Oral Presentation 25th Annual Lorne Proteomics Symposium 2020

The role of ubiquitylation on the phagosome: a tale of inflammatory responses and vesicle trafficking (#13)

Tiaan Heunis 1 , Orsolya Bilkei-Gorzo 1 , Anetta Härtlova 1 , José Marin Rubio 1 , Julien Peltier 1 , Matthias Trost 1
  1. Newcastle University, Newcastle-upon-Tyne, NOT US OR CANADA, United Kingdom

Phagocytosis is an evolutionarily conserved key process required for innate immunity and homeostasis. During phagocytosis, particles are internalised into a de novo, membranous organelle, the phagosome, which fuses with early and late endosomes and, finally, lysosomes in a process called phagosome maturation. Recently, we have shown that proinflammatory activation of macrophages by Interferon-gamma (IFN-ɣ) greatly changed phagosome functions. As phagosomes are enriched in polyubiquitylation, which is further enhanced by IFN-ɣ, we wanted to explore the role of ubiquitylation on phagosome functions. We applied a targeted mass spectrometry approach by which we quantified ubiquitin chain linkage peptides from phagosome samples. All chain linkages apart from linear chains are present on phagosomes and IFN-ɣ activation enhanced K11, K48 and K63 chains significantly. In order to identify the molecular function of this polyubiquitylation, we characterized the ubiquitinome of phagosomes of IFN-γ activated macrophages and can demonstrate that ubiquitylation is preferentially attached to proteins involved in vesicle trafficking, thereby delaying fusion with late endosomes and lysosomes. We further show that phagosomal recruitment of the E3 ligase RNF115 is enhanced upon IFN-γ stimulation, which is responsible for most of the increase of K63 phagosomal polyubiquitylation. Loss of RNF115 also affected proinflammatory cytokine production and tissue damage during in vivo infection with S. aureus. Detailed understanding of phagosomal ubiquitylation could not only increase our understanding of vesicle trafficking but could also serve as possible targets for antibacterial host-directed therapies.