KC_whole_thesis_ex_pub_mat.pdf (14.83 MB)
Exploring molecular mechanisms underlying the role of non-typeable Haemophilus influenzae in COPD
thesisposted on 2023-05-28, 12:26 authored by Rajendra KCRajendra KC
Chronic obstructive pulmonary disease (COPD) is a severe and progressive condition characterised by persistent respiratory symptoms and airflow limitation. Around 300 million people in the world have COPD. It has emerged as the third leading cause of mortality, claiming 3.2 million lives worldwide in 2017. An acute exacerbation of COPD, a sudden worsening of respiratory symptoms, is a major cause of morbidity and mortality in COPD patients. Various factors, including biomass smoke exposure and infection with bacteria trigger COPD exacerbations. Nearly half of the world's population uses biomass fuel for cooking and heating and is therefore at risk of exposure to noxious particles released from the combustion of biomass fuel. Among respiratory bacteria, nontypeable Haemophilus influenzae (NTHi) is a key pathogen implicated in colonisation and damage of airways in COPD patients. In this thesis, I assessed the impact of biomass smoke exposure on inflammation and adherence of NTHi to human bronchial epithelial cells. Due to the lack of a standard and easily accessible procedure for the preparation of biomass smoke, I first devised a simple, cost-effective, and reproducible method for the generation of biomass smoke extracts, in particular, cow dung and wood smoke extracts. Using this method, I generated quantifiable batches of biomass smoke extracts that were utilised for the assessment of cellular responses to different types of biomass smoke. I investigated the effect of biomass smoke extracts on human airway epithelial cells with respect to expression of a known receptor of NTHi, platelet-activating factor receptor (PAFR), and the pro-inflammatory cytokines interleukin 6 (IL-6) and IL-8, using quantitative polymerase chain reaction. In addition, I examined the response of bronchial epithelial cells to adherence of NTHi using immunofluorescence microscopy. I observed an increased inflammatory response in cells exposed to biomass smoke, characterised by induction of significant levels of IL-6 and IL-8 mRNA, in comparison to mock exposed cells. I demonstrated a dose-dependent increase in NTHi adhesion to epithelial cells following exposure to biomass smoke extracts. I further established an association between PAFR expression and the adhesion of NTHi in biomass smoke-exposed cells. Pre-treatment with a known PAFR antagonist, WEB-2086 inhibited biomass smoke-induced adherence of NTHi in airway cells in a dose dependent manner. In addition, pre-treatment of biomass smoke-exposed airway epithelial cells with a novel WEB-2086 analogue, C17 reduced NTHi adhesion in a dose-dependent manner. I next assessed the genomes of 568 NTHi isolates, including 40 newly sequenced clinical isolates collected from patients with different diseases, including COPD. Phylogenetic analysis based on polymorphic sites on the core genome did not provide sufficient resolution to separate COPD strains from other clinical phenotypes, suggesting a similar set of core genes are present in all clinical NTHi isolates. I applied discriminant analysis based on the presence or absence profiles of accessory genes and found a clear distinction between COPD and other disease strains. I then applied a pan genome-wide association study approach to identify the accessory genes associated with COPD. I identified a set of accessory genes that regulate metabolic functions, such as the metabolism of organic acids and oxidation-reduction reactions that regulate cellular respiration to be significantly associated with COPD strains. This result suggests that NTHi associated with COPD may exhibit genetically encoded functional variances to isolates collected from other clinical illnesses. In conclusion, this work advances our understanding of how biomass smoke could contribute to the development and progression of COPD and highlights the potential of PAFR as a therapeutic target for reducing the impact of hazardous biomass smoke exposure on respiratory health. Further, this thesis increases our understanding of gene sets shared by NTHi strains that survive and cause disease in the COPD lung.
Rights statementCopyright 2020 the author Chapter 1 appears to be the equivalent of a pre-print version of an article published as: KC, R., Shukla, S. D., Gautam, S. S., Hansbro, P. M., O'Toole, R. F., 2018. The role of environmental exposure to non-cigarette smoke in lung disease, Clinical and translational medicine, 7(1), e39. The article is included in the thesis appendices. Copyright the author(s) 2018. The article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made Another published article related to chapter 1 is: Kc R., Shukla, S. D., Walters, E. H., O'Toole, R. F. 2017. Temporal upregulation of host surface receptors provides a window of opportunity for bacterial adhesion and disease, Microbiology, 163(4), 421-30. The published article has removed from the appendices for copyright reasons Chapter 2 appears to be the equivalent of a pre-print version of an article published as: KC, R., Zosky, G. R., Shukla, S. D., O'Toole, R. F., 2018. A cost-effective technique for generating preservable biomass smoke extract and measuring its effect on cell receptor expression in human bronchial epithelial cells, Biology methods and protocols, 3(1). Copyright the author(s) 2018. Published by Oxford University Press. The article is is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. The published article is located in the appendices Chapter 4 appears to be the equivalent of a pre-print version of a published article. Material from: KC, R., Hyland, I. K., Smith, J. A., Shukla, S. D., Hansbro, P. M., Zosky, G. R., Karupiah, G., O'Toole, R. F., Cow dung biomass smoke exposure increases adherence of respiratory pathogen nontypeable Haemophilus influenzae to human bronchial epithelial cells, Exposure and health, 12, 893-895, published 2020. Reused by permission from Springer Nature Copyright 2020. The final published version is included in the appendices The author states that \A number of next-generation sequencing protocols described in Chapter 5 have been published in the Journal of biological methods\". The article is: Gautam S. S. KC R. Leong K. W. C. Aog‚àö¬8in M. M. O'Toole R. F. 2019. A step-by-step beginner's protocol for whole genome sequencing of human bacterial pathogens Journal of biological methods 6(1) e110. Copyright 2019 the authors. The article is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (https://creativecommons.org/licenses/by-nc-sa/4.0/) that allows others to share the work and/or adapt it with an acknowledgement of the work's authorship and initial publication in the journal. The published article is located in the appendices The author also states that \"a draft genome sequence of a representative isolate of nontypeable Haemophilus influenzae collected and sequenced for this work (described in Chapter 5) has been published in the Microbiology resource announcements.\" The article is: KC R. Leong K. W. C. McEwan B. Lachowicz J. Harkness N. M. Petrovski S. Karupiah G. O'Toole R. F. 2020. Draft genome sequence of an isolate of nontypeable Haemophilus influenzae from an acute exacerbation of chronic obstructive pulmonary disease in Tasmania Microbiology resource announcements 9(19) e00375-20. Copyright Copyright 2020 KC et al. It is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/) that allows others to share the work and/or adapt it with an acknowledgement of the work's authorship and initial publication in the journal. The published article is located in the appendices"