Bactericidal antibiotics are powerful agents due to their ability to convert essential bacterial functions into lethal processes. However, many important bacterial pathogens are remarkably tolerant against bactericidal antibiotics due to inducible damage repair responses. The cell wall damage response two-component system VxrAB of the gastrointestinal pathogen Vibrio cholerae promotes high-level β-lactam tolerance and controls a gene network encoding highly diverse functions, including negative control over multiple iron uptake systems. How this system contributes to tolerance is poorly understood. Here, we show that β-lactam antibiotics cause an increase in intracellular free iron levels and collateral oxidative damage, which is exacerbated in the ∆vxrAB mutant. Mutating major iron uptake systems dramatically increases ∆vxrAB tolerance to β-lactams. We propose that VxrAB reduces antibiotic-induced toxic iron and concomitant metabolic perturbations by downregulating iron uptake transporters and show that iron sequestration enhances tolerance against β-lactam therapy in a mouse model of cholera infection. Our results suggest that a microorganism's ability to counteract diverse antibiotic-induced stresses promotes high-level antibiotic tolerance and highlights the complex secondary responses elicited by antibiotics. The cholera pathogen Vibrio cholerae is highly tolerant against β-lactam antibiotics due to its sensing system VxrAB. Upon sensing antibiotic exposure, VxrAB upregulates cell wall synthesis functions and downregulates iron acquisition systems, which are both required for antibiotics tolerance. The cholera pathogen Vibrio cholerae is highly tolerant against β-lactam antibiotics due to its sensing system VxrAB. Upon sensing antibiotic exposure, VxrAB upregulates cell wall synthesis functions and downregulates iron acquisition systems, which are both required for antibiotics tolerance.

Background: Mixed phenotype acute leukemia is a rare disease characterized by an expanded blast population exhibiting multiple lineage features. It is a diagnosis of exclusion, and we and others have observed that AML-MRC or therapy-related AML cases may also exhibit similar features. We therefore sought to determine whether multi-lineage expression is present in de-novo and therapy-related MDS and whether it has an impact on clinical outcomes. Methods: We reviewed pathology, flow cytometry, cytogenetic, and molecular reports from patients seen at Memorial Sloan Kettering Cancer Center between 1996 and 2020 and identified 472 patients diagnosed with MDS using a combination of custom natural language processing tools and manual review. Cox proportional hazards modeling was performed to assess the contribution of patient characteristics, pathology, flow cytometric, cytogenetic, and molecular characteristics to overall survival (OS). Fisher's exact testing was used to assess the association of individual features. Results: We found that an abnormal myeloid lineage signature was associated with poorer OS after adjusting for age, cytogenetics, and molecular features (HR=2.3, p<0.01). In contrast, an abnormal plasma cell signature was associated with better OS (HR=0.3, p<0.01) (Figure A). Other abnormal lineages did not contribute significantly to OS although there were relatively few cases with abnormal T-cell markers (n=19). Mutations in TP53, IDH1, and EZH2 were associated with poorer OS (TP53: HR=1.5, p<0.01; IDH1: HR=2.3, p<0.01, EZH2: HR=1.9, p<0.01) and mutations in SF3B1 and IDH2 were associated with better OS (SF3B1: HR=0.35, p<0.01; IDH2: HR=0.6, p=0.05) (Figure B). TP53 mutation was enriched among patients with intermediate and poor risk cytogenetic groups (OR=11.3, p<0.01), consistent with higher risk therapy-related disease. IDH2, TET2, and ASXL1, which have been associated with clonal hematopoiesis, were enriched in the good and intermediate risk cytogenetic groups (IDH2: OR=4.3; p=0.03, TET2: OR=3.7, p<0.01; ASXL1: OR=4.3, p<0.01), although only IDH2 had a significant association with OS. Interestingly, there was a significant association of plasma cell lineage abnormalities with SRSF2 mutation (OR=3.5, p=0.05), but SRSF2 mutation was not an independent contributor to the global survival model (Figure C). Conclusion: Routine flow cytometric and molecular assessment of diagnostic bone marrows can provide added prognostic value in MDS. Identification of an abnormal myeloid lineage is associated with higher risk disease, while identification of abnormal plasma cell features is associated with lower risk disease. SRSF2 mutation is also associated with abnormal plasma cell features, but does not contribute independently to OS. Abnormal T-cell features may also provide prognostic value, although assessment in a larger cohort will be necessary. We confirm previous findings indicating that TP53 mutation is associated with higher risk disease while SF3B1 mutation is associated with better outcomes characteristic of MDS-RARS. In addition, our data suggests that IDH1 and EZH2 are associated with poorer outcomes and that IDH2 is associated with lower risk disease. Figure 1 Disclosures Tallman: Bioline rx: Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Abbvie: Research Funding; Cellerant: Research Funding; Orsenix: Research Funding; ADC Therapeutics: Research Funding; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; Glycomimetics: Research Funding; Rafael: Research Funding; Oncolyze: Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Novartis: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; KAHR: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees. Levine:Morphosys: Consultancy; Astellas: Consultancy; Janssen: Consultancy; Lilly: Consultancy, Honoraria; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Gilead: Honoraria; Novartis: Consultancy; Amgen: Honoraria; Roche: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Xiao:Stemline Therapeutics: Research Funding. Glass:Gerson Lehman Group: Consultancy.

Many bacteria are resistant to killing (tolerant) by typically bactericidal antibiotics due to their ability to counteract drug-induced cell damage. Vibrio cholerae, the cholera agent, displays an unusually high tolerance to diverse inhibitors of cell wall synthesis. Exposure to these agents, which in other bacteria leads to lysis and death, results in a breakdown of the cell wall and subsequent sphere formation in V. cholerae. Spheres readily recover to rod-shaped cells upon antibiotic removal, but the mechanisms mediating the recovery process are not well characterized. Here, we found that the mechanisms of recovery are dependent on environmental conditions. Interestingly, on agarose pads, spheres undergo characteristic stages during the restoration of rod shape. Drug inhibition and microscopy experiments suggest that class A penicillin binding proteins (aPBPs) play a more active role than the Rod system, especially early in sphere recovery. Transposon insertion sequencing (TnSeq) analyses revealed that lipopolysaccharide (LPS) and cell wall biogenesis genes, as well as the sigma E cell envelope stress response, were particularly critical for recovery. LPS core and O-antigen appear to be more critical for sphere formation/integrity and viability than lipid A modifications. Overall, our findings demonstrate that the outer membrane is a key contributor to beta lactam tolerance and suggest a role for aPBPs in cell wall biogenesis in the absence of rod-shape cues. Factors required for postantibiotic recovery could serve as targets for antibiotic adjuvants that enhance the efficacy of antibiotics that inhibit cell wall biogenesis.