Biology subjects

Walters, R. C.

Publications and source records attributed to Walters, R. C..

3 recordsLinked to original sources

bioRxiv2025

Bioenergetic metabolism restarts alongside germinant sensing and hydration in bacterial spore germination

Bacterial spore germination transforms spores from dormant bodies to vegetative cells. Initiation of germination or spore rehydration can proceed without exogenous energy sources, so bioenergetic processes in germination have been overlooked, despite many germinants being energy sources, such as sugars. Here, we apply remission spectroscopy to noninvasively measure the energy-transducing electron-transport chain of intact spores during germination. In Bacillus megaterium and Bacillus subtilis, we find energisation of cytoplasmic metabolism and the electron transport chain occurs early in germination, before or alongside rehydration. The aa3-type oxidases (Qox, Cta) accumulate nonradical ferryl intermediates of their catalytic cycle demonstrating that the electron-transport chain is operating in a regime of high membrane potential. The Yth isoform of the bd oxidase is found in abundance in both organisms, functioning to allow rapid electron transfer to O2 when the aa3-type oxidases are hindered, establishing a new role for this enzyme beyond O2-scavenging. Deletion of Yth slows rehydration, directly linking bioenergetic processes to germination. Our data lead us to propose a powered germination model, where the Ger-mediated signalling cascade and bioenergetic processes occur simultaneously and semi-independently.

biophysics↗

bioRxiv2024

Remission spectroscopy resolves the mode of action of bedaquiline within living mycobacteria

Bedaquiline, an ATP synthase inhibitor, is the spearhead of transformative therapies against drug-resistant Mycobacterium tuberculosis. Here, remission spectroscopy is used to measures the energy-transducing cytochromes within unperturbed, respiring suspensions of mycobacterial and human cells, allowing spectroscopic measurements of electron transport chains as they power living cells and respond to bedaquiline. No evidence is found for protonophoric or ionophoric uncoupling. Rather, by directly inhibiting ATP synthase, bedaquiline slows the respiratory supercomplex (Qcr:Cta; bcc:aa3) by increasing the proton-motive force, causing sub-second redirection of electron flux through the cytochrome bd oxidase (Cyd) to O2. Electron flux redirection explains the idiosyncratic bedaquiline-induced increase in O2 consumption rates previously observed. Redirection occurs as Cyd is present even in cells grown in plentiful O2. Applying the same approach to human cells did not detect bedaquiline-induced inhibition of mitochondrial function despite such inhibition being seen in isolated systems. Overall, we clarify how bedaquiline works, why different models for its action developed, and the mechanisms underlying the synergy of bedaquiline in combination regimes.

biophysics↗

bioRxiv2023

Tumour mitochondrial DNA mutations drive aerobic glycolysis to enhance checkpoint blockade

The mitochondrial genome encodes essential machinery for respiration and metabolic homeostasis but is paradoxically among the most common targets of somatic mutation in the cancer genome, with truncating mutations in respiratory complex I genes being most over-represented1. While mitochondrial DNA (mtDNA) mutations have been associated with both improved and worsened prognoses in several tumour lineages1-,3, whether these mutations are drivers or exert any functional effect on tumour biology remains controversial. Here we discovered that complex I-encoding mtDNA mutations are sufficient to remodel the tumour immune landscape and therapeutic resistance to immune checkpoint blockade. Using mtDNA base editing technology4 we engineered recurrent truncating mutations in the mtDNA-encoded complex I gene, Mt-Nd5, into murine models of melanoma. Mechanistically, these mutations promoted utilisation of pyruvate as a terminal electron acceptor and increased glycolytic flux without major effects on oxygen consumption, driven by an over-reduced NAD pool and NADH shuttling between GAPDH and MDH1, mediating a Warburg-like metabolic shift. In turn, without modifying tumour growth, this altered cancer cell-intrinsic metabolism reshaped the tumour microenvironment in both mice and humans, promoting an anti- tumour immune response characterised by loss of resident neutrophils. This subsequently sensitised tumours bearing high mtDNA mutant heteroplasmy to immune checkpoint blockade, with phenocopy of key metabolic changes being sufficient to mediate this effect. Strikingly, patient lesions bearing >50% mtDNA mutation heteroplasmy also demonstrated a >2.5-fold improved response rate to checkpoint inhibitor blockade. Taken together these data nominate mtDNA mutations as functional regulators of cancer metabolism and tumour biology, with potential for therapeutic exploitation and treatment stratification.

cancer biology↗