A Legionella pneumophila gene that promotes hemin binding

Abstract

The ability to bind and utilize hemin is a trait common to many human pathogens. Nevertheless, the relationship between Legionella pneumophila, the agent of Legionnaires' disease, and hemin has received little attention. Thus, we explored the capacity of a virulent, serogroup 1 strain of L. pneumophila to bind hemin and use it as an iron source. Hemin, but not protoporphyrin IX, restored bacterial growth in iron-limiting media, indicating that it can serve as an iron source for L. pneumophila. In support of this idea, we observed that wildtype legionellae were able to bind 50 to 60% of added hemin, a binding capacity that was comparable to those of other pathogens. To begin to identify proteins involved in hemin acquisition, we identified a Legionella locus that conferred hemin binding upon Escherichia coli. Subcloning and nucleotide sequence analysis determined that a single open reading frame, which was designated hbp for hemin-binding promotion, was responsible for this binding activity. The hbp gene was predicted to encode a secreted, 15.5-kDa protein. To ascertain the importance of this gene in L. pneumophila biology, we used allelic exchange to construct an hbp mutant. Importantly, the mutant displayed a 42% reduction in hemin binding, confirming that hbp potentiates hemin acquisition by L. pneumophila. However, the strain was unaltered in its ability to grow within macrophage-like cells and freshwater amoebae, indicating that hbp is not required for intracellular infection. Despite this, Southern hybridization analysis and database searches demonstrated that hbp is nearly exclusive to the L. pneumophila species.