Structural determinants of the interaction between the Haemophilus influenzae Hap autotransporter and fibronectin

Abstract

Haemophilus influenzae is a Gram-negative cocco-bacillus that initiates infection by colonizing the upper respiratory tract. Hap is an H. influenzae serine protease autotransporter protein that mediates adherence, invasion and microcolony formation in assays with human epithelial cells and is presumed to facilitate the process of colonization. Additionally, Hap mediates adherence to fibronectin, laminin and collagen IV, extracellular matrix (ECM) proteins that are present in the respiratory tract and are probably important targets for H. influenzae colonization. The region of Hap responsible for adherence to ECM proteins has been localized to the C-terminal 511 aa of the Hap passenger domain (HapS). In this study, we characterized the structural determinants of the interaction between HapS and fibronectin. Using defined fibronectin fragments, we established that Hap interacts with the fibronectin repeat fragment called FNIII(1-2). Using site-directed mutagenesis, we found a series of motifs in the C-terminal region of HapS that contribute to the interaction with fibronectin. Most of these motifs are located on the F1 and F3 faces of the HapS structure, suggesting that the F1 and F3 faces may be responsible for the HapS-fibronectin interaction.

Fig. 1.

Hap_S binds to FNIII_(1–2). (a) Schematic representation of the domain structure and interactions of fibronectin. (b) Far-Western dot blot assay showing binding of purified Hap_S to immobilized purified full-length fibronectin or fibronectin fragments. FN full and FN 45 are commercial full-length and 45 kDa gelatin-binding domain fragments from Sigma. (c) Far-Western dot blot assay showing binding of purified Hap_S to immobilized purified full-length fibronectin or fibronectin fragments.

Fig. 2.

The Hap ECM binding domain is responsible for bacterial binding to FNIII_(1–2). ELISA showing the binding of H. influenzae strain DB117 expressing either plasmid alone (pLS88), HapΔ26-525 or HapΔ26-725 to full-length fibronectin (black bars) or the FNIII_(1–2) fragment (white bars). Values represent the mean of three independent experiments, and error bars represent the sd.

Fig. 3.

Residues mutated in this study. (a) Hap crystal structure (Meng et al., 2011) highlighting the predicted fibronectin-binding domains mutated in this study. (b) Top-down view of the Hap prism structure. The crystal structure of Hap_S is shown in green. A triangle diagram (black) is used to highlight the prism-like surface of Hap_S with F1, F2 and F3 faces. The EIV (blue), TED (black), EN (cyan), ETD (purple), GG597 (yellow) and GG661 (red) motifs are shown as stick representations. The majority of the targeted residues exist in the F1 and F3 faces.

Fig. 4.

Gram-positive bacterial FnBP motifs contribute to Hap-fibronectin binding. (a) Amino acid sequence of Hap residues 525–725. The putative acidic fibronectin-binding residues are highlighted in grey, and glycine residues that follow are underlined. (b) ELISA showing the binding of DB117 strains expressing Hap with mutations in the putative acidic fibronectin binding pockets to full-length fibronectin. OM samples of DB117 derivatives were examined by Western analysis. (c) ELISA showing binding of DB117 derivatives expressing Hap with the combined acidic motif mutations in putative fibronectin-binding residues to full-length fibronectin. OM samples of DB117 derivatives were examined by Western analysis. (d) ELISA showing binding of DB117 derivatives expressing the Hap glycine mutants to full-length fibronectin. OM samples of DB117 derivatives were examined by Western analysis. Values represent the mean of three independent experiments, and bars represent the sd. *P≤0.05 using the unpaired t-test compared with results for HapΔ25–525.

Fig. 5.

Disruption of Hap–Hap interactions does not decrease Hap-mediated binding to fibronectin. ELISA showing binding of DB117 derivatives expressing Hap with deletions in the β-loops required for Hap–Hap interactions. Δ751–789 has a deletion of two β-loops and still mediates bacterial aggregation, while Δ751–827 has a deletion of four β-loops and does not mediate bacterial aggregation. All values represent the mean of three independent experiments, and bars represent the sd. ns, P>0.05 using the unpaired t-test compared with results for HapS243A.