Nontypable Haemophilus influenzae (NTHi) is a commensal of the human nasopharynx and also a major cause of respiratory infections.
Evidence suggests a highly specialised human host-specific adaptation for NTHi. Bacterial surface proteins are likely to play important roles in this host-pathogen interplay. However, their specific and relative contribution to colonization and/or virulence properties of NTHi has not been addressed comprehensively. We focused on the outer membrane proteins P5 and Hap, and carried out a systematic phenotypic study on isogenic single and double mutants of the genes ompP5 and/or hap generated on the pathogenic strain NTHi 375. We assessed P5 and Hap relative contribution to NTHi biofilm formation, colonization of human nasal and nasopharynx, infection of human lower airways epithelium, interplay with alveolar macrophages, and murine pulmonary infection. We show that ompP5 and hap do not contribute to bacterial biofilm growth. P5 plays a prominent role in NTHi interplay with RPMI2650 nasal cells, A549 type II pneumocytes, and MH-S alveolar macrophages, and also in mouse lung acute infection. The use of HeLa cells stably transfected with CEACAM-1 suggests that P5 involvement in host-pathogen interplay may not relate in its binding to CEACAM-1. Conversely, Hap involvement in NTHi375 interplay with the above mentioned cell types may be limited. Indeed, DompP5Dhap showed comparable phenotypes to those obtained for DompP5. Although ompP5 disruption reduced significantly hap expression, this observation could not be related to the observed DompP5-driven phenotypes. Mechanistically, the use of a specific blocking anti-a5 integrin antibody and the synthetic RGD peptide excluded a role of both P5 and Hap as bacterial ligands for a5 integrin. Altogether, this work provides a context for the relative importance of Hap and P5 outer membrane proteins to a range of phenotypic traits, providing a greater understanding of their contribution to NTHi interplay with the host.