Biology subjects

Tait, J. R.

Publications and source records attributed to Tait, J. R..

4 recordsLinked to original sources

bioRxiv2026

Direct observation of ATP-driven ubiquitin chain handling by Cdc48

Cdc48 (p97 or VCP in metazoans) targets polyubiquitin to selectively disassemble and degrade proteins. Cdc48 is believed to move along the ubiquitin chain towards linked proteins, but this has not been directly observed. By following single molecules in time, we find that the polypeptide branch points of ubiquitin chains are repeatedly inserted and rejected from the Cdc48 pore in ATP-driven manner, in bursts lasting up to seconds. This non-processive mode either ends by terminal substrate rejection, or advances to processive action, which drives ubiquitin unfolding in two steps, branch point translocation, and the extrusion of ubiquitin and linked protein as polypeptide loops in trans. Final retrograde movement can bring polypeptide segments back to cis. Our results establish the dynamics of ubiquitin chain handling by Cdc48, reveal key hallmarks of kinetic proofreading. We speculate that Cdc48 translocation may play a role in ubiquitin chain selection.

biophysics↗

bioRxiv2025

Bioactive Cationic Lipidated Oligomers (CLOs) as Antimicrobial Materials: Metabolomic Insights into MRSA Membrane Disruption

The escalating incidence of antimicrobial resistance in Staphylococcus aureus, particularly the methicillin-resistant strain (MRSA), necessitates the development of novel therapeutic strategies. Cationic lipidated oligomers (CLOs) have emerged as promising membrane-active antimicrobial agents; however, their mechanisms of action remain insufficiently understood. In this study, untargeted metabolomics was employed to systematically profile the temporal metabolic perturbations induced by two structurally distinct CLOs, C12-o-DMEN-10 and C12-o-BEDA-10, in MRSA across four defined time points (0.25, 0.5, 1, and 3 hours). These CLOs previously demonstrated differential antibacterial activity, as evidenced by dose-dependent propidium iodide (PI) uptake and growth inhibition assays. Metabolomic analysis revealed pronounced and sustained disruptions in bacterial membrane lipid metabolism, including significant depletion of phosphatidylglycerols ([≥] -2.5 log2FC, p < 0.05), alongside elevated levels of phosphatidylethanolamines, lysophospholipids, and fatty acid-derived metabolites indicative of membrane destabilization and lipid remodelling. Although both CLOs affected overlapping metabolic pathways, they differed in the extent and temporal dynamics of their effects. These findings provide mechanistic insights into CLO-mediated antibacterial activity and highlight the value of metabolomics in elucidating both direct and downstream cellular responses, which extend beyond the scope of conventional membrane integrity assays, such as PI fluorescence.

systems biology↗

bioRxiv2024

Providing insight into the mechanism of action of Cationic Lipidated Oligomers (CLOs) using metabolomics

The increasing resistance of clinically relevant microbes against current commercially available antimicrobials underpins the urgent need for alternative and novel treatment strategies. Cationic lipidated oligomers (CLOs) are innovative alternatives to antimicrobial peptides, and have reported antimicrobial potential. An understanding of their antimicrobial mechanism of action is required to rationally design future treatment strategies for CLOs, either in monotherapy or synergistic combinations. In the present study, metabolomics was used to investigate the potential metabolic pathways involved in the mechanisms of antibacterial activity of one CLO, C12-o-(BG-D)-10, which we have previously shown to be effective against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. The metabolomes of MRSA ATCC 43300 at 1, 3 and 6 h following treatment with C12-o-(BG-D)-10 (48 {micro}g/mL i.e., 3x MIC) were compared to those of the untreated controls. Our findings reveal that the studied CLO, C12-o-(BG-D)-10, disorganized the bacterial membrane as the first step towards its antimicrobial effect, as evidenced by marked perturbations in the bacterial membrane lipids and peptidoglycan biosynthesis observed at early time points i.e., 1, and 3 h. Central carbon metabolism, and biosynthesis of DNA, RNA, and arginine were also vigorously perturbed, mainly at early time points. Moreover, bacterial cells were under osmotic and oxidative stress across all time points, evident by perturbations of trehalose biosynthesis and pentose phosphate shunt. Overall, this metabolomics study has, for the first time, revealed that the antimicrobial action of C12-o-(BG-D)-10 may potentially stem from the dysregulation of multiple metabolic pathways. ImportanceAntimicrobial resistance poses a significant challenge to healthcare systems worldwide. Novel anti-infective therapeutics are urgently needed to combat drug-resistant microorganisms. Cationic lipidated oligomers (CLOs) show promise as new antibacterial agents against Gram-positive pathogens like Staphylococcus aureus (MRSA). Understanding their molecular mechanism(s) of antimicrobial action may help design synergistic CLO treatments along with monotherapy. Here, we describe the first metabolomics study to investigate the killing mechanism(s) of CLOs against MRSA. The results of our study indicate that the CLO, C12-o-(BG-D)-10, had a notable impact on the biosynthesis and organization of the bacterial cell envelope. C12-o-(BG-D)-10 also inhibits arginine, histidine, central carbon metabolism, and trehalose production, adding to its antibacterial characteristics. This work illuminates the unique mechanism of action of C12-o-(BG-D)-10 and opens an avenue to design innovative antibacterial oligomers/polymers for future clinical applications.

microbiology↗

bioRxiv2024

Elucidating effects of single and multiple resistance mechanisms on bacterial response to meropenem by quantitative and systems pharmacology modeling and population genomics

Meropenem is commonly used against Pseudomonas aeruginosa. Traditionally, the time unbound antibiotic concentration exceeds the MIC (fT>MIC) is used to select carbapenem regimens. We aimed to: characterize the effects of different baseline resistance mechanisms on bacterial killing and resistance emergence; evaluate whether fT>MIC can predict these effects; and, develop a novel quantitative and systems pharmacology (QSP) model to describe effects of baseline resistance mechanisms on the time-course of bacterial response. Seven isogenic P. aeruginosa strains with a range of resistance mechanisms and MICs were used in 10-day hollow-fiber infection model studies. Meropenem pharmacokinetic profiles were simulated for various regimens (t1/2,meropenem=1.5h). All viable counts on drug-free, 3xMIC and 5xMIC meropenem-containing agar across all strains, five regimens and control (n=90 profiles) were simultaneously subjected to QSP modeling. Whole genome sequencing was completed for total population samples and emergent resistant colonies at 239h. Regimens achieving [≥]98%fT>1xMIC suppressed resistance emergence of the mexR knockout strain. Even 100%fT>5xMIC failed to achieve this against the strain with OprD loss and the ampD and mexR double-knockout strain. Baseline resistance mechanisms affected bacterial outcomes, even for strains with the same MIC. Genomic analysis revealed that pre-existing resistant subpopulations drove resistance emergence. During meropenem exposure, mutations in mexR were selected in strains with baseline oprD mutations, and vice versa, confirming these as major mechanisms of resistance emergence. Secondary mutations occurred in lysS or argS, coding for lysyl and arginyl tRNA synthetases, respectively. The QSP model well characterized all bacterial outcomes of the seven strains simultaneously, which fT>MIC could not.

microbiology↗