Periodic Reporting for period 1 - DRIMMS (Lipid droplets as innate immunity hubs)
Okres sprawozdawczy: 2023-01-01 do 2024-06-30
Successful defence against invaders is critical for survival of eukaryotic cells. Microbes have developed many strategies to subvert host organisms which, in turn, evolved several innate immune responses to counterattack. As major lipid storage organelles of eukaryotes, lipid droplets (LDs) are an attractive source of nutrients for invaders. Pathogens induce and physically interact with LDs and the current view is that they ‘hijack’ LDs to draw on substrates for host colonisation. We challenged this dogma by demonstrating that LDs are endowed with a regulated protein-mediated antibiotic activity. We introduced the new concept that dependence on host nutrients is an ‘Achilles’ heel’ of intracellular pathogens and LDs an innate immunity chokepoint harnessed to organise a front-line defence.
We have formed a multidisciplinary group combining complementary knowledge and transdisciplinary expertise to investigate the hypothesis that LDs are innate immunity hubs sensing infection and directly confronting invaders. We will characterise how LDs efficiently coordinate and precisely execute a plethora of immune responses such as killing, signalling and inflammation. Proteomics and lipidomics will identify defensive players. 3D electron tomography and confocal microscopy will be combined with proximity labelling strategies to get unprecedented understanding of host LD-pathogen dynamics. The successful pathogen Mycobacterium tuberculosis will be used to test the medical significance of our findings and unravel bacterial mechanisms of resistance to LD-mediated defences.
Characterisation of these novel in nate immune systems will be paradigm-shifting in immunology, physiology and cell biology. In the age of antimicrobial resistance and viral pandemics, unravelling how eukaryotic LDs fight and defeat dangerous microorganisms will inspire new anti-infective therapies.
We have formed a multidisciplinary group combining complementary knowledge and transdisciplinary expertise to investigate the hypothesis that LDs are innate immunity hubs sensing infection and directly confronting invaders. We will characterise how LDs efficiently coordinate and precisely execute a plethora of immune responses such as killing, signalling and inflammation. Proteomics and lipidomics will identify defensive players. 3D electron tomography and confocal microscopy will be combined with proximity labelling strategies to get unprecedented understanding of host LD-pathogen dynamics. The successful pathogen Mycobacterium tuberculosis will be used to test the medical significance of our findings and unravel bacterial mechanisms of resistance to LD-mediated defences.
Characterisation of these novel in nate immune systems will be paradigm-shifting in immunology, physiology and cell biology. In the age of antimicrobial resistance and viral pandemics, unravelling how eukaryotic LDs fight and defeat dangerous microorganisms will inspire new anti-infective therapies.
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).
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).
DRIMMS has the potential to change our fundamental understanding of innate immunity to open a brand-new field in the priority study area of infectious disease research (with a critical social impact). We will develop unique quantitative methods and combinations of light and cryoelectron microscopy to provide an unprecedented level of understanding of this process at the tissue, organelle, individual protein level and submolecular resolution. We will span length scales to produce unique details of how LD sense, interact and kill intracellular bacteria. The iterative information’s flow combined with our rapid screening methods and high-throughput proximity labelling assays will allow to identify novel proteins, micropeptides and lipids with crucial roles in defence and determine signalling pathways and protein platforms triggering and organising this previously neglected cellular defences. Furthermore, we will extend all these findings and resources to tackle a highly relevant medical problem. We will make use of the entire team and apply, for the first time to study tuberculosis, techniques already developed to study LD metabolism and cell biology. We will provide novel understanding of Mtb’s virulence and hopefully extend our conclusions to other highly pathogenic bacteria. DRIMMS has been designed to generate the first physical/functional map of protein networks and cellular systems functioning on LDs as first responders of eukaryotic innate immunity and thus, with the potential of changing the textbooks.