Biology subjects

Fearns, A.

Publications and source records attributed to Fearns, A..

7 recordsLinked to original sources

bioRxiv2026

ESX-1 remodels the Mycobacterium tuberculosis antigen storage compartment in macrophages

Mycobacterium tuberculosis (Mtb) secretes antigenic proteins that drive protective T cell responses, yet how Mtb intracellular localization affects the trafficking and spatiotemporal distribution of mycobacterial antigens in macrophages remains elusive. Here, we developed a split fluorescent protein reporter to monitor the dynamics of the TB vaccine lead antigen Ag85B in infected human macrophages. We discovered that Mtb triggers the formation and remodeling of two distinct types of Mtb Antigen Storage Compartment (MASC) that retains Ag85 within macrophage membranes. Using subcellular spatial proteomics, we profiled the composition of the MASC, identifying both the Mtb and macrophage proteins that constitute these compartments. Unexpectedly, MASCs are defined by metabolic compartmentalization and rewiring to lipid metabolism in both the macrophage and Mtb. Thus, Mtb builds via the ESX-1 system a metabolically distinct antigen storage compartment in immune cells, with potential implications for antigen selection and delivery in TB vaccine development.

cell biology↗

bioRxiv2025

Autologous human iPSC-derived Alveolus-on-Chip reveals early pathological events of M. tuberculosis infection

Immunocompetent and experimentally accessible alveolar systems to study human respiratory diseases are lacking. Here, we developed a single donor human induced pluripotent stem cell (iPSC)-derived Lung-on-Chip (iLoC) containing Type II and I alveolar epithelial cells, vascular endothelial cells, and macrophages in a microfluidic device that mimic lung 3D mechanical stretching and air-liquid interface. Imaging and scRNA-seq analysis revealed that the iLoC recapitulated cellular profiles present in the human distal lung. Infection of the iLoC with the human pathogen Mycobacterium tuberculosis (Mtb) showed that both macrophages and epithelial cells were infected and showed limited bacterial replication. Stochastically, large macrophage clusters containing necrotic core-like structure and Mtb replication were observed. A genetically engineered autophagy deficient iLoC revealed that after Mtb infection, macrophage necrosis was higher upon ATG14 deficiency without bacterial replication. Altogether, we report an autologous, genetically tractable human alveolar model to study lung diseases and therapies.

bioengineering↗

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↗

bioRxiv2020

Intracellular localisation of Mycobacterium tuberculosis affects antibiotic efficacy

To be effective, chemotherapy against tuberculosis (TB) must kill the intracellular population of Mycobacterium tuberculosis (Mtb). However, how host cell environments affect antibiotic accumulation and efficacy remains elusive. Pyrazinamide (PZA) is a key antibiotic against TB, yet its behaviour is not fully understood. Here, by using correlative light, electron, and ion microscopy to image PZA at the subcellular level, we investigated how human macrophage environments affect PZA activity. We discovered that PZA accumulates heterogeneously between individual bacteria in multiple host cell environments. Crucially, Mtb phagosomal localisation and acidification increase PZA accumulation and efficacy. By imaging two antibiotics commonly used in combined TB therapy, we showed that bedaquiline (BDQ) significantly enhances PZA accumulation by a host cell mediated mechanism. Thus, intracellular localisation and specific microenvironments affect PZA accumulation and efficacy; explaining the potent in vivo efficacy compared to its modest in vitro activity and the critical contribution to TB combination chemotherapy.

microbiology↗

bioRxiv2020

Correlative Light Electron Ion Microscopy reveal in vivo localisation of bedaquiline in Mycobacterium tuberculosis infected lungs

Correlative light, electron and ion microscopy (CLEIM) offers huge potential to track the intracellular fate of antibiotics, with organelle-level resolution. However, a correlative approach that enables subcellular antibiotic visualisation in pathogen-infected tissue is lacking. Here, we developed CLEIM in tissue (CLEIMiT), and used it to identify the cell-type specific accumulation of an antibiotic in lung lesions of mice infected with Mycobacterium tuberculosis. Using CLEIMiT, we found that the anti-TB drug bedaquiline is localised not only in foamy macrophages in the lungs during infection but also accumulate in polymorphonuclear (PMN) cells.

microbiology↗

bioRxiv2020

Macrophage-specific responses to human- and animal-adapted tubercle bacilli reveal pathogen and host factors driving multinucleated cell formation

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. However, the host and pathogen determinants driving host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed both blood-derived primary human and bovine macrophages (hM{phi} or bM{phi}, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hM{phi} whereas both Mbv and Mtb efficiently replicate in bM{phi}. Specifically, we show that out of the four infection combinations, only the infection of bM{phi} with Mbv promoted the formation of multinucleated cells (MNCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bM{phi} promote macrophage multi-nucleation. Importantly, we extend our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNCs. Our findings implicate MNC formation in the contrasting pathology between Mtb and Mbv for the bovine host, and identify MPB70 from Mbv and extracellular vesicles from bM{phi} as mediators of this process.

microbiology↗

bioRxiv2019

Mycobacterium tuberculosis cording in the cytosol of live lymphatic endothelial cells

The ability of Mycobacterium tuberculosis to form serpentine cords is intrinsically related to its virulence, but specifically how M. tuberculosis cording contributes to pathogenesis remains obscure. We show that several M. tuberculosis clinical isolates form intracellular cords in primary human lymphatic endothelial cells (hLEC) in vitro and also in the lymph nodes of patients with tuberculosis. We identified via RNA-seq a transcriptional programme in hLEC that activates cellular pro-survival and cytosolic surveillance of intracellular pathogens pathways. Consistent with this, cytosolic access of hLEC is required for intracellular M. tuberculosis cording; and cord formation is dependent on the M. tuberculosis ESX-1 type VII secretion system and the mycobacterial lipid PDIM. Finally, we show that M. tuberculosis cording is a novel size-dependent mechanism used by the pathogen to evade xenophagy in the cytosol of endothelial cells. These results provide a mechanism that explains the long-standing association between M. tuberculosis cording and virulence.

cell biology↗