Conroy, T. | bioRxiv Science

bioRxiv2026

Filament formation is a conserved mechanism of Thoeris SIR2 effector activation

Thoeris defence systems protect bacteria from phages via abortive infection. In type I Thoeris systems, ThsA effectors containing silent information regulator 2 (SIR2) and SMF/DprA-LOG (SLOG) domains are activated by the cyclic ADP-ribose (ADPR) isomer 3'cADPR, triggering abortive infection via nicotinamide adenine dinucleotide (NAD+) depletion. 3'cADPR activates the NADase activity of Bacillus subtilis ThsA tetramers via filament formation of its SIR2 domains, but the molecular details of how 3'cADPR triggers this process remain incompletely understood. Here, we demonstrate that ThsA activation by 3'cADPR-induced SIR2 filament formation is conserved in type I Thoeris systems from Streptococcus equi and Entercoccus facium. We present cryo-electron microscopy structures of the S. equi ThsA filament bound to 3'cADPR and the non-cleavable NAD+ analog carba-NAD+, and of the S. equi ThsA tetramer bound to 3'cADPR. These structures reveal that SIR2 filament formation is required to stabilise an active site conformation that can bind and hydrolyse NAD+. The structures also show that 3'cADPR induces quaternary alterations in the SLOG dimers and consequently the SIR2 tetramer to enable ThsA filament formation. Collectively, our study provides a comprehensive understanding of 3'cADPR-induced activation of type I Thoeris effectors.

microbiology↗

bioRxiv2025

Prediction of Gemcitabine sensitivity in resectable Pancreatic Cancer using a Glycation Stress Transcriptomic Signature

BACKGROUND & AIMSPancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with limited response to systemic therapy. Gemcitabine (GEM) benefits only a subset of patients. Methylglyoxal (MG), a glycolysis byproduct, has been linked to tumor behavior and therapy response in PDAC, suggesting potential as a stratification marker. METHODSWe developed a metabolically informed gene signature (MG-GEM) integrating MG- related glycolytic stress with clinical outcomes. Using the Puleo cohort (n=309), differential expression analysis between tumors with high and low MG stress identified 365 genes. LASSO Cox regression selected 16 prognostic genes, combined into a weighted risk score for patient stratification. MG GEM was validated in internal and external cohorts, including PRODIGE 24/CCTG PA6. Molecular, transcriptomic, and immune features were compared between high and low MG GEM groups. Finally, predictive performance was evaluated against the GemPred signature. RESULTSMG GEM divided PDAC patients into distinct risk groups with marked differences in overall and disease-free survival among GEM treated patients (OS 11.7 vs 27.2 months; DFS 7.6 vs 17.8 months, both p<0.0001). High MG-GEM tumors showed enrichment for KRAS G12D and SMAD4 mutations, basal and activated stroma subtypes, glycolytic metabolism, and reduced immune infiltration. Low MG-GEM tumors showed KRAS G12V, classical and immune subtypes, cholesterogenic metabolism, and adaptive immune favourable signatures. MG-GEM independently predicted GEM-specific clinical outcomes, irrespective of GemPred signature, and significantly enhanced patient stratification when combined with it. Within the PRODIGE-24/TGCC PA6 cohort, MG-GEM exhibited prognostic relevance and selectively identified patients who derived a survival benefit from adjuvant GEM, but not from FOLFIRINOX. CONCLUSIONSThe 16 gene MG GEM signature predicts prognosis in resected PDAC, reflects glycolytic stress driven chemoresistance, surpassing conventional molecular classifications. As a metabolically informed signature, MG-GEM holds promise for guiding chemotherapy selection and informing KRAS-targeted combination strategies, meriting further prospective clinical validation.

cancer biology↗