Biology subjects

Roche, D.

Publications and source records attributed to Roche, D..

5 recordsLinked to original sources

bioRxiv2025

Peripheral blood cell-type and sex-specific signatures of alcohol misuse revealed by single-cell transcriptomics

Alcohol use disorder (AUD) is a complex condition with diverse molecular underpinnings. Chronic alcohol exposure is associated with alterations in both innate and adaptive immune cell populations that in turn contribute to AUD susceptibility and severity, suggesting a bidirectional gene-environment relationship. Yet, most clinical AUD studies of this system have relied on indirect measurement of immune function and/or assessment of only a small number of cell types or immune markers, approaches which cannot capture the immune systems inherent complexity and cellular heterogeneity. Therefore, in order to better characterize immune dysregulation in AUD, the goal of this study was to use single cell RNA sequencing (scRNA-seq) in peripheral blood mononuclear cells (PBMCs) to compare immune cell proportions and differential gene expression between individuals with AUD and healthy controls. Our findings highlight a distinct disproportionality of lymphocytes and monocytes in individuals with AUD and healthy controls as well as sex and dose-dependent alterations immune cell functional characteristics. These peripheral blood cell-type proportions and dose-dependent signatures of alcohol exposure may aid in developing more targeted and effective pharmacological interventions for AUD. Results also highlight the importance in including sex as a biological variable in AUD research, particularly when examining immune function.

genomics↗

bioRxiv2025

Global Transcriptomics and Targeted Metabolite Analysis Reveal the Involvement of the AcrAB Efflux Pump in Physiological Functions by Exporting Signaling Molecules in Photorhabdus laumondii

In Gram-negative bacteria, resistance-nodulation-division-type efflux pumps, particularly AcrAB-TolC, play a critical role in mediating resistance to antimicrobial agents and toxic metabolites, contributing to multidrug resistance. Photorhabdus laumondii is an entomopathogenic bacterium that has garnered significant interest due to its production of bioactive specialized metabolites with anti-inflammatory, antimicrobial, and scavenger deterrents properties. In previous work, we demonstrated that AcrAB confers self-resistance to stilbenes in P. laumondii TT01. Here, we explore the pleiotropic effects of AcrAB in this bacterium. RNA sequencing of {Delta}acrA compared to wild-type revealed growth-phase-specific gene regulation, with stationary-phase cultures showing significant downregulation of genes involved in stilbenes, fatty acid, and anthraquinone pigment biosynthesis, as well as genes related to cellular clumping and fimbrial pilin formation. Genes encoding putative LuxR regulators, type VI secretion systems, two-partner secretion systems, and contact dependent growth inhibition systems were upregulated in {Delta}acrA. Additionally, exponential-phase cultures revealed reduced expression of genes related to motility in {Delta}acrA. The observed transcriptional changes were consistent with phenotypic assays, demonstrating that the {Delta}acrA mutant had altered bioluminescence and defective orange pigmentation due to disrupted anthraquinone production. These findings confirm the role of stilbenes as signaling molecules involved in gene expression, thereby shaping these phenotypes. Furthermore, we showed that AcrAB contributes to swarming and swimming motilities independently of stilbenes. Collectively, these results highlight that disrupting acrAB causes transcriptional and metabolic dysregulation in P. laumondii, likely by impeding the export of key signaling molecules such as stilbenes, which may serve as a ligand for global transcriptional regulators. ImportanceRecent discoveries have highlighted Photorhabdus laumondii as a promising source of novel anti-infective compounds, including non-ribosomal peptides and polyketides. One key player in the self-resistance of this bacterium to stilbene derivatives is the AcrAB-TolC complex, which is also a well-known contributor to multidrug-resistance. Here, we demonstrate the pleiotropic effects of the AcrAB efflux pump in P. laumondii TT01, impacting secondary metabolite biosynthesis, motility, and bioluminescence. These effects are evident at transcriptional, metabolic, and phenotypic levels and are likely mediated by the efflux of signaling molecules such as stilbenes. These findings shed light on the multifaceted roles of efflux pumps and open avenues to better explore the complexity of RND pump-mediated signaling pathways in bacteria, thereby aiding combat multidrug resistant infections.

microbiology↗

bioRxiv2022

A selective bottleneck during host entry drives the evolution of new legume symbionts

During the emergence of new host-microbe symbioses, multiple selective pressures-acting at the different steps of the microbial life cycle-shape the phenotypic traits that jointly determine microbial fitness. However, the relative contribution of these different selective pressures to the adaptive trajectories of microbial symbionts are still poorly known. Here we characterized the dynamics of phenotypic adaptation and its underlying genetic bases during the experimental evolution of a plant pathogenic bacterium into a legume symbiont. We observed that fast adaptation was predominantly driven by selection acting on competitiveness for host entry, which outweighed selection acting on within-host proliferation. Whole-population sequencing of evolved bacteria revealed that phenotypic adaptation was supported by the continuous accumulation of new mutations and the sequential sweeps of cohorts of mutations with similar temporal trajectories. The identification of adaptive mutations within the fixed mutational cohorts showed that all of them improved competitiveness for host entry, while only a subset of those also improved within host proliferation. Computer simulations predict that this effect emerges from the presence of a strong selective bottleneck at host entry. Together, these results show how selective bottlenecks can alter the relative influence of selective pressures acting during bacterial adaptation to multistep infection processes.

evolutionary biology↗

bioRxiv2021

Emergence of NADP+-reducing enzymes in Escherichia coli central metabolism via adaptive evolution

The nicotinamide cofactor specificity of enzymes plays a key role in regulating metabolic processes and attaining cellular homeostasis. Multiple studies have used enzyme engineering tools or a directed evolution approach to switch the cofactor preference of specific oxidoreductases. However, whole-cell adaptation towards the emergence of novel cofactor regeneration routes was not previously explored. To address this challenge, we used an Escherichia coli NADPH-auxotroph strain. We continuously cultivated this strain under selective conditions. After 500-1100 generations of adaptive evolution using different carbon sources, we isolated several strains capable of growing without an external NADPH source. Most isolated strains were found to harbor a mutated NAD-dependent malic enzyme (MaeA). A single mutation in MaeA was found to switch cofactor specificity while lowering enzyme activity. Most mutated MaeA variants also harbored a second mutation that restored the catalytic efficiency of the enzyme. Remarkably, the best MaeA variants identified this way displayed overall superior kinetics relative to the wildtype variant with NAD+. In other evolved strains, the dihydrolipoamide dehydrogenase (Lpd) was mutated to accept NADP+ thus enabling the pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase complexes to regenerate NADPH. Interestingly, no other central metabolism oxidoreductase seems to evolve towards reducing NADP+, which we attribute to several biochemical constraints such as unfavorable thermodynamics. This study demonstrates the potential and biochemical limits of evolving oxidoreductases within the cellular context towards changing cofactor specificity, further showing that long-term adaptive evolution can optimize enzyme activity beyond what is achievable via rational design or directed evolution using small libraries. ImportanceIn the cell, NAD(H) and NADP(H) cofactors have different functions. The former mainly accepts electrons from catabolic reactions and carries them to respiration, while the latter provides reducing power for anabolism. Correspondingly, the ratio of the reduced to the oxidized form differs for NAD (low) and NADP (high), reflecting their distinct roles. We challenged the flexibility of E. colis central metabolism in multiple adaptive evolution experiments using an NADPH-auxotroph strain. We found several mutations in two enzymes, changing the cofactor preference of malic enzyme and dihydrolipoamide dehydrogenase. Upon deletion of their corresponding genes we performed additional evolution experiments which did not lead to the emergence of any additional mutants. We attribute this restricted number of mutational targets to intrinsic thermodynamic barriers: The high ratio of NADPH to NADP+ limits metabolic redox reactions which can regenerate NADPH, mainly by mass action constraints.

microbiology↗

bioRxiv2019

Brain cooling marginally increases maximum thermal tolerance in Atlantic cod

The physiological mechanisms determining thermal limits in fishes are debated but remain elusive. It has been hypothesised that loss of motor function observed as a loss of equilibrium during an acute thermal challenge is due to direct thermal effects on brain neuronal function. To test this hypothesis, we mounted cooling plates on the head of Atlantic cod (Gadus morhua) and quantified whether local cooling of the brain increased whole-organism critical thermal maxima (CTmax). Brain cooling reduced brain temperature by 2-6{degrees}C and increased CTmax by 0.5-0.7{degrees}C relative to instrumented and uninstrumented controls, suggesting that direct thermal effects on brain neurons might contribute to setting upper thermal limits in fish. However, the improvement in CTmax with brain cooling was small relative to the difference in brain temperature, demonstrating that other mechanisms (e.g., failure of spinal and peripheral neurons, or muscle) may also contribute to controlling acute thermal tolerance in fishes.\n\nSummary statementWe tested whether brain temperature sets the upper thermal limit in a fish. Selectively cooling the brain during whole-organism thermal ramping marginally increased thermal tolerance.

physiology↗