Structural Basis for the Differential Binding Affinities of the HsfBD1 and HsfBD2 Domains in the Haemophilus influenzae Hsf Adhesin

Haemophilus influenzae is a human-specific gram-negative coccobacillus that causes a variety of human infections ranging from localized respiratory infections to invasive diseases. Hsf is the major nonpilus adhesin in encapsulated strains of H. influenzae and belongs to the trimeric autotransporter family of proteins. The Hsf protein contains two highly homologous binding domains, designated HsfBD1 and HsfBD2. In this study we characterized the differential binding properties of HsfBD1 and HsfBD2. In assays using HeLa cells, we found that bacteria expressing either full-length Hsf or HsfBD1 by itself adhered at high levels, while bacteria expressing HsfBD2 by itself adhered at low levels. Immunofluorescence microscopy and a cellular enzyme-linked immunosorbent assay using purified proteins revealed that the binding affinity was significantly higher for HsfBD1 than for HsfBD2. Purified HsfBD1 was able to completely block adherence by bacteria expressing either HsfBD1 or HsfBD2, while purified HsfBD2 was able to block adherence by bacteria expressing HsfBD2 but had minimal activity against bacteria expressing HsfBD1. Conversion of the residue at position 1935 in the HsfBD1 binding pocket from Asp to Glu resulted in HsfBD2-like binding properties, and conversion of the residue at position 569 in the HsfBD2 binding pocket from Glu to Asp resulted in HsfBD1-like binding properties, as assessed by adherence assays with recombinant bacteria and by immunofluorescence microscopy with purified proteins. This work demonstrates the critical role of a single amino acid in the core of the binding pocket in determining the relative affinities of the HsfBD1 and HsfBD2 binding domains.Haemophilus influenzae is a gram-negative coccobacillus that causes both serious invasive diseases and localized respiratory tract infections in humans (10, 17, 19). Isolates of H. influenzae can be separated into encapsulated and nonencapsulated or so-called nontypeable strains (12). Most strains recovered from patients with invasive disease are encapsulated and express the type b capsule, while the majority of strains associated with respiratory tract infections are nontypeable (19).The pathogenesis of disease due to H. influenzae type b begins with colonization of the upper respiratory tract (4, 8, 11, 13, 16, 19). Most type b strains are capable of expressing hemagglutinating pili, which mediate bacterial attachment to oropharyngeal epithelial cells, extracellular matrix proteins, and mucin and promote colonization. Mutant strains that lack hemagglutinating pili are also capable of adherence and colonization, highlighting the fact that nonpilus adhesive factors also exist (4, 5, 8, 20). In recent work, we have demonstrated that the major nonpilus adhesin in H. influenzae type b is a large protein called Hsf, which forms short fibers visible by electron microscopy (15).The Hsf adhesin is encoded by the hsf locus and is a trimeric autotransporter protein that shares significant homology with Hia, a trimeric autotransporter adhesin that is present in ∼25% of nontypeable H. influenzae strains. Hsf contains an N-terminal signal sequence, an internal passenger domain with two binding domains, and a C-terminal outer membrane pore-forming domain, analogous to Hia (3, 6). The binding domains in Hsf are called HsfBD1 and HsfBD2 and share high-level homology with each other and with the two binding domains in Hia (2, 14). HsfBD1 and HsfBD2 interact with the same host cell receptor structure on Chang epithelial cells, although with different affinities (3). Based on in vitro experiments using purified proteins and Chang epithelial cells, HsfBD1 has a dissociation constant (K_d) of ∼0.2 nM and HsfBD2 has a K_d of ∼2.5 nM.In previous work using X-ray crystallography and site-directed mutagenesis, we established that both HiaBD1 and HiaBD2 are trimeric structures with acidic binding pockets formed by contiguous IsNeck and Trp-ring domains (9, 21). Using structural modeling and site-directed mutagenesis, we determined that HsfBD1 and HsfBD2 possess the same fold and trimeric assembly as HiaBD1 and HiaBD2, with conservation of the residues that are essential for HiaBD1 adhesive activity (3).In the current study we examined the structural basis for the different binding affinities of HsfBD1 and HsfBD2. In initial experiments, we found that the differences between HsfBD1 and HsfBD2 were easier to observe with HeLa cells than with Chang cells, reflecting the fact that the receptor density is lower on HeLa cells. Our results demonstrated the critical role of a single amino acid in the core of the binding pocket in determining the relative affinities of HsfBD1 and HsfBD2.