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Regulation of phagosome maturation and bacterial killing by the TPL-2 complex

Breyer, Felix; (2020) Regulation of phagosome maturation and bacterial killing by the TPL-2 complex. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

A fundamental role of the innate immune response involves the killing of phagocytosed pathogens, such as bacteria, by macrophages. During phagocytosis, bacteria are internalised into membrane-bound vacuoles called phagosomes. The nascent phagosome undergoes a complex maturation process, which involves sequential membrane fusion and fission events with the endosomal compartment and ultimately with lysosomes to form a phagolysosome. The mature phagolysosome is an acidic, hydrolytic and highly oxidative organelle, which efficiently degrades internalised bacteria. Toll-like receptor (TLR) activation of mitogen-activated protein (MAP) kinases in macrophages is mediated by tumour progression locus 2 (TPL-2), a MAP 3 kinase that is critical for inflammatory immune responses to bacteria, viruses, and fungi. In unstimulated macrophages, TPL-2 forms a ternary complex with NF-kB1 p105 and A20-binding inhibitor of NF-kB-2 (ABIN-2). TLR-induced activation of the IkB kinase (IKK) complex leads to p105 phosphorylation and its proteasomal degradation. This liberates TPL-2 to activate MAP kinase signalling, which results in gene expression of numerous inflammatory mediators. ABIN-2 is also released, however, its physiological function in innate immune responses has remained unclear. I discovered that the TPL-2 complex promotes bead phagosome maturation in macrophages. Genetic inactivation of TPL-2 catalytic activity or ABIN-2 ubiquitin binding substantially altered the composition of the phagosome proteome in primary mouse macrophages. Further, I found that TPL-2 catalytic activity induced phagosome proteolytic activity and phagosome acidification, while ABIN-2 ubiquitin binding promoted phagosome proteolysis without affecting phagosome acidification. My genetic and pharmacological experiments indicated that TPL-2 regulates phagosome function independently of its known ability to activate MAP kinases. I demonstrated that induction of phagosome maturation was mediated by TPL-2-dependent regulation of V-ATPase function via serine 1903 phosphorylation of DMXL1, a V-ATPase-interacting regulatory protein. Importantly, I showed that TPL-2 catalytic activity also induced phagosome maturation independently of MAP kinase signalling in primary human macrophages, establishing the clinical relevance of my findings. Furthermore, my results revealed that ubiquitin binding to ABIN-2 promotes phagosome proteolytic activity independently of TPL-2 regulation of MAP kinase signalling. Consistent with these findings analysing bead phagosomes, I discovered that TPL-2 catalytic activity and ABIN-2 ubiquitin binding are required for efficient killing of internalised Staphylococcus aureus by macrophages. Genetic inactivation of TPL-2 catalytic activity or ABIN-2 ubiquitin binding impaired maturation of S. aureus phagosomes. I also found that TPL-2 catalytic activity was required for optimal killing of phagocytosed Citrobacter rodentium by inducing phagosomal acidification. Moreover, I demonstrated that ABIN-2 ubiquitin binding was essential for efficient killing of Salmonella typhimurium. Together, these discoveries demonstrated that both TPL-2 catalytic activity and ABIN-2 ubiquitin binding are important for the killing of several bacterial species, Gram-negative and Gram-positive as well as extracellular and intracellular microbes. In conclusion, my research identified novel signalling pathways that promote phagosome maturation and pathogenic bacterial killing by macrophages. This work increases our understanding of a critical process in innate immune responses and may lead to development of novel therapeutic approaches for acute bacterial infections.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Regulation of phagosome maturation and bacterial killing by the TPL-2 complex
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2020. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
URI: https://discovery.ucl.ac.uk/id/eprint/10117819
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