The role of non-typeable haemophilus influenzae biofilms in host-microbial and microbial-microbial interactions in chronic obstructive pulmonary disease

Abstract

Non-typeable Haemophilus influenzae is a commensal-turned-pathogen that has been implicated in several COPD patient cohorts and is associated with acute exacerbations and increased hospitalisation. It is currently unclear why NTHi represents such a challenging pathogen to treat, causing recurrent and chronic infections. Recently, there is growing evidence for the existence of NTHi in the biofilm lifestyle, conferring increased persistence, immune system and antimicrobial evasion and complex inter-specific interactions with other lung pathogens. Herein, I have developed a small airway epithelium NTHi coculture model system in order to investigate the host pathogen interactions that occur in the COPD small airways, as in improvement to current model systems which lack physiological relevance. I first demonstrated the detection of biofilm-like aggregations of NTHi by fluorescent in-situ hybridisation (FISH) in COPD lung biopsies and sputum expectorates. I adopted a bioinformatic approach to identify and shortlist 18 biofilm genes of interest (GOIs) in novel NTHi strains isolated from the COPD and healthy lung, including ST14, ST408 and ST253. These included genes functioning in quorum sensing, adhesion, surface modifications, immune system effectors, production of EPS matrix components and antimicrobial tolerance. I began to model NTHi biofilm growth and composition in a standard submerged model and progressed onto coculture NTHi with hSABCi-NS1.1 small airway epithelial (SAE) cells cultured at an air-liquid-interface (ALI), before applying these optimised methods to a primary donor small airways model of COPD. Using a range of fluorescent staining, SCLM and SEM techniques I categorised NTHi as an eDNA- and protein-rich EPS matrix biofilm-producing bacteria which aggregates at the epithelial surface; a mode of intracellular and paracellular invasion. RT-qPCR has shown these biofilms elicit sophisticated NTHi-strain dependent gene expression responses as well as host-dependent gene expression responses, and in particular the adhesins Hia, OMP P5, PilA as well as biofilm EPS matrix gene comE and antibiotic resistance gene β-lactamase are upregulated by NTHi in response to coculture with the small airway, highlighting targets for future therapeutic intervention. This small airway model was validated through a series of cytokine and viability assays which demonstrated that both models could launch a robust proinflammatory response against NTHi biofilms whilst maintaining barrier impermeability and low cytotoxicity. In addition, the model was successful in generating NTHi-derived outer membrane vesicles, which have emerged as an important vector of cell-cell communication, immune system modulation and potential transmission of biofilm-associated gene expression and coordination. Overall, I propose supporting evidence for NTHi as a biofilm in the COPD small airway based on these model systems. Thus, NTHi biofilms may explain the prevalence of chronic, recurrent infection.

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Identifiers

ORCID for Jake Weeks: orcid.org/0000-0002-1593-0169

ORCID for Cosma Mirella Spalluto: orcid.org/0000-0001-7273-0844

ORCID for Karl Staples: orcid.org/0000-0003-3844-6457

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