The role of phagocytosis-dependent activation of TFEB in the clearance of salmonella

Erika Ospina Escobar

doi:10.32920/ryerson.14655972

Abstract

During phagocytosis, macrophages engulf and sequester pathogens into phagosomes. Phagosomes then fuse with acidic and degradative lysosomes to degrade the internalized pathogen. We previously demonstrated that phagocytosis of IgG-opsonized particles and non-opsonized E.coli causes activation of the Transcription Factor EB (TFEB), which enhances the expression of lysosomal genes, increases the degradative capacity of lysosomes and boosts bactericidal activity. However, pathogens like Salmonella typhimurium have evolved mechanisms to evade and/or alter phagosome maturation to promote their own survival. We investigated: i) whether pathogens like Salmonella can alter TFEB activation and ii) whether phagocytosis-dependent activation of TFEB can counteract the pathogenicity of microorganisms. Here, we show that non-viable (heat-killed) S. typhimurium, pathogenic (EHEC and UPEC) and non-pathogenic E.coli (DH5α) all caused TFEB nuclear translocation in RAW macrophages, while strikingly live S. typhimurium maintained TFEB in the cytosol in the first hours post-infection. By contrast, Salmonella mutants for ΔsifA, ΔsopD2, ΔphoP all triggered TFEB activation in the first hour of infection. However, Salmonella infection eventually triggered a steady increase in nuclear TFEB after 4 h of infection, suggesting a more complex interplay between TFEB and Salmonella infection. We dissected the importance of TFEB activation towards Salmonella survivability by pre-activating TFEB before infection within WT macrophages and macrophages with a CRISPR-based deletion of TFEB. Our work suggests that Salmonella actively interferes with TFEB signaling in order to enhance its own survival. These results could provide insight into using TFEB as a target for the clearance of infections.

Highlights

1.1 Macrophages and the immune systemAll living organisms are prone to attacks by disease-causing agents present in the environment
In order to understand the importance of transcription factor EB (TFEB) in the killing of intracellular pathogens, we first assessed how TFEB responded to phagocytosis of non-pathogenic (DH5α) and pathogenic E.coli (UPEC and Escherichia coli (EHEC)), both of which are unable to prevent lysosomal degradation in murine macrophages (Horvath et al, 2011; Poirier et al, 2008)
Using the macrophage cell line RAW 264.7, which has been widely used in the study of phagocytosis and lysosomal degradation, we examined TFEB translocation in macrophages expressing TFEB-GFP after phagocytosis of nonopsonized live uropathogenic Escherichia coli (UPEC), EHEC and DH5α (D’Costa et al, 2015; Horvath et al, 2011; Kuhle & Hensel, 2004; Pastore et al, 2016; Wisner et al, 2012; Zhao et al, 2015)

Summary

Introduction

All living organisms are prone to attacks by disease-causing agents present in the environment To tackle this problem, humans have a complex group of cells and tissues that form the immune system and work together to protect us against dangerous and foreign bodies like antigens, pathogens, cancer cells and toxins(Janeway, 2001). If the physical barrier gets penetrated, the second line of defense is activated and consists of a group of cells that are recruited to the site of infection in what is called an immune response (Warrington, Watson, Kim, & Antonetti, 2011). Various types of cells are involved in the immune response including dendritic cells (DCs), neutrophils and macrophages (Warrington et al, 2011) These cells are called phagocytes due to their ability to engulf and kill microbes, remove dead cells, and debris. While neutrophils are the most abundant and the first cells to arrive to the site of infection, they are short-lived compared to macrophages and DCs (Warrington et al, 2011)

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