Relative contribution of P5 and Hap outer membrane proteins to nontypable Haemophilus influenzae host colonisation and pathogenesis

Abstract

Nontypable Haemophilus influenzae (NTHi) is a commensal of the human nasopharynx and also an opportunistic pathogen, being a major cause of respiratory infections. Evidence indicates that NTHi is highly adapted to the human airways, and endowed with genes present in all isolates analysed to date encoding surface proteins likely to be involved in this host-pathogen interface. However, NTHi interplay with upper and lower airways cells, and the specific and relative contribution of its outer membrane proteins to colonization and/or virulence properties of this pathogen have not been comprehensively addressed. In this study, we systematically compared NTHi infection of nasal, nasopharyngeal, bronchial and alveolar epithelial cells, which revealed significant differences in NTHi ability to adhere to- and invade this range of cell types. Moreover, 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 NTHi375. By using well established in vitro cultured cells and in vivo infection model systems, we assessed P5 and Hap relative contribution to NTHi biofilm formation, colonization of nasal and nasopharynx epithelium, infection of bronchial and alveolar epithelium, interplay with alveolar macrophages, and murine pulmonary infection. Although ompP5 and hap do not contribute to bacterial biofilm growth, P5 plays a prominent role in NTHi interplay with nasal cells, type II pneumocytes, and alveolar macrophages, and also in NTHi mouse lung acute infection. Mechanistically, P5-host interplay does not seem to be mediated by P5 binding to the host cell receptors CEACAM-1 or α5 integrin. Conversely, Hap involvement in NTHi375 interplay with the above mentioned cell types was shown to be limited, despite its ability to promote bacterial cell adhesion when expressed in the heterologous strain H. influenzae Rd KW20. 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.