Biology subjects

Bernard, E. M.

Publications and source records attributed to Bernard, E. M..

5 recordsLinked to original sources

bioRxiv2025

Sterile- or pathogen-induced endolysosomal damage activate the NLRP6 inflammasome in human intestinal epithelial cells

NLRP6 controls host defense against bacteria and viruses in the gastrointestinal tract by a poorly understood mechanism. Here, we report that NLRP6 forms an inflammasome upon endolysosomal damage caused by sterile triggers or bacterial pathogens such as Listeria monocytogenes in human intestinal epithelial cells (IECs). NLRP6 activation required Listeriolysin O-dependent cytosolic invasion of L. monocytogenes and drove IEC pyroptosis via ASC/Caspase-1-mediated GSDMD cleavage. NLRP6 inflammasome formation was independent of bacterial PAMPs, such as lipoteichoic acid or dsRNA, which were previously reported to activate NLRP6. L. monocytogenes mutants deficient in cell-to-cell spread or escape from secondary vacuoles induced less cell death, linking bacteria-induced endolysosomal damage to NLRP6 activation. Finally, sterile endolysosomal damage recapitulated pathogen-induced NLRP6 activation and drove IEC pyroptosis. In summary, our study reveals that NLRP6 allows IECs to detect endolysosomal damage, thereby not only making it responsive to pathogens but more generally to wide-ranging sources of pathological endolysosomal damage.

immunology↗

bioRxiv2024

Ninjurin-1 mediated plasma membrane rupture is a two-step process requiring cell swelling

Ninjurin-1 (NINJ1) executes cell lysis by polymerizing into filaments, yet how these form membrane lesions is unknown. Integrating cell-based assays with molecular dynamics simulations, we demonstrate that NINJ1-driven membrane rupture starts with the lateral association of NINJ1 dimers into double filaments. Subsequently, swelling of necrotic cells increases plasma membrane tension, leading to separation of the filament interface and NINJ1 lesion opening. Inhibiting cell swelling prevents this increase in tension, blocking NINJ1-mediated plasma membrane rupture without disrupting NINJ1 oligomerization, implicating membrane tension as key for opening NINJ1 lesions. The NINJ1 homolog NINJ2 oligomerizes but fails to form membrane lesions during cell death due to the greater stability of its dimer. Finally, using atomic force microscopy of NINJ1-containing proteoliposomes, we show that NINJ1 forms large pores by stabilizing membrane edges. In summary, our findings support a model whereby membrane tension drives a zipper-like opening of NINJ1 double filaments, initiating necrotic cell lysis.

immunology↗

bioRxiv2022

Lysosomal damage drives mitochondrial proteome remodelling and reprograms macrophage immunometabolism

Transient lysosomal damage after infection with cytosolic pathogens or silica crystals uptake results in protease leakage. Whether limited leakage of lysosomal contents into the cytosol affects the function of cytoplasmic organelles is unknown. Here, we show that sterile and non-sterile lysosomal damage triggers a cell death independent proteolytic remodelling of the mitochondrial proteome in macrophages. Mitochondrial metabolic reprogramming required lysosomal leakage of Cathepsin B and Cathepsin L and was independent of proteasome degradation and mitophagy. In a mouse model of endomembrane damage, metabolic analysis confirmed that in vivo, live lung macrophages that internalised crystals displayed impaired mitochondrial function and increased glycolytic and lipid metabolism. Single-cell RNA-sequencing analysis of bronchoalveolar lavage revealed that lysosomal damage skewed metabolic and immune responses primarily in CD36+/LIPA+ and Krt79+/Car4+ subsets of alveolar macrophages. Importantly, modulation of macrophage metabolism with 2-Deoxy- d- glucose and oxamate impacted the host response to Mycobacterium tuberculosis (Mtb) infection in an endomembrane damage dependent manner. This work uncovers a new inter-organelle communication pathway, providing a general mechanism by which macrophages undergo mitochondrial metabolic reprograming after endomembrane damage.

cell biology↗

bioRxiv2021

Visualizing pyrazinamide action by live single cell imaging of phagosome acidification and Mycobacterium tuberculosis pH homeostasis

The intracellular population of Mycobacterium tuberculosis (Mtb) is dynamically segregated within multiple subcellular niches with different biochemical and biophysical properties that, upon treatment, may impact antibiotic distribution, accumulation, and efficacy. However, it remains unclear whether fluctuating intracellular microenvironments alter mycobacterial homeostasis and contribute to antibiotic enrichment and efficacy. Here, we describe a dual-imaging approach that allows quantitative monitoring of host subcellular acidification and Mtb intrabacterial pH profiles by live-fluorescence microscopy in a biosafety level 3 laboratory. By combining this live imaging approach with pharmacological and genetic perturbations, we show that Mtb can maintain its intracellular pH independently of the surrounding pH in primary human macrophages. Importantly, we show that unlike bedaquiline (BDQ), isoniazid (INH) or rifampicin (RIF), the front-line drug pyrazinamide (PZA) displays antibacterial efficacy by acting as protonophore which disrupts intrabacterial pH homeostasis in cellulo. By using Mtb mutants with different intra-macrophage localisation, we confirmed that intracellular acidification is a prerequisite for PZA efficacy in cellulo. We anticipate this dual imaging approach will be useful to identify host cellular environments that affect antibiotic efficacy against intracellular pathogens. HighlightsO_LIMtb maintains its intrabacterial pH inside both acidic and neutral subcellular microenvironments of human macrophages C_LIO_LIPyrazinamide, but not other frontline antibiotics, acts as a protonophore in cellulo C_LIO_LIPyrazinamide-mediated intrabacterial pH homeostasis disruption and antibacterial efficacy requires host endolysosomal acidification C_LIO_LICytosolic localisation mediated by ESX-1 contributes to pyrazinamide antibacterial activity resistance C_LIO_LIPyrazinamide conversion into pyrazinoic acid by the pyrazinamidase/nicotinamidase PncA is essential for its protonophore activity and efficacy in cellulo C_LI

microbiology↗

bioRxiv2019

The antibiotic bedaquiline activates host macrophage innate immune resistance to bacterial infection

Antibiotics are widely used in the treatment of bacterial infections. Although known for their microbicidal activity, antibiotics may also interfere with the hosts immune system. Here we analyzed the effects of bedaquiline (BDQ), an inhibitor of the mycobacterial ATP synthase, on human macrophages. Genome-wide gene expression analysis revealed that BDQ reprogramed macrophages into potent bactericidal phagocytes. We found that 1,495 genes were differentially expressed in M. tuberculosis-infected macrophages incubated with the drug, with an over-representation of genes involved in metabolism, lysosome biogenesis and activation. BDQ treatment triggered a variety of antimicrobial defense mechanisms, including nitric oxide production, phagosome-lysosome fusion, and autophagy. These effects were associated with activation of transcription factor EB (TFEB), involved in the transcription of lysosomal genes, resulting in enhanced intracellular killing of different bacterial species that were naturally insensitive to BDQ. Thus, BDQ could be used as a host-directed therapy against a wide range of bacterial infections.

immunology↗