Relative Contribution of P5 and Hap Surface Proteins to Nontypable Haemophilus influenzae Interplay with the Host Upper and Lower Airways

Abstract

Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.

Genome Expression Profiling-Based Identification and Administration Efficacy of Host-Directed Antimicrobial Drugs against Respiratory Infection by Nontypeable Haemophilus influenzae

“…A549 cells were maintained, seeded, and infected for 2 h, as described previously (10,34). Cells were washed three times with PBS and incubated for 6 h in RPMI 1640 medium containing 10% FCS, 10 mM HEPES, and gentamicin (100 g/ml).…”

“…A CD1 mouse model of NTHi respiratory infection was used as described previously (7,10,34,35). NTHi375 was used for lung infection, and mice were randomly divided into the following six groups (n ϭ 5): (i) control A (intraperitoneally [i.p.]…”

“…Rolipram treatment was performed at a dose of 10 mg/kg of body weight in 0.1 ml of PBS-ethanol (2:1) and administered by oroesophageal gavage (Popper & Sons, Inc.) (38). NTHi intranasal infection was performed, and the lungs were processed as previously described (7,10,34,35) at 12 or 24 hpi. Each lung homogenate was serially 10-fold diluted in PBS, and 100 l of each dilution was spread in triplicate on sBHI-agar (detection limit, Ͻ10 CFU/lung).…”

“…Our current understanding of the molecular mechanisms involved in NTHi infection remains limited, but identical strains have been repeatedly isolated from the lungs of COPD patients in serial clinic visits, suggesting that NTHi presents features that promote chronic infection (7,8). NTHi is a facultative intracellular patho-gen (9)(10)(11)(12)(13)(14)(15)(16), and epithelial invasion may allow bacterial cells to temporarily evade the immune system and/or therapeutic interventions. Indeed, a correlation between the occurrence of intracellular NTHi and persistent infection has been proposed (17,18).…”

Evidence of the presence of nucleic acids and β-glucan in the matrix of non-typeable Haemophilus influenzae in vitro biofilms

“…Several proteins are present among the EPS of in vitro NT Hi biofilms, namely, the adhesins Hap and HMW1/HMW2, and the IgA1 protease36. The requirement for the surface protein Hap in biofilm formation has, however, recently been questioned37. In an independent study involving liquid chromatography coupled with tandem mass spectrometry, more than 200 proteins were identified in the extracellular matrix of NT Hi biofilms formed on Millipore filters38.…”