During these early phases, we have been focused on initial Tasks defining experimental models and generate seminal data.
Obj1. Systematic identification of defensive LD proteins, micropeptides and lipids. Why are there 317 proteins upregulated by danger signals on/near LDs? Are LD hubs selected by innate immunity to rapidly integrate a variety of defensive responses (Research Question (RQ) 1.1)? What proteins and lipids are key for the defence (RQ 1.2)? Are LD-associated smORFs defensive eukaryotic molecules (RQ 1.3)? Obj1 aims to determine the immune role(s) of LDs through the identification of defensive LD proteins/lipids (Task 1a, Task 1b, Task 1c, Task 1d).
Obj2. Functional analysis of LD-phagolysosome interactions in model cell/animal systems. Our light and 3D electron microscopy provided evidence of the existence of sensing, trafficking and docking mechanisms that facilitate the engagement of antibacterial LD proteins with pathogens. This analysis revealed that in contact sites the LD monolayer produces an apparent discontinuity in the phagolysosomal membrane enclosing bacterial. We also demonstrated that professional intramacrophage pathogens, such as Salmonella, avoid interaction with LDs to escape from this antimicrobial mechanism. Likewise, the scarce published data suggest that only a minor proportion of Mtb-containing phagosomes makes contacts with LDs in foamy macrophages. These observations raise numerous crucial questions. How do LDs specifically associate with the membrane enclosing the pathogen (RQ 2.1)? Does this defence involve formation of a pore across the phagolysosomal membrane and interaction of LD proteins with bacteria? What specific protein-protein, protein-lipid and lipid-lipid complexes are implicated in the process (RQ 2.2)? Does the cytoskeleton play a role (RQ 2.3)? And importantly, how do some bacteria escape this surveillance (RQ 2.4)? (Task 2a, Task 2b, Task 2c, Task 2d, Task 2e, Task 2f)
Obj3. Identification of mechanisms of bacterial resistance to the LD defence – the Mtb model. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, offers the best example of coevolution with humans leading to a highly successful pathogen that today infects about a quarter of the world’s population being the leading cause of death from a single infectious agent. The exceptionally high ability of Mtb to survive intracellularly in macrophages and dependence on host cell lipids make it an ideal candidate to dissect LD-mediated antibacterial defences and exploitation by intracellular pathogens. It has been recently described that the LDs of Mtb-infected macrophages participate in the production of defensive cytokines and eicosanoids. Moreover, our pilot studies have revealed that proteins recruited to LDs display potent toxicity to mycobacteria. Yet, lipid-loaded macrophages failed to better contain the growth of Mtb, supporting the view that Mtb has evolved mechanisms of resistance to LD-mediated innate immune responses. Therefore, many important questions have yet to be answered. Is Mtb avoiding the LD-mediated defence characterised in Obj1 and 2 (RQ 3.1)? What are the Mtb virulence factors produced to manipulate LDs and host lipids (RQ 3.2)? Is Mtb controlling host metabolism by hijacking LDs (RQ 3.3)? Here, we will make use of the entire team and the techniques already developed in previous objectives to study LD metabolism and cell biology during Mtb infection. These studies will not only provide the first description of Mtb’s impact on LD immunobiology but produce unprecedented morphological and mechanistic insights into LD interactions with intracellular Mtb. Together, the inclusion of several examples of bacterial pathogens in studies of Obj1 and 2, and our in-depth study of host LD-Mtb interactions in Obj3, will undoubtedly reveal novel immune and virulence strategies (Task 3a, Task 3b, Task 3.c,Task 3d).
