Biology subjects

Hunt, D. M.

Publications and source records attributed to Hunt, D. M..

5 recordsLinked to original sources

bioRxiv2025

Pre-clinical validation of a novel AAV-mediated gene therapy for KCNV2 retinopathy improves visual function in a mouse model and expression in patient organoids

Voltage-gated (Kv) potassium channels are critical for neuronal physiology, and their dysfunction can lead to serious consequences. For example, mutations in the silent modulatory Kv8.2 subunit are known to cause irreversible inherited blindness (KCNV2 retinopathy). This is a currently incurable condition that causes lifelong visual loss, reduced visual acuity, photoaversion, night blindness and abnormal colour vision, alongside a distinctive supernormal electrophysiological (ERG) retinal response to light. In this study, we demonstrate that AAV-mediated gene replacement therapy delivering a codon-optimised human KCNV2 gene subretinally into Kv8.2 knock-out mice significantly restores retinal function. Treated mice exhibited improved ERG responses and correct expression of KCNV2 and its encoded Kv8.2 protein in photoreceptors. Recovery of visually guided scotopic and photopic optomotor responses to wildtype levels was achieved at lower vector doses, highlighting dose-dependent efficacy. Furthermore, treatment of human retinal organoids derived from a KCNV2 patient iPSC line resulted in substantial Kv8.2 protein rescue. This work provides the first preclinical proof-of-concept for the safety and therapeutic potential of gene therapy for KCNV2 retinopathy, laying a strong foundation for future clinical trials.

neuroscience↗

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

Divergent downstream biosynthetic pathways are supported by L-cysteine synthases of Mycobacterium tuberculosis

Mycobacterium tuberculosiss (Mtb) autarkic lifestyle within the host involves rewiring its transcriptional networks to combat host-induced stresses. With the help of RNA-seq performed under various stress conditions, we identified that genes belonging to Mtb sulfur metabolism pathways are significantly upregulated during oxidative stress. Using an integrated approach of microbial genetics, transcriptomics, metabolomics, animal experiments, chemical inhibition, and rescue studies, we investigated the biological role of non-canonical L-cysteine synthases, CysM and CysK2. While transcriptome signatures of Rv{Delta}cysM and Rv{Delta}cysK2 appear similar under regular growth conditions, we observed unique transcriptional signatures when subjected to oxidative stress. We followed pool size and labelling (34S) of key downstream metabolites, viz. mycothiol and ergothioneine, to monitor L-cysteine biosynthesis and utilization. This revealed the significant role of distinct L-cysteine biosynthetic routes on redox stress and homeostasis. CysM and CysK2 independently facilitate Mtb survival by alleviating host-induced redox stress, suggesting they are not fully redundant during infection. With the help of genetic mutants and chemical inhibitors, we show that CysM and CysK2 serve as unique, attractive targets for adjunct therapy to combat mycobacterial infection.

microbiology↗

bioRxiv2018

Nitrogen utilisation by the metabolic generalist pathogen Mycobacterium tuberculosis

Bacterial metabolism is fundamental to pathogenesis and has a dominant effect on bacterial killing by antibiotics. Here we explore how Mycobacterium tuberculosis utilises amino acids as nitrogen sources, using a combination of bacterial physiology and stable isotope tracing coupled to liquid chromatography mass spectrometry metabolomics methods. Our results define core properties of the nitrogen metabolic network from M. tuberculosis, such as: (i) the lack of homeostatic control of amino acid pool sizes; (ii) similar rates of utilisation of different amino acids as sole nitrogen sources; (iii) improved nitrogen utilisation from amino acids compared to ammonium; and (iv) co-metabolism of nitrogen sources. Finally, we discover that alanine dehydrogenase, is involved in ammonium assimilation in M. tuberculosis, in addition to its essential role in alanine utilisation. This study represents the first in-depth analysis of nitrogen source utilisation by metabolic generatlist M. tuberculosis and reveals a flexible metabolic network with characteristics that are likely product of evolution in the human host.

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↗