The Unique Structure of Haemophilus influenzae Protein E Reveals Multiple Binding Sites for Host Factors

Abstract

cHaemophilus influenzae protein E (PE) is a multifunctional adhesin involved in direct interactions with lung epithelial cells and host proteins, including plasminogen and the extracellular matrix proteins vitronectin and laminin. We recently crystallized PE and successfully collected X-ray diffraction data at 1.8 Å. Here, we solved the structure of a recombinant version of PE and analyzed different functional regions. It is a dimer in solution and in the asymmetric unit of the crystals. The dimer has a structure that resembles a flattened ␤-barrel. It is, however, not a true ␤-barrel, as there are differences in both the hydrogen-bonding pattern and the shape. Each monomer consisted of a 6-stranded antiparallel ␤-sheet with a rigid ␣-helix at the C terminus tethered to the concave side of the sheet by a disulfide bridge. The laminin/plasminogen binding region (residues 41 to 68) is exposed, while the vitronectin binding region (residues 84 to 108) is partially accessible in the dimer. The dimerized PE explains the simultaneous interaction with laminin and vitronectin. In addition, we found this unique adhesin to be present in many bacterial genera of the family Pasteurellaceae and also orthologues in other, unrelated species (Enterobacter cloacae and Listeria monocytogenes). Peptides corresponding to the surface-exposed regions PE 24 to 37, PE 74 to 89, and PE 134 to 156 were immunogenic in the mouse. Importantly, these peptide-based antibodies also recognized PE at the bacterial surface. Taken together, our detailed structure of PE explains how this important virulence factor of H. influenzae simultaneously interacts with host vitronectin, laminin, or plasminogen, promoting bacterial pathogenesis.

Haemophilus influenzae stores and distributes hemin by using Protein E

“…This is to our knowledge the first experimental evidence on distribution of hemin between H. influenzae and sheds light upon the mechanisms that are related to nutrient sharing. Surface representation of PE crystal structure Singh et al, 2013) with a hypothetical model of the PE-hemin complex. Insert: zoom in projection of a putative hemin-binding pocket in dimeric PE.…”

“…E. coli harboring pET26bhpe or pET16bhpe (Hallstrom et al, 2009;Singh et al, 2013) were grown in LB broth in the presence of 50 µg/ml kanamycin or 100 µg/ml ampicillin, respectively.…”

“…Protein E was recombinantly expressed in E. coli and purified from the soluble fraction or inclusion bodies by using a HisTrap Ni ++ NTA resin column (GE Healthcare Biosciences) as described previously Singh et al, 2013). Hemin (Sigma) was prepared as a 5 mM stock solution in 1 M NaOH, and thereafter diluted 200-fold in Tris-HCl, pH 7.8 containing 100 mM NaCl (TS buffer) and stored at -20 °C.…”

“…In addition, PE simultaneously binds to extracellular matrix proteins and plasminogen, interactions that all contribute to bacterial virulence (Barthel et al, 2012;Hallstrom et al, 2009;Hallstrom et al, 2011). We recently crystallized PE and showed that it is a dimer with multiple binding sites for proteins derived from the host (Hallstrom et al, 2011;Singh et al, 2012;Singh et al, 2013). In the present paper, we suggest that H. influenzae contains a PE-dependent storage pool of hemin, and this may result in a hemin supply to the H. influenzae population when there, for example, is a paucity of hemin in the environment.…”

Haemophilus influenzae surface fibril (Hsf) is a unique twisted hairpin-like trimeric autotransporter

“…Two boosters were administered after 5 and 6 weeks. Finally, blood was drawn from immunized rabbits and antibodies (Abs) from sera were purified by standard affinity purification protocol (Singh et al, 2013). BD and PD domains were aligned and modeled to identify the conserved surface exposed regions (Fig.…”