Biology subjects

Rodgers, A.

Publications and source records attributed to Rodgers, A..

11 recordsLinked to original sources

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↗

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↗

bioRxiv2023

Nine-residue low-complexity disordered peptide as a model system, an NMR/CD study

Disordered proteins and protein segments can be crucial for biological function. In this work we present a detailed biophysical characterization of the low-complexity nine-residue peptide with the sequence GGKGMGFGL. Based on proton solution NMR chemical shifts, circular dichroism measurements, as well as the analysis of concentration dependence of NMR linewidth, proton longitudinal relaxation times, hydrogen-deuterium exchange measurements, and 15N rotating frame NMR relaxation measurements, we conclude that the peptide is fully disordered and monomeric in solution. The peptide will serve as a model system for future structural and dynamics studies of biologically relevant disordered peptides in solution and solid states.

biochemistry↗

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

Cell-type specific induction of cyclo-oxygenase-2 in layer II/III prefrontal cortical neurons mediates stress-induced anxiety phenotypes in mice.

The ability of the medial prefrontal cortex (mPFC) to exert top-down control of behavior is affected by stress. The molecular response of mPFC to stress is incompletely understood, however, in part because of the regions cellular heterogeneity. Here we used single nucleus RNA sequencing (snRNAseq) to map specific molecular cell types within the mPFC and to detect cell-type specific transcriptional changes to foot-shock stress. We identified Ptgs2, encoding cyclo-oxygenase 2, as an important candidate that is upregulated in layer II/III excitatory neurons after stress. Specifically, Ptgs2 was transiently upregulated with shock-induced fear learning and fear expression, along with Bdnf, Nptx2, and Lingo1, in a layer II/III neuronal population marked by the neuronal excitatory gene Slc17a7 and cell-type specific neuropeptide Penk. These dynamic cell-type specific expression patterns identified with snRNAseq were validated with quantitative fluorescent in situ hybridization. Using a pharmacological approach, we found that systemic lumiracoxib, a selective Ptgs2-inhibitor, led to a significant reduction in fear expression. Furthermore, genetic ablation of Ptgs2 in excitatory Camk2a-expressing neurons led to reduced stress-induced anxiety-like behaviors. Together these findings suggest that Ptgs2 is expressed in a dynamic, cell-type specific way in Layer II/III Penk+ neurons in mPFC, and that its role in prostaglandin and /or endocannabinoid regulation within these neurons may be an important mediator of stress-related behavior.

neuroscience↗

bioRxiv2021

Solving the Sample Size Problem for Resource Selection Analysis

O_LISample size sufficiency is a critical consideration for conducting Resource-Selection Analyses (RSAs) from GPS-based animal telemetry. Cited thresholds for sufficiency include a number of captured animals M [≥] 30 and as many relocations per animal N as possible. These thresholds render many RSA-based studies misleading if large sample sizes were truly insufficient, or unpublishable if small sample sizes were sufficient but failed to meet reviewer expectations. C_LIO_LIWe provide the first comprehensive solution for RSA sample size by deriving closed-form mathematical expressions for the number of animals M and the number of relocations per animal N required for model outputs to a given degree of precision. The sample sizes needed depend on just 2 biologically meaningful quantities: habitat selection strength and a novel measure of landscape complexity, which we define rigorously. The mathematical expressions are calculable for any environmental dataset at any spatial scale and are applicable to any study involving resource selection (including sessile organisms). We validate our analytical solutions using globally relevant empirical data including 5,678,623 GPS locations from 511 animals from 10 species (omnivores, carnivores, and herbivores living in boreal, temperate, and tropical forests, montane woodlands, swamps, and arctic tundra). C_LIO_LIOur analytic expressions show that the required M and N must decline with increasing selection strength and increasing landscape complexity, and this decline is insensitive to the definition of availability used in the analysis. Our results contradict conventional wisdom by demonstrating that the most biologically relevant effects on the utilization distribution (i.e. those landscape conditions with the greatest absolute magnitude of resource selection) can often be estimated with far fewer data than is commonly assumed. C_LIO_LIWe identify several critical steps in implementing these equations, including (i) a priori selection of expected model coefficients, and (ii) sampling intensity for background (absence/pseudo-absence) data within a given definition of availability. We show that random sampling of background data violates the underlying mathematics of RSA, leading to incorrect values for necessary M and N and potentially incorrect RSA model outputs. We argue that these equations should be a mandatory component for all future RSA studies. C_LI

ecology↗

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

The Mycobacterium tuberculosis sRNA F6 regulates expression of groEL/S

Almost 140 years after the identification of Mycobacterium tuberculosis as the etiological agent of tuberculosis, important aspects of its biology remain poorly described. Little is known about the role of post-transcriptional control of gene expression and RNA biology, including the role of most of the small RNAs (sRNAs) identified to date. We have carried out a detailed investigation of the M. tuberculosis sRNA, F6, and shown it to be dependent on SigF for expression, and significantly induced in starvation conditions in vitro and in a mouse model of infection. Further exploration of F6 using an in vitro starvation model of infection indicates that F6 affects the expression of the essential chaperonins, GroEL2 and GroES. Our results point towards a role for F6 during periods of low metabolic activity typically associated with long-term survival of M. tuberculosis in human granulomas.

microbiology↗

bioRxiv2018

Immune profiling of cord blood after prolonged rupture of membranes.

We hypothesised that foetal immune responses to an infectious challenge may be detected by genome-wide transcriptional profiling of cord blood. In order to test this hypothesis, we sought to identify transcriptomic changes in post-natal cord blood samples following prolonged pre-labour rupture of membranes (PROM) as a surrogate for increased risk of infection. By comparison to controls we found increased levels of blood transcripts in a subset of prolonged PROM cases, significantly enriched for innate immune system signalling pathways. These changes were idiosyncratic, suggesting qualitative and quantitative variation in foetal immune responses which may reflect differences in exposure and/or in host genetics. Our data support the view that PROM represents an infection risk to the foetus. In addition, we propose that cord blood transcriptional profiling offers exciting opportunities to identify immune correlates of clinical outcome following potential in utero exposures to infection. These may be used to elucidate the mechanisms of immunological protection and pathology in the foetus and identify biomarkers to stratify the risk of adverse outcomes.

immunology↗

bioRxiv2018

Antibiotic resistance evasion is explained by rare mutation frequency and not by lack of compensatory mechanisms

Drug resistant infections represent one of the most challenging medical problems of our time. D-cycloserine is an antibiotic used for decades without appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We investigated why Mycobacterium tuberculosis fails to become resistant to D-cycloserine. To address this question we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysis in vitro, in macrophages and in mice. Altogether, our results suggest that the ultra-low mutation frequency associated with D-cycloserine resistance is the dominant factor delaying the appearance of clinical resistance to this antibiotic in bacteria infecting humans, and not lack of potential compensatory mechanisms.\n\nOne Sentence SummaryWe show that the lack of D-cycloserine resistance in Mycobacterium tuberculosis is due its ultra-low mutation frequency rather than lack of compensatory mechanisms.

microbiology↗

bioRxiv2018

Discovery of a novel stereospecific β-hydroxyacyl-CoA lyase/thioesterase shared by three metabolic pathways in Mycobacterium tuberculosis

The vast number of poorly characterised enzymes in Mycobacterium tuberculosis (Mtb) is one of the key barriers precluding a better understanding of the biology that underpins pathogenesis. Here, we investigated the Mtb orphan enzyme Rv2498c to delineate its physiological role. Our results from in vitro enzymatic assays, phylogenetic analysis, X-ray crystallography and in vivo Mtb experiments, de-orphan Rv2498c as a multi-functional {beta}-hydroxyacyl-CoA lyase/thioesterase ({beta}-HAClyase/thioesterase) that participates in three different metabolic pathways: L-leucine catabolism, itaconate dissimilation, and glyoxylate shunt. Moreover, the deletion of the rv2498c gene from the Mtb genome resulted in attenuation in the mouse model compared to infection with the parent strain. To the best of our knowledge, this is the first report of an (R)-3-hydroxyl-3-methylglutaryl-CoA for leucine catabolism and an itaconate-specific resistance mechanism in Mtb.

biochemistry↗