Biology subjects

Gutierrez, M. G.

Publications and source records attributed to Gutierrez, M. G..

14 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↗

bioRxiv2026

Endolysosomal damage promotes intraluminal protein condensate formation that limits cytosolic leakage

Endolysosomal membrane damage is a detrimental process in mammalian cells that results in leakage of the luminal contents into the cytosol. However, the nature and extent of the leakage during membrane damage is unknown. Here, we show that endomembrane damage induces the rapid formation of intraluminal condensates in endolysosomes. A subset of resident luminal proteins undergo spatially coordinated condensation upon endomembrane damage. Electron microscopy reveals distinct luminal morphology, and cryo-electron tomography confirms the condensed ultrastructure in their native state. Condensate formation occurs across mechanistically distinct modes of membrane injury and is reversibly dissociated upon lysosomal recovery. Remarkably, these condensates impose a previously unrecognised barrier to endolysosomal escape of therapeutic oligonucleotides. Despite endomembrane damage, luminal oligonucleotide therapeutics are sequestered in damaged endolysosomes through condensate-mediated biophysical immobilisation. Targeting condensate sequestration could represent a novel strategy to improve oligonucleotide-based therapeutics.

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↗

bioRxiv2024

Mycobacterium tuberculosis overcomes phosphate starvation by extensively remodelling its lipidome with phosphorus-free lipids

Tuberculosis (TB) is the biggest cause of death from infectious disease worldwide [1-4]. The causative agent, Mycobacterium tuberculosis (Mtb), possesses a complex cell envelope that determines many of the key physiologic and virulence properties that facilitate infection, which comprises multiple classes of unique lipids [5-7]. The macrophage phagosome is a key reservoir of infection in pulmonary TB and multiple studies have shown that inorganic phosphate (Pi) is limiting in this environment [8-11]. The ability of Mtb to sense and respond to phosphate starvation is required for virulence in animal models and replication in human macrophages in vitro [12-14]. Here, we show that during Pi restriction the Mtb lipidome is markedly remodelled such that phospholipids are replaced with multiple classes of phosphorus-free lipids, some of which have not been documented before. Further, we discover that Mtb can metabolise phospholipid polar heads derived from host pulmonary surfactant as an alternative phosphate source, which sustains cell division for several generations during Pi restriction. These dual manipulations of phospholipid metabolism provide Mtb with an escape from phosphate restriction specific to the infection of alveolar macrophages, one of the earliest steps in establishing pulmonary tuberculosis. The changes in envelope lipidome remodelling, akin to that observed in some marine and terrestrial bacteria [15-20] suggests that standard Mtb culture conditions that use media with high concentrations of Pi do not reflect the physiologic environment during infection, thereby potentially undermining vaccine and drug development for tuberculosis. Moreover, the distinct Mtb phosphate-free lipids and the metabolic pathways that generate them could provide new antibiotic targets.

microbiology↗

bioRxiv2024

Ancient Borrelia genomes document the evolutionary history of louse-borne relapsing fever

Several disease-causing bacteria have transitioned from tick-borne to louse-borne transmission, a process associated with increased virulence and genome reduction. However, the historical time frame and speed of such evolutionary transitions have not been documented with ancient genomes. Here, we discover four ancient cases of Borrelia recurrentis, the causative agent of louse-borne relapsing fever, in Britain between [~]600 and 2,300 years ago, and sequence whole genomes up to 29-fold coverage. We estimate a recent divergence from the closest tick-borne ancestor, likely within the last [~]8,000 years. We reconstruct a chronology of gene losses and acquisitions using the pan-genome of related species, and show that almost all of the reductive evolution observed in B. recurrentis had occurred by [~]2,000 years ago, and was thus a rapid process after divergence. Our observations provide a new understanding of the origins of B. recurrentis and document complex reductive evolution in a specialist vector-borne pathogen.

evolutionary biology↗

bioRxiv2024

Physiologic media renders human iPSC-derived macrophages permissive for M. tuberculosis by rewiring organelle function and metabolism

In vitro studies are crucial for our understanding of the human macrophage immune functions. However, traditional in vitro culture media poorly reflect the metabolic composition of blood, potentially affecting the outcomes of these studies. Here, we analysed the impact of a physiological medium on human induced pluripotent stem cell (iPSC)-derived macrophages (iPSDM) function. Macrophages cultured in a human plasma-like medium (HPLM) were more permissive to Mycobacterium tuberculosis (Mtb) replication and showed decreased lipid metabolism with increased metabolic polarisation. Functionally, we discovered that HPLM-differentiated macrophages showed different metabolic organelle content and activity. Specifically, HPLM-differentiated macrophages displayed reduced lipid droplet and peroxisome content, increased lysosomal proteolytic activity, and increased mitochondrial activity and dynamics. Inhibiting or inducing lipid droplet formation revealed that lipid droplet content is a key factor influencing macrophage permissiveness to Mtb. These findings underscore the importance of using physiologically relevant media in vitro for accurately studying human macrophage function.

cell biology↗

bioRxiv2023

LRRK2 kinase dependent and independent function on endolysosomal repair promotes macrophage cell death

LRRK2 is commonly mutated in Parkinsons disease and has cell type-specific mechanisms of activation and function. In macrophages, LRRK2 is associated with lysosomes and is activated following lysosomal damage. However, effects of pathogenic LRRK2-G2019S in macrophages are unknown. Here, using primary mouse and human iPSC-derived macrophage (iPSDM) models of LRRK2-G2019S, we defined the substrates of LRRK2 after lysosomal damage. Using phosphoproteomics we found that LRRK2-G2019S and wild-type macrophages showed similar levels of Rab phosphorylation after lysosomal damage, with the exceptions of Rab12 and Rab35, which were increased and decreased, respectively, in LRRK2-G2019S. LRRK2-G2019S macrophages showed a LRRK2 kinase activity-independent deficit in lysosomal membrane repair which resulted in more cell death and increased apoptosis. Importantly, we recapitulated this phenotype in iPSDM from patients carrying the G2019S mutation, but not in isogenic control iPSDM. Altogether, we define here the signaling downstream of G2019S in macrophages and identify susceptibility to cell death after lysosomal damage as an important phenotype of this mutation.

cell biology↗

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↗

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

LRRK2-dependent Rab GTPase phosphorylation in response to endolysosomal damage depends on macrophage differentiation

The Parkinsons Disease (PD) kinase LRRK2 is highly expressed in immune cells such as macrophages. In these cells, LRRK2 regulates innate immune pathways and it is activated after membrane damage leading to the phosphorylation of the Rab GTPases Rab8A and Rab10. Due to their wide-range of functions in immunity and tissue remodelling, macrophages in vivo are phenotypically heterogeneous. In vitro systems are used to differentiate these cells into diverse macrophage subsets to mimic the populations observed in vivo. M-CSF and GM-CSF differentiated human blood monocytes are often used to generate monocyte-derived macrophages as a model for tissue macrophages. However, how LRRK2 is activated in different macrophage subsets after membrane damage is unknown. Here, we report that bone marrow derived macrophages and human monocyte-derived macrophages differentiated with either M-CSF or GM-CSF show different levels of LRRK2 activation after membrane damage. Notably, the membrane damaging agent LLOMe triggered LRRK2-dependent Rab8A and Rab10 phosphorylation primarily in GM-CSF differentiated macrophages. Moreover, LRRK2 and Rab8A were recruited to damaged endolysosomes in GM-CSF differentiated macrophages. Strikingly, GM-CSF differentiated macrophages recruited significantly more CHMP4B and Galectin-3 into damaged endolysosomes. These results suggest that LRRK2-regulated pathways of endolysosomal membrane damage and repair differ between macrophage subsets.

cell biology↗

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↗

bioRxiv2019

Elevated LRRK2 and α-synuclein levels in CSF of infectious meningitis patients

Neurodegenerative diseases such as Parkinsons (PD) have a complex aetiology consisting of an interplay of genetic and environmental factors. Inflammation and infection are proposed external factors that trigger disease progression. Tuberculous and cryptococcal meningitis frequently lead to long-term neurological sequelae but their association with the development of PD are unexplored. In this study, we protein profiled the CSF from 76 patients with or without infectious meningitis and found that proteins commonly associated with PD (LRRK2, tau and alpha-synuclein) were significantly elevated, establishing a link between neuroinflammation and infection. Importantly, these findings suggest that LRRK2, tau and alpha-synuclein could represent biomarkers of neuroinflammation.

immunology↗

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↗