The Haemophilus influenzae Hap Autotransporter Binds to Fibronectin, Laminin, and Collagen IV

Abstract

Nontypeable Haemophilus influenzae (NTHI) initiates infection by colonizing the upper respiratory tract mucosa. NTHI disease frequently occurs in the context of respiratory tract inflammation, where organisms encounter damaged epithelium and exposed basement membrane. In this study, we examined interactions between the H. influenzae Hap adhesin and selected extracellular matrix proteins. Hap is an autotransporter protein that undergoes autoproteolytic cleavage, with release of the adhesive passenger domain, Hap s , from the bacterial cell surface. We found that Hap promotes bacterial adherence to purified fibronectin, laminin, and collagen IV and that Hap-mediated adherence is enhanced by inhibition of autoproteolysis. Adherence is inhibited by pretreatment of bacteria with a polyclonal antiserum recognizing Hap s . Purified Hap s binds with high affinity to fibronectin, laminin, and collagen IV but not to collagen II. Binding of Hap s to fibronectin involves interaction with the 45-kDa gelatin-binding domain but not the 30-kDa heparin-binding domain of fibronectin. Taken together, these observations suggest that interactions between Hap and extracellular matrix proteins may play an important role in NTHI colonization of the respiratory tract.Extracellular matrix (ECM) consists of a diverse group of proteins that form the scaffolding responsible for the development, growth, and maintenance of mammalian tissues. In epithelial tissues, certain ECM proteins function together with cells to form barriers intended to prevent penetration of these tissues by microorganisms. Both pathogenic and commensal bacteria have evolved mechanisms designed to subvert epithelial barriers (3,5,14,23,28). These mechanisms include bacterial surface proteins called adhesins that bind to ECM and bacterial proteases that degrade ECM components and permit migration of bacteria to deeper tissue spaces where they may gain easier access to nutrients and safe harbor from the host immune response.Haemophilus influenzae is a gram-negative bacterium that is often found as a commensal inhabitant of the respiratory tract in healthy adults but also represents a common cause of both localized respiratory tract and invasive systemic disease (25). In studies examining interactions between H. influenzae clinical isolates and human respiratory tract tissue, bacteria were often associated with damaged epithelium and exposed ECM (16,17). Furthermore, examination of bronchial biopsies from patients with persistent H. influenzae bronchitis revealed organisms in the subepithelial compartment, suggesting that this pathogen is capable of penetrating the basement membrane (11). Additional analysis of interactions between clinical isolates and purified ECM components demonstrated that many H. influenzae strains were capable of binding to fibronectin, laminin, and various collagens (2, 27). In recent work, Virkola and coworkers found that H. influenzae hemagglutinating pili mediated attachment to both fibronectin and heparin-binding growth-associated molecule, alt...

Haemophilus influenzae Surface Fibrils Contribute to Serum Resistance by Interacting with Vitronectin

“…H. influenzae pili, which hemagglutinate human erythrocytes and adhere to human oropharyngeal epithelial cells (10 -16), exhibit adherence to fibronectin and heparin-binding ECM proteins. The nonpilus adhesin Haemophilus adhesion and penetration protein was reported as a binder of fibronectin, laminin, and collagen IV (8).…”

“…Consequently, the bacteria reach the basement membrane and the extracellular matrix (ECM), and may penetrate into deeper tissue layers and consequently into the circulation. Studies on the interaction of H. influenzae and tissue samples from the human respiratory tract show that H. influenzae has been associated with disrupted epithelial cells and exposed ECM proteins such as fibronectin, collagen, vitronectin, and laminin (7)(8)(9).…”

Chromosomal Expression of the Haemophilus influenzae Hap Autotransporter Allows Fine-Tuned Regulation of Adhesive Potential via Inhibition of Intermolecular Autoproteolysis

“…In previous work, we established that the H. influenzae Hap autotransporter promotes adherence to cultured respiratory epithelial cells and selected extracellular matrix proteins and facilitates bacterial aggregation and microcolony formation (5,7,16). Hap adhesive activity is mediated by the surface-associated Hap S passenger domain, which also has protease activity and directs autoproteolytic cleavage of itself from the membrane-associated Hap ␤ translocator domain (6).…”

Temporal upregulation of host surface receptors provides a window of opportunity for bacterial adhesion and disease

“…Adherence of NTHi to fibronectin, laminin and type IV collagen is mediated by an autotransporter, Haemophilus adhesion and penetration protein (Hap) [37]. Recently, NTHi lipoprotein P4 demonstrated effective binding to nasopharyngeal, type II alveolar, and bronchial epithelial cells via fibronectin [7].…”