Autophagy-Related Proteins Target Ubiquitin-Free Mycobacterial Compartment to Promote Killing in Macrophages.

Aïcha Bah,Isabelle Vergne,Camille Lacarrière

doi:10.3389/fcimb.2016.00053

Abstract

Autophagy is a lysosomal degradative process that plays essential functions in innate immunity, particularly, in the clearance of intracellular bacteria such as Mycobacterium tuberculosis. The molecular mechanisms involved in autophagy activation and targeting of mycobacteria, in innate immune responses of macrophages, are only partially characterized. Autophagy targets pathogenic M. tuberculosis via a cytosolic DNA recognition- and an ubiquitin-dependent pathway. In this report, we show that non-pathogenic M. smegmatis induces a robust autophagic response in THP-1 macrophages with an up regulation of several autophagy-related genes. Autophagy activation relies in part on recognition of mycobacteria by Toll-like receptor 2 (TLR2). Notably, LC3 targeting of M. smegmatis does not rely on membrane damage, ubiquitination, or autophagy receptor recruitment. Lastly, M. smegmatis promotes recruitment of several autophagy proteins, which are required for mycobacterial killing. In conclusion, our study uncovered an alternative autophagic pathway triggered by mycobacteria which involves cell surface recognition but not bacterial ubiquitination.

Highlights

Macroautophagy, hereafter referred to as autophagy, is a eukaryotic lysosomal degradative process involved in removal and recycling of cytoplasmic components
Autophagic response to non-pathogenic mycobacteria, such as M. smegmatis, has been studied in murine RAW264.7 macrophages (Zullo and Lee, 2012). To determine whether this response is conserved in human cells, we investigated autophagy in another cellular model commonly used to study mycobacteriamacrophage interaction, THP-1 macrophages (Riendeau and Kornfeld, 2003)
Addition of Bafilomycin A1 (BafA1) increases both LC3-II/actin ratios in non-infected and M. smegmatis-infected cells, with LC3-II/actin ratio remaining two times higher in infected cells (Figures 1A,B). These results indicate that basal and functional autophagy is present in THP1 macrophages and that M. smegmatis triggers an upregulation of this process upon infection

Summary

Introduction

Macroautophagy, hereafter referred to as autophagy, is a eukaryotic lysosomal degradative process involved in removal and recycling of cytoplasmic components. The process is initiated by activation of Ulk1/Atg13/FIP200/Atg101 and Beclin-1/hVps34/Atg complexes which result in the formation of an isolation membrane. This membrane elongates to engulf cytoplasmic cargo and fuses with itself to form a double-membrane bound organelle called autophagosome. At this stage, two ubiquitin-like conjugation systems are required: (i) the covalent linkage of Atg with Atg in complex with Atg16L1; (ii) LC3 lipidation with phosphatidylethanolamine. Once the autophagosome formed, Atg5-12/Atg16L1 protein complex is released while LC3 remains on the autophagosomal inner membrane.

Methods

Results

Conclusion