Mapping of Binding Domains of Nontypeable Haemophilus influenzae HMW1 and HMW2 Adhesins

Nontypeable Haemophilus influenzae is a major cause of localized respiratory tract disease, including otitis media, sinusitis, conjunctivitis, bronchitis, and pneumonia (33). The initial step in the pathogenesis of disease involves colonization of the nasopharynx (19). Colonization with a particular strain may persist for weeks to months, usually without producing symptoms (25, 32). However, in the setting of viral respiratory infection or allergic disease, the organism will often spread contiguously to the middle ear, the sinuses, the conjunctiva, or the lungs. Successful colonization requires that the organism overcome the mucociliary escalator, and adherence to respiratory epithelium plays a fundamental role in achieving this goal. Among diverse strains of nontypeable H. influenzae, both pilus and nonpilus adhesins have been defined. Approximately 75% of clinical isolates produce proteins that are immunologically and functionally related to the HMW1 and HMW2 adhesins produced by H. influenzae strain 12 (3). HMW1 and HMW2 are nonpilus proteins that were originally identified as the predominant targets of the host immune response during acute otitis media (2). Adherence assays with strain 12 mutants deficient in expression of HMW1 or HMW2 or both suggested that these proteins have adhesive properties, and examination of Escherichia coli transformants expressing either HMW1 or HMW2 confirmed this conclusion (30). Overall, HMW1 and HMW2 share 71% identity and 80% similarity (3). Interestingly, despite their sequence similarity, these proteins exhibit different cellular binding specifities (10, 29, 30). For example, HMW1 promotes high-level binding to Chang conjunctival cells and HaCaT keratinocytes, while HMW2 promotes low-level or undetectable binding to Chang cells but high-level binding to HaCaT cells. This observation suggests that the two proteins may recognize different receptors on the host cell surface and thus may contain different binding domains. Recent experiments demonstrated that HMW1-mediated binding to Chang cells involves interaction with a glycoprotein receptor containing α2,3-linked sialic acid (27). This binding is inhibited by the lectin Maackia amurensis agglutinin (MAA), which specifically recognizes sialic acid in an α2,3 configuration. Information regarding the host cell receptor with which HMW2 interacts is presently lacking. HMW1 and HMW2 are encoded by separate chromosomal loci, hmw1 and hmw2, respectively, each consisting of three genes, including hmwA, which codes for the structural protein (HMW1 or HMW2), followed by hmwB and hmwC (4). The hmw1B and hmw2B genes encode the HMW1B and HMW2B proteins, which are 99% identical, while the hmw1C and hmw2C genes encode the HMW1C and HMW2C proteins, which share 97% identity. In order to facilitate interaction with host cells, HMW1 and HMW2 are localized on the surface of the organism in a process that involves cleavage of a 441-amino-acid N-terminal fragment (3). In recent work, we demonstrated that the HMWB and HMWC proteins are required for normal processing and secretion of the structural proteins and established that HMW1B-HMW1C and HMW2B-HMW2C are functionally interchangeable (29). The HMW1 preprotein contains 1,536 amino acids, while the HMW2 preprotein consists of 1,477 amino acids. Comparison of the amino acid sequences reveals ∼99% identity over the first 441 residues and nearly 80% identity over the C-terminal 306 residues (Fig. ​(Fig.1).1). The middle portions of the proteins share much less homology and are marked by a 62-amino-acid segment in HMW1 that is absent from HMW2 (Fig. ​(Fig.1).1). Of note, with both HMW1 and HMW2, the cleaved N-terminal 441-amino-acid fragment displays all of the predicted homology to related bacterial proteins, including Bordetella pertussis filamentous hemaglutinin (FHA), Serratia marcescens ShlA (20), Proteus mirabilis HpmA (34), and Vibrio cholerae OmpU (24), among others. Studies of HMW1, FHA, and ShlA indicate that the region of homology plays a critical role in secretion (8, 12, 23, 29). FIG. 1 Schematic representation of the HMW1 and HMW2 proteins, the HMW1-HMW2 chimeric proteins, and the HMW1 and HMW2 MBP fusion proteins. (A) Alignment of the predicted amino acid sequences of HMW1 and HMW2, with a summary of levels of identity and similarity ... To begin to explore the biological significance of the differing binding specificities of HMW1 and HMW2, in the present study we sought to define the regions of these proteins involved in interaction with the host cell surface. Generation of a series of chimeric proteins and examination of E. coli DH5α producing the resulting proteins suggested that an N-terminal domain of mature HMW1 is responsible for HMW1-mediated adherence and that the corresponding region of HMW2 directs HMW2-mediated adherence. Experiments with purified maltose-binding protein (MBP) fusion proteins confirmed these results, which are relevant to vaccine development and provide insight into the evolution of H. influenzae.