acid sequence that displays similarity to H. influenzae hemin binding protein HbpA, hemin-binding motifs present in eukaryotic transcription activator heme-activated protein, and the heme containing proteins hemoglobin (alpha-chain) and cytochrome C3, suggesting that this region may be involved in hemin binding and/or transport.