Biology subjects

Barlow, C.

Publications and source records attributed to Barlow, C..

9 recordsLinked to original sources

bioRxiv2026

Quinone-transporting filaments extend the respiratory chain of Gram positive bacteria

Cellular respiration depends on transferring electrons to hydrophobic quinones in membrane bilayers, constraining capacity to available surface area. Expanding this capacity is thought to have driven cellular complexity and eukaryogenesis, with Gram-negative bacteria evolving internal invaginations and eukaryotes using membrane-bound organelles. Whether Gram-positive bacteria, which lack such membranes, evolved alternatives was unknown. Here, we show that Bacillus subtilis forms a quinone-transporting pseudomembrane composed of filaments of the NADH dehydrogenase Ndh and the quinone-transporting protein Ncp. Cryo-EM, lipidomics, and molecular dynamics reveal that Ndh and Ncp co-assemble with phospholipids into a complex containing a solvent-excluded hydrophobic lumen that sequesters quinones. These complexes further assemble into filaments, linking chambers into a continuous conduit that amplifies quinone reduction while occupying minimal membrane space. Phylogenetic analysis suggests this recent innovation is widespread in Bacillota. Quinone-transporting filaments thus reveal a third strategy for overcoming surface-area limits and provide principles for engineering synthetic energy systems.

biochemistry↗

bioRxiv2026

Non-enzymatic assimilation of organosulfur compounds at the interface of geochemistry and biochemistry

Dimethyl sulfide (DMS) and dimethyl sulfoxide (DMSO) have important microbial-driven roles in global nutrient cycling, chemotaxis and/or climate regulation. Microbial DMSO and DMS assimilation were thought to require their enzymatic reduction to methanethiol. However, reactive oxygen species were recently shown to mediate methane production from DMS and DMSO. Here, we show that this oxidative mechanism can sustain microbial growth without the need for enzymes to directly reduce DMS or oxidize DMSO. Both, light and heat were effective geochemical drivers of this mechanism that yielded methanesulfonic acid and sulfite, and which mimicked the stepwise oxidation of DMSO by monooxygenases that only enzymatically enhance this oxidation reaction. Furthermore, we demonstrated intracellular DMSO degradation, in which sulfur and the simultaneously released carbon were both utilized by microbes. This study emphasizes that metabolic pathways are not necessarily enzyme-driven, with putatively ancient, non-enzymatic reaction networks taking place at a transition between geochemistry and biochemistry.

biochemistry↗

bioRxiv2025

Characterisation of SpoT-disruption induced metabolic shift in bacteria

SpoT, a pivotal stringent response regulatory enzyme, serves a dual function of both synthesizing and hydrolyzing the stress alarmones ppGpp and pppGpp, collectively known as (p)ppGpp. These alarmones act as global regulators, governing bacterial metabolic and physiological adaptation to diverse environmental stresses. Our previous investigation revealed that multidrug-resistant Acinetobacter baumannii notably upregulates SpoT in response to polymyxin treatment. Intriguingly, disrupting spoT gene enhances polymyxin killing. To comprehend SpoT metabolic regulatory function in mediating polymyxin tolerance, we conducted untargeted metabolomics, comparing metabolic perturbations upon polymyxin B (PMB) treatment between a spoT-disrupted A. baumannii mutant and its wild-type counterpart. Depletion of guanine-based purines GTP and GDP in the PMB-treated spoT-disrupted mutant suggests impaired (p)ppGpp hydrolysis, potentially leading to lethal accumulation of these molecules. PMB also induced more pronounced depletion of carbon sources (i.e., phosphoenolpyruvate, succinate, coenzyme A), energy metabolites (i.e., NADH biosynthesis pathway, ADP), amino acids, and the antioxidative system (i.e., glutathione, gamma-L-glutamyl-L-cysteine, (R)-S-lactoylglutathione) in the spoT-disrupted mutant. Interestingly, a distinctive time-dependent perturbation of fatty acyls was also observed following polymyxin treatment, with multiple fatty acyl conjugates significantly elevated in the spoT-disrupted mutant at 1 hour. However, this situation reversed at 4 hours, with more elevated fatty acyl groups in the wild-type compared to the spoT-disrupted mutant, indicating greater and more rapid PMB-induced membrane disruption in the spoT-disrupted mutant. Collectively, our findings suggest a potential role for SpoT in intricately coordinating A. baumannii energy expenditure and metabolite repertoire to ensure optimal functionality in stress tolerance and repair machineries (e.g., glutathione system, fatty acid regulation) following polymyxin treatment. IMPORTANCEPolymyxins, the last-resort antibiotics for multidrug-resistant Gram-negative bacteria, face increasing challenges due to growing tolerance. To tackle Acinetobacter baumannii polymyxin tolerance, we turned to SpoT, a (p)ppGpp synthetase/hydrolase protein. Our study focused on spoT-disrupted mutant, which exhibited heightened polymyxin antibacterial killing, yet the underlying mechanisms remained elusive. Through metabolomics, we unraveled key insights. Enhanced polymyxin killing in spoT-disrupted mutant likely resulted from lethal accumulation of stress alarmones (p)ppGpp, perturbations in carbon and energy metabolism, thiol-based antioxidant system depletion, and disrupted lipidic membrane repair. These findings illuminate the intricate metabolic signaling networks driving polymyxin tolerance in A. baumannii. By untangling pathogen-directed stress tolerance mechanisms, we deepen our understanding and identify potential therapeutic targets. Our discoveries offer new avenues to combat polymyxin resistance, paving the way for more effective treatments against superbugs. Through continued exploration, we can harness this knowledge to develop innovative strategies and overcome the challenges posed by polymyxin tolerance.

microbiology↗

bioRxiv2024

Valine restriction extends survival in a Drosophila model of short-chain enoyl-CoA hydratase 1 (ECHS1) deficiency

Short-chain enoyl-CoA hydratase 1 deficiency (ECHS1D) is a rare genetic disorder caused by biallelic pathogenic variants in the ECHS1 gene. ECHS1D is characterised by severe neurological and physical impairment that often leads to childhood mortality. Therapies such as protein and single nutrient-restricted diets show poor efficacy, whereas development of new treatments is hindered by the low prevalence of the disorder and a lack of model systems for treatment testing. Here we report on the establishment of a Drosophila model of ECHS1D. Flies carrying mutations in Echs1 (CG6543) were characterised for their physical and metabolic phenotypes, and dietary intervention to improve fly model health was explored. The Echs1 null larvae recapitulated human ECHS1D phenotypes including elevated biomarkers (S-(2-carboxypropyl)cysteamine and 2,3-dihydroxy-2-methylbutyric acid), poor motor behaviour and early mortality, and could be rescued by expression of a human ECHS1 transgene. We observed that both restriction of valine in isolation, or all branched-chain amino acids (BCAAs - leucine, isoleucine, and valine) together, extended larval survival, supporting the idea that reducing BCAA pathway catabolic flux is beneficial in this disorder. Further, metabolic profiling revealed substantial changes to carbohydrate metabolism, suggesting that Echs1 loss causes widespread metabolic dysregulation beyond valine metabolism. The similarities between Drosophila and human ECHS1D suggest that the fly model is a valuable animal system in which to explore mechanisms of pathogenesis and novel treatment options for this disorder.

genetics↗

bioRxiv2023

An obligate aerobe adapts to hypoxia by hybridising fermentation with carbon storage

In soil ecosystems, obligately aerobic bacteria survive oxygen deprivation (hypoxia) by entering non-replicative persistent states. Little is known about how these bacteria rewire their metabolism to stay viable in these states. The model obligate aerobe Mycobacterium smegmatis maintains redox homeostasis during hypoxia by mediating fermentative hydrogen production. However, the fate of organic carbon during fermentation, and the associated remodeling of carbon metabolism, is unresolved. Here we systematically profiled the metabolism of M. smegmatis during aerobic growth, hypoxic persistence, and the transition between these states. Using differential isotope labelling, and paired metabolomics and proteomics, we observed rerouting of central carbon metabolism through the pentose phosphate pathway and Entner-Doudoroff pathway during hypoxia. We show that M. smegmatis excretes high levels of hydrogen concurrently with upregulating triacylglyceride synthases and accumulating glycerides as carbon stores. Using electron cryotomography (cryo-ET), we observed the presence of large spheroid structures consistent with the appearance of lipid droplets. Thus, in contrast to obligately and facultative anaerobic fermentative bacteria, M. smegmatis stores rather than excretes organic carbon during hypoxia. This novel hybrid metabolism likely provides a competitive advantage in resource-variable environments by allowing M. smegmatis to simultaneously dispose excess reductant during hypoxia and maintain carbon stores to rapidly resume growth upon reoxygenation.

microbiology↗

bioRxiv2022

G2019S selective LRRK2 kinase inhibitor abrogates mitochondrial DNA damage

Pathogenic mutations in LRRK2 cause Parkinsons disease (PD). The G2019S variant is the most common, which results in abnormally high kinase activity. Compounds that target LRRK2 kinase activity are currently being developed and tested in clinical trials. We recently found that G2019S LRRK2 causes mitochondrial DNA (mtDNA) damage and treatment with multiple classes of LRRK2 kinase inhibitors at concentrations associated with dephosphorylation of LRRK2 reversed mtDNA damage to healthy control levels. Because maintaining the normal function of LRRK2 in heterozygous G2019S LRRK2 carriers while specifically targeting the G2019S LRRK2 activity could have an advantageous safety profile, we explored the efficacy of a G2019S mutant selective LRRK2 inhibitor to reverse mtDNA damage in G2019S LRRK2 models and patient cells relative to non-selective LRRK2 inhibitors. Potency of LRRK2 kinase inhibition by EB-42168, a G2019S mutant LRRK2 kinase inhibitor, and MLi-2, a nonselective inhibitor, was determined by measuring phosphorylation of LRRK2 at Ser935 and/or Ser1292 using quantitative western immunoblot analysis. The Mito DNADX assay, a novel system that allows for the accurate real-time quantification of mtDNA damage in a 96-well platform, was performed in parallel. We confirmed that EB-42168 selectively inhibits LRRK2 phosphorylation on G2019S LRRK2 relative to wild-type LRRK2. On the other hand, MLi-2 was equipotent for wild-type and G2019S LRRK2. Acute treatment with EB-42168 inhibited LRRK2 phosphorylation and also restored mtDNA damage to healthy control levels. Precision medicine is a common approach in modern day cancer research that is not yet routinely applied to neurodegenerative diseases. Abrogation of mtDNA damage with mutant selective tool inhibitor EB-42168 demonstrates the promise of a precision medicine approach for LRRK2 G2019S PD. Levels of mtDNA damage may serve as a potential pharmacodynamic biomarker of altered kinase activity that could be useful for small molecule development and clinical trials.

neuroscience↗

bioRxiv2022

Energy extraction from air: structural basis of atmospheric hydrogen oxidation

Diverse aerobic bacteria use atmospheric H2 as an energy source for growth and survival. This recently discovered yet globally significant process regulates the composition of the atmosphere, enhances soil biodiversity, and drives primary production in certain extreme environments. Atmospheric H2 oxidation has been attributed to still uncharacterised members of the [NiFe]-hydrogenase superfamily. However, it is unresolved how these enzymes overcome the extraordinary catalytic challenge of selectively oxidizing picomolar levels of H2 amid ambient levels of the catalytic poison O2, and how the derived electrons are transferred to the respiratory chain. Here we determined the 1.52 [A] resolution CryoEM structure of the mycobacterial hydrogenase Huc and investigated its mechanism by integrating kinetics, electrochemistry, spectroscopy, mass spectrometry, and molecular dynamics simulations. Purified Huc is an oxygen-insensitive enzyme that couples the oxidation of atmospheric H2 at its large subunit to the hydrogenation of the respiratory electron carrier menaquinone at its small subunit. The enzyme uses a narrow hydrophobic gas channel to selectively bind atmospheric H2 at the expense of O2, while three [3Fe-4S] clusters and their unusual ligation by a D-histidine modulate the electrochemical properties of the enzyme such that atmospheric H2 oxidation is energetically feasible. Huc forms an 833 kDa complex composed of an octamer of catalytic subunits around a membrane-associated central stalk, which extracts and transports menaquinone a remarkable 94 [A] from the membrane, enabling its reduction. These findings provide a mechanistic basis for the biogeochemically and ecologically critical process of atmospheric H2 oxidation. Through the first characterisation of a group 2 [NiFe]-hydrogenase, we also uncover a novel mode of energy coupling dependent on long-range quinone transport and pave way for the development of biocatalysts that oxidize H2 in ambient air.

biochemistry↗

bioRxiv2022

Disease-modifying effects of sodium selenate in a model of drug-resistant, temporal lobe epilepsy.

There are no pharmacological disease-modifying treatments that can mitigate the seizures and comorbidities associated with established chronic temporal lobe epilepsy (TLE). This study evaluated the effect of sodium selenate in the post-status epilepticus (SE) rat model of chronic drug resistant TLE. Wistar rats underwent kainic acid-induced SE or sham. Ten-weeks post-SE, rats were randomly assigned to receive either sodium selenate, levetiracetam, or vehicle treatments continuously for 4 weeks. To evaluate the effects of the treatments, 1 week of continuous video-EEG was acquired before, during, and 4, 8 weeks post-treatment, followed by behavioral tests. Targeted and untargeted proteomics and metabolomics were performed on post-mortem brain tissue to identify potential pathways associated with modified disease outcomes. Telomere length was investigated as a novel surrogate marker of disease severity. Sodium selenate treatment was able to mitigate disease severity, reducing the number of spontaneous seizures (p< 0.05), cognitive dysfunction (p< 0.05 in both novel object placement and recognition tasks), and sensorimotor deficits (p< 0.01) 8 weeks post-treatment cessation. Moreover, increased protein phosphatase 2A (PP2A) expression, reduced hyperphosphorylated tau, and reversed telomere length shortening caused by SE (p< 0.05). Network medicine integration of multi-omics/ pre-clinical outcomes identified protein-metabolite modules positively correlated with the TLE phenotype. Our results provide evidence that treatment with sodium selenate results in a sustained disease modifying effect in chronically epileptic rats in the post-KA SE model of TLE, including improved comorbid learning and memory deficits.

neuroscience↗

bioRxiv2022

Orally active mGluR2/3 metabotropic antagonist pro-drug mimics the beneficial effects of physical exercise on neurogenesis, behavior, and exercise-related molecular pathways in an Alzheimer's disease mouse model

Modulation of physical activity represents an important intervention that may delay, slow, or prevent mild cognitive impairment (MCI) or dementia due to Alzheimers disease (AD). One mechanism proposed to underlie the beneficial effect of physical exercise involves the apparent stimulation of adult hippocampal neurogenesis (AHN). BCI-838 is a pro-drug whose active metabolite BCI-632 is an antagonist at the group II metabotropic glutamate receptor (mGluR2/3). We previously demonstrated that administration of BCI-838 to a mouse model of cerebrovascular accumulation of oligomeric A{beta}E22Q (APPE693Q = "Dutch APP") reduced learning behavior impairment and anxiety, both of which are associated with the phenotype of the Dutch APP mice. Here we show that (i) administration of BCI-838, physical exercise, or a combination of BCI-838 and physical exercise enhanced AHN in a four-month old mouse model of AD amyloid pathology (APPKM670/671NL/ PSEN1{Delta}exon9 = APP/PS1), (ii) administration of BCI-838 alone or associated with physical exercise led to stimulation of AHN and improvement in both spatial and recognition memory, (iii) significantly, the hippocampal dentate gyrus transcriptome of APP/PS1 mice following BCI-838 treatment up-regulated brain-derived neurotrophic factor (BDNF), PIK3C2A of the PI3K-MTOR pathway, and metabotropic glutamate receptors, and down-regulated EIF5A of ketamine-modulating mTOR activity, and finally (iv) qPCR findings validate a significantly strong association between increased BDNF levels and BCI-838 treatment. Our study points to BCI-838 as a safe and orally active compound capable of mimicking the beneficial effect of exercise on AHN, learning behavior, and anxiety in a mouse model of AD neuropathology.

neuroscience↗