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

Begoña Euba,Igor Ruiz De Los Mozos,Jaione Valle,Javier Moleres,Junkal Garmendia,José Antonio Bengoechea,Cristina Viadas

doi:10.1371/journal.pone.0123154

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.

Highlights

Nontypable Haemophilus influenzae (NTHi) is a Gram negative coccobacillus that is a common commensal in the nasopharynx of both children and adults, and an important cause of localized respiratory tract infections such as acute otitis media, otitis media with effusion, community-acquired pneumonia, and exacerbations of chronic bronchitis and chronic obstructive pulmonary disease (COPD) [1]
We could not detect expression of the ompP5 gene in NTHi375ΔompP5 and NTHi375ΔhapΔompP5 strains, neither expression of the hap gene in NTHi375Δhap and NTHi375ΔhapΔompP5 strains. These mutants did not exhibit growth defects compared to the wild-type strain when OD600 was monitored over time during growth in sBHI liquid culture (Fig 1B)
Our systematic use of microfermenters, respiratory cultured cells and mouse respiratory infection model systems has allowed for the first time to comprehensively compare single and double P5 and Hap defective mutants generated in a pathogenic genome sequenced strain, NTHi strain 375 (NTHi375) [37]

Summary

Introduction

Nontypable (non-capsulated) Haemophilus influenzae (NTHi) is a Gram negative coccobacillus that is a common commensal in the nasopharynx of both children and adults, and an important cause of localized respiratory tract infections such as acute otitis media, otitis media with effusion, community-acquired pneumonia, and exacerbations of chronic bronchitis and chronic obstructive pulmonary disease (COPD) [1]. P5 has been shown to be an adhesin to human oropharyngeal cells [7], mucin [8], chinchilla eustachian tube mucus [9], and respiratory syncytial virus infected type II pneumocytes [10]. P5 may be important for optimal NTHi growth in rich medium [12], and it may be a bacterial ligand for the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) [14], playing a role in nasopharynx colonisation in the chinchilla model [15,16]. NTHi stimulates the expression of intercellular adhesion protein 1 (ICAM-1) on respiratory epithelial cells [17], and P5 has been shown to be a ligand for ICAM-1 [18]

Methods

Results

Conclusion