Biology subjects

Harding, C.

Publications and source records attributed to Harding, C..

7 recordsLinked to original sources

bioRxiv2025

Global translational and metabolic remodelling during iron deprivation in Toxoplasma gondii

Iron is required to support essential cellular processes. Due to diverse and dynamic host environments, the obligate intracellular parasite Toxoplasma gondii must adapt to iron limited conditions. To investigate the adaptations critical to parasite survival under these conditions, we conducted proteomic and metabolomic profiling of Toxoplasma cultured in iron depleted conditions. We find that iron depletion results in remodelling of the parasite proteome and triggers swift translational repression. This occurs prior to downregulation of the iron-regulated translation factor ABCE1, indicating an upstream, ABCE1-independent mechanism. In the context of repressed translation, we also observe a significant rewiring of energy metabolism. Iron depleted Toxoplasma have altered mitochondrial morphology and a profound reduction in mitochondrial respiration. Untargeted metabolomic analysis revealed tricarboxylic acid cycle (TCA) cycle dysregulation, characterised by accumulation of citrate and fumarate, both substrates of iron-dependent TCA cycle enzymes, and accumulation of glycolytic intermediates. We found iron deprived parasites continue to take up glucose and maintain glycolytic output, comparable to iron replete conditions. Limiting glucose availability either in culture media or by genetic ablation of glucose uptake caused a significant increase in sensitivity to iron restriction. Conversely, limitation of mitochondrially metabolised glutamine improved parasite fitness in iron depleted conditions. Together, our results establish iron as a key regulator of parasite translation and metabolic flexibility and demonstrate carbon source availability as important in Toxoplasma adaptation to iron deprivation. ImportanceThis study determines the effects of iron deprivation on the parasite Toxoplasma gondii. Using proteomics and metabolomics, we reveal iron as a novel regulator of both translation and energy metabolism in Toxoplasma, underpinning the importance of this nutrient for essential cellular processes. We find that iron depletion introduces a metabolic bottleneck whereby parasites are dependent on glucose as their major carbon source. By modulating the parasites metabolism by altering carbon source availability, we identify nutrient conditions that improve parasite survival under iron restriction. These data reveal a key role for adaptive plasticity of Toxoplasma central carbon metabolism to drive survival under iron limited conditions. Understanding the interactions between parasite nutrient availability and metabolism allows us both to map the metabolic flexibility of these parasites, and to identify potential vulnerabilities.

microbiology↗

bioRxiv2024

A modular system for programming multistep activation of endogenous genes in stem cells

Although genomes encode instructions for mammalian cell differentiation with rich syntactic relationships, existing methods for genetically programming cells have modest capabilities for stepwise regulation of genes. Here, we developed a sequential genetic system that enables transcriptional activation of endogenous genes in a preprogrammed, stepwise manner. The system relies on the removal of an RNA polymerase III termination signal to induce both the transcriptional activation and the DNA endonuclease activities of a Cas9-VPR protein to effect stepwise progression through cascades of gene activation events. The efficiency of the cascading system enables a new dimension for cellular programming by allowing the manipulation of the sequential order of gene activation for directing the differentiation of human stem cells. One-Sentence SummaryDevelopment of a synthetic biology system for preprogrammed, stepwise activation of endogenous genes.

synthetic biology↗

bioRxiv2022

Exploring the in-situ evolution of Nitrofurantoin resistance in clinically derived Uropathogenic Escherichia coli isolates.

BACKGROUNDNitrofurantoin has been re-introduced as a first-choice antibiotic to treat uncomplicated acute urinary tract infections in England and Wales. Its mode of action involves initial reduction by nitroreductases, to generate electrophilic intermediates that inhibit protein and nucleic acid synthesis. Highly effective against common uropathogens such as Escherichia coli, its use is accompanied by a low incidence (<10%) of antimicrobial resistance. Resistance to Nitrofurantoin is predominantly via the acquisition of loss-of-function, step-wise mutations in the nitroreductase genes nfsA and nfsB. OBJECTIVETo explore the in situ evolution of NitR, longitudinal uropathogenic E. coli isolates recovered from two rUTI patients. RESULTSGrowth rate analysis identified a 2-10% slower doubling time for Nitrofurantoin resistant strains, but statistically, these data suggested there was no fitness advantage of evolved strains over their sensitive predecessor (ANOVA P-value = 0.13). Genetic manipulation of E. coli to mimic nitrofurantoin resistance evolution, again confirmed no fitness advantages (ANOVA P-value = 0.22). Rather, further analysis argued that a first-step mutant gained a selective advantage, at sub-MIC (4-8 mg/L) nitrofurantoin concentrations. CONCLUSIONCorrelation of these findings to Nitrofurantoin pharmacokinetic data suggests that the low incidence of E. coli NitR, within the community, is driven by urine-based nitrofurantoin concentrations that selectively inhibit the growth of E. coli strains carrying the key first-step loss-of-function mutation.

microbiology↗

bioRxiv2021

Evasion of Toll-like Receptor Recognition by Escherichia coli is mediated via Population Level Regulation of Flagellin Production

Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract infections. Analysis of the innate immune response in immortalised urothelial cells suggests that the bacterial flagellar subunit, flagellin, is key in inducing host defences. A panel of 40 clinical uro-associated Escherichia coli isolates recovered from either asymptomatic bacteruria (ASB), cystitis or pyelonephritis patients, were characterised for motility and their ability to induce an innate response in urothelial cells stably transfected with a NF{kappa}B luciferase reporter. Twenty-four isolates (60%) were identified as motile with strains recovered from cystitis patients exhibiting a bipolar motility distribution pattern (P < 0.005) and associated with a 2-5 fold increase in NF{kappa}B signalling. Although two isolates were associated with swarm sizes of >7 cm and NF{kappa}B activities of >30 fold (P = 0.029), data overall suggested bacterial motility and the NF{kappa}B signalling response were not directly correlated. To explore whether the signalling response reflected antigenic variation flagellin was purified from 11 different isolates and the urothelial cell challenges repeated. Purified flagellin filaments generated comparable (30.4{+/-}1.8 to 46.1{+/-}2.5 fold, P = NS) NF{kappa}B signalling responses, irrespective of either the source of the isolate or H-serotype. These data argued against any variability between isolates being related to flagellin itself. To determine the roles, if any, of flagellar abundance in inducing these responses flagellar hook numbers of a range of cystitis and ABU isolates were quantified using a plasmid encoded flagellar hook gene flgEA240C. Foci data suggested isolates were averaging between 1 and 2 flagella per cell, while only 10 to 60% each isolates population exhibited foci. These data suggested selective pressures exist in the urinary tract that allow uro-associated E. coli strains to maintain motility exploiting population heterogeneity to prevent host TLR5 recognition.

microbiology↗

bioRxiv2020

Daytime light enhances the amplitude of circadian output in a diurnal mammal

Mammalian circadian rhythms are orchestrated by a master pacemaker in the hypothalamic suprachiasmatic nuclei (SCN), which receives information about the 24 h light:dark cycle from the retina. The accepted function of this light signal is to reset circadian phase in order to ensure appropriate synchronisation with the celestial day. Here, we ask whether light also impacts another key property of the circadian oscillation, its amplitude. To this end, we measured rhythms in behavioural activity and body temperature, and SCN electrophysiological activity in the diurnal murid rodent Rhabdomys pumilio following stable entrainment to 12:12 light:dark cycles at 4 different daytime intensities (ranging from 12.77 to 14.80 log melanopsin effective photons/cm2/s). Rhabdomys showed strongly diurnal activity and body temperature rhythms in all conditions, but measures of rhythm robustness were positively correlated with daytime irradiance under both entrainment and subsequent free run. Whole-cell and extracellular recordings of electrophysiological activity in ex vivo SCN revealed substantial differences in electrophysiological activity between dim and bright light conditions. At lower daytime irradiance, daytime peaks in SCN spontaneous firing rate and membrane depolarisation were substantially depressed, leading to an overall marked reduction in the amplitude of circadian rhythms in spontaneous activity. Our data reveal a previously unappreciated impact of daytime light intensity on SCN physiology and the amplitude of circadian rhythms, and highlight the potential importance of daytime light exposure for circadian health.

neuroscience↗

bioRxiv2019

Genetic screens reveal a central role for heme biosynthesis in artemisinin susceptibility

Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria, however, resistance threatens to undermine global control efforts. To explore artemisinin resistance in apicomplexan parasites broadly, we used genome-scale CRISPR screens recently developed for Toxoplasma gondii to discover sensitizing and desensitizing mutations. Using a sublethal concentration of dihydroartemisinin (DHA), we uncovered the putative porphyrin transporter Tmem14c whose disruption increases DHA susceptibility. Screens performed under high doses of DHA provided evidence that mitochondrial metabolism can modulate resistance. We show that disruption of a top candidate from the screens, the mitochondrial protease DegP2, lowered levels of free heme and decreased DHA susceptibility, without significantly altering fitness in culture. Deletion of the homologous gene in P. falciparum, PfDegP, similarly lowered heme levels and DHA susceptibility. These results expose the vulnerability of the heme biosynthetic pathway for genetic perturbations that can lead to survival in the presence of DHA. We go on to show that chemically reducing heme biosynthesis can decrease the sensitivity of both T. gondii and P. falciparum to DHA, suggesting guidelines for developing combination therapies.

microbiology↗

bioRxiv2018

Evidence for phospholipid export from the gram-negative inner membrane: time to rethink the Mla pathway?

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of 3 components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here we solve the crystal structure of MlaC in its phospholipid free closed apo conformation, revealing a novel pivoting {beta}-sheet mechanism which functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC we provide evidence that the Mla pathway functions in an anterograde rather than a retrograde direction by showing the inner membrane MlaFEDB machinery exports phospholipids and transfers them to MlaC in the periplasm. We confirm that the lipid export process occurs through the MlaD component of the MlaFEDB complex. This lipid export process is shown to be independent of ATP. Our data provides, for the first time, evidence of an apparatus for lipid export to the outer membrane.

biophysics↗