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Physical Extraction and Fast Protein Liquid Chromatography for Purifying Flagella Filament from Uropathogenic Escherichia Coli for Immune Assay

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Physical Extraction and Fast Protein Liquid Chromatography for Purifying Flagella Filament from Uropathogenic Escherichia Coli for Immune Assay Bacterial Flagella as Triggers of the Innate Immune System and IL-10 Production During Acute Urinary Tract Infection Author Acharya, Dhruba Published 2020 Thesis Type Thesis (PhD Doctorate) School School of Medical Science DOI https://doi.org/10.25904/1912/3786 Copyright Statement The author owns the copyright in this thesis, unless stated otherwise. Downloaded from http://hdl.handle.net/10072/391068 Griffith Research Online https://research-repository.griffith.edu.au Bacterial Flagella as Triggers of the Innate Immune System and IL-10 Production During Acute Urinary Tract Infection Dhruba Acharya BSc. MLT, MSc. (Immunology) School of Medical Sciences Griffith University Submitted in fulfilment of the requirements of degree of Doctor of Philosophy June 2019 Abstract Urinary Tract Infections (UTIs) are a huge public health problem affecting 150 million people each year worldwide. Expenditures aimed at the management of UTI account for approximately $3.5 billion USD in medical costs annually. More than 80% of all UTIs are caused by uropathogenic Escherichia coli (UPEC). Studies have shown key roles for virulence factors of UPEC, such as flagella, autotransporters, capsule, fimbriae, toxins, lipopolysaccharide (LPS), and siderophores, in UTI disease pathogenesis. UPEC flagella contribute to the pathogenesis of UTI in several ways, such as through flagella-mediated motility, colonization, adhesion, and biofilm formation. The flagella filament is synthesized as a polymerized product of >20,000 protein monomers termed flagellin or FliC (encoded by the gene fliC). The contribution of flagella to innate immune stimulation is only partially defined, for example gaps exist in our understanding of how flagella engage host responses in the bladder during acute UTI. Extracellular FliC is recognized by Toll-Like Receptor 5 (TLR5), whereas intracellular FliC is recognized by Naip5 and Ipaf. Through MyD88-dependent and independent pathways, FliC causes upregulation of various proinflammatory, regulatory cytokines, and growth factors. A detailed understanding of how FliC from UPEC engages innate immunity in the bladder during UTI is lacking. A major focus of this thesis was the regulatory cytokine, IL-10. IL-10 plays pleiotropic roles in defence against infection; depending on the illness and the causal pathogen. Frequently, IL-10 facilitates immune suppression to moderate inflammatory mechanisms that can damage the host. IL-10 is rapidly produced in the bladder in response to UPEC early during UTI. However, the mechanism of engagement of innate immunity by UPEC that leads to elicitation of IL-10 in the bladder is unknown. One facet in understanding the role of IL-10 in infectious disease is the elucidation of microbial products that elicit production of this key regulator of host innate immune responses. The overarching aim of this thesis was to define the role of flagella (particularly FliC) in immune modulation during acute UTI. A plethora of methods have been previously described for the extraction and purification of FliC. However, there is limited literature utilizing highly purified, non-denatured FliC, offering any insight into host-pathogen interactions. In this study, UPEC CFT073, and a derivative strain containing four mutations in genes encoding fimbriae and pili (CFT073Δ4) were used to generate a deletion in the flagellar filament, fliC. This enabled the concurrent preparation of a iii suitable carrier control to be applied in downstream assays. The flhDC gene, which regulates flagella biosynthesis, was used in an IPTG inducible plasmid (pMG600) to overexpress flagella. Extraction and purification of FliC to homogeneity was performed using a sequential extraction method based on mechanical shearing, ultracentrifugation, size exclusion chromatography, protein concentration and endotoxin removal. Protein purity and integrity was assessed using SDS-PAGE, western blots with anti-flagellin antisera, and native-PAGE. This study established a new, carefully optimized method to extract and purify FliC from the reference UPEC strain CFT073 to be suitable for immune assays. The biological activity of FliC was assessed by measuring cytokine responses in both mouse macrophages (J774A.1) and human monocytes(U937). Macrophage and monocyte responses to purified FliC included significant levels of several cytokines, including TNF-α, IL-6, IL12, INF-γ, consistent with prior literature reports. This thesis sought to identify the role of the major UPEC flagella filament protein, FliC, in triggering IL-10 synthesis in the bladder. Analysis of IL-10 induction in response to various UPEC CFT073 derivatives and purified FliC showed a role for FliC in the induction of IL-10 in both human and mouse cell cultures. The effect of FliC on other cytokines, chemokines, and growth factors indicated several pro-inflammatory, and cell recruitment/ migratory factors are also shown to be induced by host cells in response to this protein. This study also explored the FliC induced immune responses, including IL- 10 induction in the bladder in vivo. ELISA and multiplex assays revealed that FliC, when injected via transurethral catheterization, induced a rapid IL-10 response in the mouse bladder. Characterization of the genome-wide innate immunological context of FliC- induced cytokines in the bladder using RNA-sequencing also identified 1400-gene network of transcriptional and antibacterial defences, that were differently expressed compared to carrier control. Of the FliC-responsive bladder transcriptome, the changes in expression of il10 and other genes were dependent on TLR5, according to the -/- comparative analysis of TLR5-deficient mice (Tlr5 ). Exploration of the potential of FliC, and its associated innate immune signature in the bladder, revealed a significant benefit for the control of infection in the mice that received purified FliC, prophylactically or therapeutically after transurethral UPEC infection. Collectively, this thesis shows that detection of FliC through TLR5 triggers rapid IL-10 synthesis in the bladder, and FliC therefore represents a potential immune modulator for the treatment or prevention of UTI. iv Statement of Originality This work has not previously been submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself. Professional English language editing of this thesis was completed by a professional scientific editing company called Scribendi (www.scribendi.com). Thesis editing was done in accordance with the Griffith University HDR policy, ‘Guidelines for editing research thesis’. _____________ Dhruba Acharya v Acknowledgement of Assistance I am profoundly grateful to my primary supervisor, Professor Glen C. Ulett, for the continuous support during my PhD study. I appreciate his advice, motivation, enthusiasm, and guidance to develop and advance my research through different stages of this project. I would like to acknowledge my co-supervisor Dr. Matthew J. Sullivan for his constant guidance from bench to book. He has been a true mentor and a wonderful friend of mine throughout this degree. I would like to express my deepest gratitude to Dr. Harry Sakellaris, without whom, I would not have started my career in Australia. Many thanks to Professor Mark Schembri, (The University of Queensland) for his intellectual advice to shape and plan my experiments. I appreciate his insightful contribution in manuscript writing. To the fellow past and present members of my lab, Dr. Deepak S. Ipe, Dr. Kelvin Goh, Dean Gosling, Lahiru Katupitiya, Michelle Chamoun, and Darren Prince. They are all amazing colleagues and friends and I would like to thank them for creating an excellent environment around working stations. Dr. Prabin Gyawali; Heartfelt thanks to you brother for being there in my highs and lows, thick and thin, and pleasure and setbacks. I am deeply indebted to these beautiful souls: Dr. Narayan Gyawali and family, Nirajan Shrestha, Sabina Shrestha, Bimbishar Bhattarai, Shikha Karki, Indra Shrestha, Chandani Shrestha, Manasvi and Shriyans Shrestha. I would like to remember my father Late Shree Jhabindra Acharya. I take this opportunity to acknowledge my mother Saraswoti Devi Acharya and rest of “The Acharya’s”: Chandra Prakash Acharya, Bhawana Thapaliya Acharya, Emil and Milee Acharya, Narendra Acharya, Archana Pokharel Acharya, Nevan and Niyan Acharya. “My soulmate Kabita Kuikel Acharya, this would not have been possible without your support, love and care. You are the best thing that has ever happened in my life and our shared DNA, Adhvik Acharya, the greatest gift of all”. Finally, I would like to thank the Government of Australia and Griffith University for the scholarship, support and the people of Australia for giving us a home away from home. vi Table of Contents Abstract ...................................................................................................................... iii Statement of Originality ............................................................................................. v Acknowledgement of Assistance ................................................................................ vi Table of Contents ...................................................................................................... vii List of Figures ...........................................................................................................
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