M. tuberculosis infection of human iPSC-derived macrophages reveals complex membrane dynamics during xenophagy evasion.

Elliott M Bernard,Pierre Santucci,Claudio Bussi,Antony Fearns,Lucy M Collinson,Maximiliano G Gutierrez,Rachel J Lai,Christopher J Peddie

doi:10.1242/jcs.252973

Elliott M Bernard, Pierre Santucci + Show 6 more

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https://doi.org/10.1242/jcs.252973

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Abstract

ABSTRACTXenophagy is an important cellular defence mechanism against cytosol-invading pathogens, such as Mycobacterium tuberculosis (Mtb). Activation of xenophagy in macrophages targets Mtb to autophagosomes; however, how Mtb is targeted to autophagosomes in human macrophages at a high spatial and temporal resolution is unknown. Here, we use human induced pluripotent stem cell-derived macrophages (iPSDMs) to study the human macrophage response to Mtb infection and the role of the ESX-1 type VII secretion system. Using RNA-seq, we identify ESX-1-dependent transcriptional responses in iPSDMs after infection with Mtb. This analysis revealed differential inflammatory responses and dysregulated pathways such as eukaryotic initiation factor 2 (eIF2) signalling and protein ubiquitylation. Moreover, live-cell imaging revealed that Mtb infection in human macrophages induces dynamic ESX-1-dependent, LC3B-positive tubulovesicular autophagosomes (LC3-TVS). Through a correlative live-cell and focused ion beam scanning electron microscopy (FIB SEM) approach, we show that upon phagosomal rupture, Mtb induces the formation of LC3-TVS, from which the bacterium is able to escape to reside in the cytosol. Thus, iPSDMs represent a valuable model for studying spatiotemporal dynamics of human macrophage–Mtb interactions, and Mtb is able to evade capture by autophagic compartments.

Highlights

Macrophages are arguably the most important cell type during infection with M. tuberculosis (Mtb), one of the most successful bacterial pathogens globally
We further investigated the spatiotemporal dynamics of autophagic targeting of Mtb and revealed a remarkable ESX-1 mediated, phagosome membrane damage driven relocalisation of LC3B to tubulovesicular structures (LC3-TVS) in the vicinity of the bacilli and the ability of Mtb to escape from LC3B positive tubulovesicular autophagosomes (LC3-TVS) into the cytosol
In order to better understand the role of xenophagy of Mtb in human macrophages, we established an embryonic body (EB)-based protocol for induced pluripotent stem cell derived macrophages (iPSDM) differentiation where haematopoiesis is induced under serum-free conditions

Summary

Introduction

Macrophages are arguably the most important cell type during infection with M. tuberculosis (Mtb), one of the most successful bacterial pathogens globally. Mtb initially resides within phagosomes before damaging the phagosomal membrane to access the cytosol. This is achieved through the co-action of the Type VII secretion system ESX-1, encoded within the virulence-related RD1 region, and the presence of cell wall-associated PDIM lipids (Augenstreich et al, 2017; Barczak et al, 2017; Lerner et al, 2017; Lerner et al, 2018; Quigley et al, 2017; van der Wel et al, 2007). Cytosolic access is important for selective recognition of Mtb by the autophagy machinery and induction of immune responses driven by Type I interferon (IFN) (Watson et al, 2015)

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