Architecture and Adhesive Activity of the Haemophilus influenzae Hsf Adhesin

Abstract

Haemophilus influenzae type b is an important cause of meningitis and other serious invasive diseases and initiates infection by colonizing the upper respiratory tract. Among the major adhesins in H. influenzae type b is a nonpilus protein called Hsf, a large protein that forms fiber-like structures on the bacterial surface and shares significant sequence similarity with the nontypeable H. influenzae Hia autotransporter. In the present study, we characterized the structure and adhesive activity of Hsf. Analysis of the predicted amino acid sequence of Hsf revealed three regions with high-level homology to the HiaBD1 and HiaBD2 binding domains in Hia. Based on examination of glutathione S-transferase fusion proteins corresponding to these regions, two of the three had adhesive activity and one was nonadhesive in assays with cultured epithelial cells. Structural modeling demonstrated that only the two regions with adhesive activity harbored an acidic binding pocket like the binding pocket identified in the crystal structure of HiaBD1. Consistent with these results, disruption of the acidic binding pockets in the adhesive regions eliminated adhesive activity. These studies advance our understanding of the architecture of Hsf and the family of trimeric autotransporters and provide insight into the structural determinants of H. influenzae type b adherence.

Extraction and Purification of Haemophilus influenzae Type b Lipooligosac‌charide by Modified Phenol Method

“…Although the capsular polysaccharide is the key virulence determinant, this pathogen also expresses an O-deacylated lipooligosaccha-ride (LOS) consisting of a mainly conserved triheptose backbone attached to a core region, covalently linked to lipid A [4][5][6][7][8]. Lipid A is a natural ligand for the MD2-Toll-like receptor 4 (TRL4) complex with a pivotal role in producing inflammatory responses [8,9].…”

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

“…Previously, on the basis of interactions with Chang conjunctival epithelial cells, three binding domains (BD2 529-652 , BD3 1206-1337and BD1 1896-2022 ) have been characterized in the Hsf molecule (Cotter et al, 2005b). All binding domains consist of an N-terminal Neck domain and a C-terminal Trp-ring domain "N-Neck : Trp ring-" (Fig.…”

“…S1). The binding domain numbering of Hsf was assigned on the basis of the homology with Hia (Cotter et al, 2005b). Following the previous nomenclature, we named the adjacent PDs with a similar numbering (Fig.…”

“…Among several known bacterial trimeric autotransporters, Hsf is unique for its structural appearance (Cotter et al, 2005b). It is not a trimeric straight fiber like protein, rather a folded and twisted molecule at the bacterial surface.…”

“…In contrast, Haemophilus surface fibril (Hsf) is present in all typeable strains (St Geme et al, 1996;Rodriguez et al, 2003;Watson et al, 2013). Notwithstanding homologous, Hia and Hsf are relatively different sizewise; Hia has a size of ≈114 kDa (≈342 kDa as a trimer), whereas Hsf is almost twice in size with a monomer of approximately 243 kDa that builds up a ≈750 kDa trimer (Cotter et al, 2005b). These two TAAs are highly homologous at their N-and C-termini with an overall 81% similarity and 72% identity.…”