Biology subjects

Hardy, L.

Publications and source records attributed to Hardy, L..

7 recordsLinked to original sources

bioRxiv2025

The tRNA epitranscriptomic landscape and RNA modification enzymes in Vibrio cholerae

Transfer RNAs (tRNAs) are central to protein synthesis, ensuring precise decoding of the genetic code by delivering aminoacids to the ribosome. Among all RNA species, tRNAs are the most heavily and diversely modified, with modifications playing critical roles in stability, folding, and function. Here, we present a comprehensive, isodecoder-level map of tRNA modifications in the human pathogen Vibrio cholerae. This map was generated by chemical-based sequencing methods, comparing wild-type and deletion strains. By assigning specific tRNA modifications to their cognate enzymes, we defined a comprehensive modification landscape in Vibrio cholerae and confirmed species-specific features, such as the presence of a functional TrmK enzyme, largely restricted to Gram-positive bacteria. Additionally, we detected a modification at U55 that occurs independently of TruB. To assess the biological significance of these modifications, we evaluated fitness under both standard conditions and subinhibitory antibiotic stress, and examined how modifications in the anticodon stem-loop region influence codon decoding efficiency and accuracy. Based on a comparative analysis of E. coli and V. cholerae, we discuss how species-specific differences in tRNA isodecoder gene repertoires may influence the functional impact and biological importance of tRNA modifications. This work provides the first experimentally validated, genome-wide map of tRNA modifications in V. cholerae, serving as a reference for future research into RNA modifications, translation regulation, and pathogen biology. Author summaryThis study charts the first genome-wide map of transfer RNA (tRNA) modifications in the cholera pathogen, Vibrio cholerae, revealing how chemical marks on tRNAs shape translation and stress responses. Using complementary chemical sequencing methods and a panel of targeted gene deletions, we assigned specific modifications to their enzymes across individual tRNA isodecoders. This integrative approach validates conserved features (e.g., {Psi}55 and T54), and specific ones, such as an active TrmK that installs m{superscript 1}A22 despite being considered largely restricted to Gram-positive bacteria, and uncovers enzyme interplay among dihydrouridine synthases. By testing mutant strains in standard and sub-inhibitory antibiotic conditions, we show that several modifications are dispensable for basal growth but become critical under proteotoxic stress, influencing fitness and translation accuracy, including stop-codon readthrough. Codon-specific reporter assays further demonstrate that modifications at wobble position 34 and at position 37 modulate decoding of distinct codon families, linking epitranscriptomic changes to gene expression programs. Comparative analysis with Escherichia coli suggests that species-specific tRNA isodecoder repertoires tune the functional impact of modifications. Our map provides an additional reference for studying RNA modification biology in pathogens and how it contributes to stress adaptation and virulence.

microbiology↗

bioRxiv2025

High precision fluorescence tomography-guided system for pre-clinical radiation research: system design and validation

PurposeCone-beam computed tomography (CBCT), commonly used for image guidance in pre-clinical studies, is limited in soft tissue localization and lacks the functional information needed for image-guided radiation studies and treatment assessment. To address these limitations, we developed 3D fluorescence tomography (FT) for high precision functional image-guided research and integrated it with small animal irradiators. MethodsThe FT system, integrated with a commercial bioluminescence-guided platform in a standalone configuration, enabling compact multi-projection and multi-spectral imaging. A dual-axis galvo mirror scanner was used for laser spots scanning to excite internal fluorophore. A transportable mouse bed allows animals imaged in the optical system and transferred to irradiators for CBCT imaging and FT-guided irradiation. Spatial geometry-based methods were developed to map the laser spots and fluorescence images onto the animal numerical mesh surface, generated from the CBCT image, to serve as input data for FT reconstruction. A self-calibration method using Born-ratio data, fluorescence normalized to the excitation image, was adopted for reconstruction. Mouse phantom embedded with QD800 reporter and orthotopic glioblastoma (GBM) model injected with IRDye 800CW labelled U87-EGFR cells were used to verify the FT-guided system in target localization. ResultsThe laser spot mapping accuracy are within 0.7 mm maximum deviation. Our system can reconstruct the target QD800 as deep as 17 mm in the mouse phantom with single projection fluorescence imaging at localization accuracy < 1 mm deviation. Using the Born ratio approach, we effectively eliminated excitation light leakage and autofluorescence, enabling our FT system to localize IRDye 800CW-labeled EGFR-overexpressing GBM cells in the mouse brain with approximately 1 mm accuracy. ConclusionWe developed a compact FT system integrated with a small animal irradiator, achieving sub-millimeter localization accuracy in phantom and in vivo models. Its precision, flexibility, and compatibility position it as a powerful tool for advancing functional imaging-guided pre-clinical radiation research.

bioengineering↗

bioRxiv2024

Canine Mammary Tumours (CMTs) exploit Mitochondrial Cholesterol for aggressive reprogramming

In human breast cancer the mitochondrial translocator protein (TSPO) aids pro-survival cellular response by facilitating the formation of mitochondrial contact sites with the nucleus termed Nucleus Associated Mitochondria (NAM). Here, we show that TSPO positively associates with the aggressiveness of tissues and cells isolated from Canine Mammary Tumours (CMTs). TSPO is also readily upregulated in reprogrammed mammary tumour cells following long-term deprivation of oestrogen or exposure to the endocrine chemotherapeutic (ET) agent Tamoxifen. The latter triggers mitochondrial handling of cholesterol which is facilitated by TSPO whose upregulation reduces susceptibility to Tamoxifen. TSPO binding ligands boost, on the other hand, the efficacy of Tamoxifen and Chemotherapy agents. In aggressive canine mammary tumour cells, TSPO repression impairs the NF-kB pattern thus confirming the pro-survival role of the NAM uncovered in the human counterpart. Mitochondrial cholesterol handling via TSPO emerges therefore as a signature in the aggressive reprogramming of CMTs thus advancing our understanding of the molecular mechanisms underpinning this pathology. A novel target mechanism to improve bio-marking and therapeutic protocols is here proposed.

cancer biology↗

bioRxiv2024

YraN is a helicase-associated nuclease fostering extended recombination events by natural transformation

Natural transformation is a widespread mechanism driving genetic exchanges in bacteria. It proceeds by the capture and internalization of exogenous DNA in linear single strands, ultimately integrated in the genome by homologous recombination. It is unknown how the RecA-directed D-loop intermediate of this dedicated recombination pathway is processed. We report that resolution of the transformation D-loop depends on two endonucleases of opposing phylogenetic distribution in bacteria. One is YraN, which has co-evolved and interacts with the ComM helicase, known to extend DNA recombination at the transformation D-loop. The other is CoiA, which is restricted to the Bacillota. CoiA is shown to be a resolvase of the transformation D-loop, extended by the RadA helicase in these species. We demonstrate that both YraN and CoiA act synergistically with their cognate helicases. These findings reveal that bacteria have evolved two helicase/nuclease pairs for the maturation and recombination extension of the transformation D-loop.

microbiology↗

bioRxiv2021

Persistent DNA damage promotes microglial dysfunction in Ataxia-telangiectasia

The autosomal recessive genome instability disorder Ataxia-telangiectasia, caused by mutations in ATM kinase, is characterised by the progressive loss of cerebellar neurons. We find that DNA damage associated with ATM loss results in dysfunctional behaviour of human microglia, immune cells of the central nervous system. Microglial dysfunction is mediated by the pro-inflammatory RELB/p52 non-canonical NF-{kappa}B transcriptional pathway and leads to excessive phagocytic clearance of neurites. Pathological phagocytosis of neuronal processes by microglia has also been observed in multiple sclerosis, Alzheimers and progranulin deficiency, suggesting a common mechanism that promotes neuronal damage. Activation of the RELB/p52 pathway in ATM-deficient microglia is driven by persistent DNA damage and is dependent on the NIK kinase. These results provide insights into the underlying mechanisms of aberrant microglial behaviour in Ataxia-telangiectasia, potentially contributing to neurodegeneration.

cell biology↗

bioRxiv2020

Transposon-insertion sequencing in a clinical isolate of Legionella pneumophila identifies essential genes and determinants of natural transformation

Legionella pneumophila is a Gram-negative bacterium ubiquitous in freshwater environments which, if inhaled, can cause a severe pneumonia in humans. The emergence of L. pneumophila is linked to several traits selected in the environment, the acquisition of some of which involved intra- and interkingdown horizontal gene transfer events. Transposon-insertion sequencing (TIS) is a powerful method to identify the genetic basis of selectable traits as well as to identify fitness determinants and essential genes, possible antibiotic targets. TIS has not yet been used to its full power in L. pneumophila, possibly because of difficulty to obtain a high-saturation transposon insertion library. Indeed, we found that ST1 isolates, to which belong the commonly used laboratory strains, are poorly permissive to saturating mutagenesis by conjugation-mediated transposon delivery. In contrast, we obtained high-saturation libraries in non-ST1 clinical isolates, offering the prospect of using TIS on unaltered L. pneumophila strains. Focusing on one of them, we therefore used TIS to identify essential genes in L. pneumophila. We also revealed that TIS could be used to identify genes controlling vertical transmission of mobile genetic elements. We then applied TIS to identify all the genes required for L. pneumophila to develop competence and undergo natural transformation, defining the set of major and minor Type IV pilins that are engaged in DNA uptake. This work paves the way for the functional exploration of the L. pneumophila genome by TIS and the identification of the genetic basis of other life traits of this species. ImportanceLegionella pneumophila is the etiologic agent of a severe form of nosocomial and community-acquired pneumonia in humans. L. pneumophila is found in man-made and freshwater environments which are the causing source of the infection. The environmental life traits of L. pneumophila, such as its abilities to form biofilms, resist biocides and unicellular predators, are essential to its ability to accidentally infect humans. A comprehensive identification of the genetic basis of these life traits could be obtained through the use of transposon-insertion sequencing. Yet, this powerful approach, had not been fully implemented in L. pneumophila. Here we described the successful implementation of the transposon-sequencing approach in a clinical isolate of L. pneumophila. We identify essential genes, potential drug targets, and genes required for horizontal gene transfer by natural transformation. This work represents an important step towards identifying the genetic basis of the many life traits of this environmental and pathogenic species.

microbiology↗

bioRxiv2018

Mitochondria form cholesterol-rich contact sites with the nucleus during retrograde response

Mitochondria drive cellular adaptation to stress by retro-communicating with the nucleus. This process is known as Mitochondrial Retrograde Response (MRR) and is induced by mitochondrial dysfunctions which perturb cell signalling. MRR results in the nuclear stabilization and activation of pro-survival transcription factors such as the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). Here we demonstrate that MRR is facilitated by the formation of contact sites between mitochondria and the nucleus which establish microdomains of communication between the two organelles. The 18kD Translocator Protein (TSPO), which de-ubiquitylates and stabilizes the mitochondrial network preventing its mitophagy-mediated segregation, is required for this interaction. The tethering TSPO enacts is mediated by the complex formed with the Protein Kinase A via the A-kinase anchoring protein Acyl-CoA Binding Domain Containing 3 (ACBD3) and allows the redistribution of cholesterol which sustains the pro-survival response by blocking NF-kB de-acetylation. This work proposes a new paradigm in the mitochondrial retro-communication by revealing the existence of contact sites between mitochondrial and the nucleus and a signalling role for cholesterol.

cell biology↗