Session: Session 1

A PROTEOMIC AND FUNCTIONAL VIEW OF THE BIOGENESIS OF INTRABACTERIAL LIPID INCLUSIONS IN MYCOBACTERIA

Tonia DARGHAM^2,3, Luc CAMOIN¹, Wassim DAHER^5,6, Jean-François CAVALIER², Laurent KREMER^5,6, Stéphane CANAAN², John-Jairo AGUILERA-CORREA⁶, Romain AVELLAN², Ivy MALLICK², Léa CELIK², Geal BRASSEUR⁴, Isabelle PONCIN², Vanessa POINT², Stéphane AUDEBERT¹

¹Marseille Protéomique - CRCM - IPC - INSERM, Marseille, France
²Aix-Marseille Univ., CNRS, LISM UMR 7255, IMM FR3479, IM2B, Marseille, France
³IHU Méditerranée Infection, Aix-Marseille Univ., Marseille, France
⁴Aix-Marseille Univ., CNRS, LCB UMR 7283, IMM FR3479, IM2B, Marseille, France
⁵INSERM, IRIM, Montpellier, France
⁶Centre National de la Recherche Scientifique UMR 9004, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, Montpellier, France

Introduction: During granuloma formation, extracellular and phagocytised mycobacteria accumulate triacylglycerol (TAG) in the form of intracytoplasmic lipid inclusions (ILIs). These organelles enable bacilli to escape the host immune system and serve as a carbon source for bacterial persistence, particularly in foamy macrophages. Despite the importance of these organelles in the life cycle of mycobacteria, few studies have been carried out to determine the proteins involved in ILI formation.

Objectives: In this context, identifying the proteins associated with ILIs during their formation could lead to the discovery of new therapeutic targets for manipulating the growth and persistence of pathogenic mycobacteria.

Methods: Using APEX2, a proximity labelling technique combined with proteomics, we uncovered the ILI proxisome during the mycobacterial life cycle using an in vitro model that mimics ILI accumulation in M. abscessus, as a model strain.

Results: A total of 228 candidate proteins were detected after 24-48h of ILI accumulation. We showed that this was a dynamic process involving protein exchanges depending on ILI size. We identified 123 proteins at 24 and 143 proteins at 48h, of which 38 are present at both times. 10 potential targets were selected. Their location on the ILI surface at different time points has been validated and their physiological role in ILI formation has been determined. Based on a 50% TAG reduction, MAB_3486 and MAB_4532c have been found as potential actors in TAG accumulation during ILI biogenesis.

Conclusions: Our data clearly suggest that the ILI metabolism is a highly dynamic and evolutive complex process involving several families of proteins, including acetyltransferases, acyl-CoA dehydrogenase, enoyl-CoA hydratases, and some membrane proteins like LprG and PKS. Overall, this study represents a first step in deciphering the ILI metabolism in pathogenic mycobacteria and opens new perspectives to control mycobacterial-related diseases.