Engineering commensal bacteria and probiotics for lactate conversion and treatment of sepsis

The gut microbiota and its metabolites play a crucial role in the clinical progression of sepsis, a leading cause of morbidity and mortality worldwide. Early evidence shows that engineered live bacterial therapeutics (LBTs) can be used to treat impaired systemic metabolism and immunity. Attempts at probiotic therapies for sepsis have been limited to traditional probiotic formulations with mixed results, in part due to poor engraftment and unclear mechanisms of action. Lactic acidosis is frequently observed in many conditions including sepsis, where it is strongly associated with poor clinical outcomes. Recent evidence indicates that circulating lactate enters the gut, where it can be fermented by commensal bacteria into beneficial byproducts which support organ function. The influence of the gut microbiota makes sepsis patients an ideal target population for synthetic probiotic therapy approaches, and the prevalence and availability of lactate during sepsis makes it an ideal substrate for the production of metabolites with therapeutic benefit. To leverage these factors, I propose to optimize and evaluate a probiotic platform to which optimizes gut microbial conversion of lactate to pyruvate, leading to the enrichment of molecules shown to be beneficial in sepsis models. Preliminary data indicates that 1) anti-anaerobic antibiotics worsen patient outcomes and hinder rates of systemic lactate conversion and that 2) oral administration of Bacillus subtilis engineered to express lactate oxidase (LOX) can accelerate systemic lactate conversion in murine models of lactic acidosis. These exciting findings prompt evaluation of the therapeutic efficacy of LOX expressing bacteria in the context of sepsis. With that said, the existing B. subtilis model is limited by poor engraftment, potentially leading to suboptimal efficacy and translation to chronic conditions. In this application, I propose to utilize the cecal slurry mouse model to evaluate the ability of LOX expressing bacteria to curtail sepsis severity, promote survival, and mitigate inflammation and organ damage (Aim 1). Concurrently, I will address limitations in the current B. Subtilis model by integrating LOX into native Escherichia coli with superior engraftment. This new model will contain circuity for lactate import and pyruvate export along with a CRISPR- based kill switch to ensure safety and biocontainment (Aim 2). The resultant strain will be evaluated for safety, colonization, and ability to curtail sepsis (Aims 1 and 2). Finally, both the B. subtilis and E. coli models will be used for in vivo ‘pulse’ experiments utilizing radiolabeled lactate to identify metabolic pathways influenced by their administration, identifying additional disease states for future applications (Aim 2). Building on existing patient data which identified a candidate mechanism of action, these projects seek to bridge the translational gap between microbiome engineering and human critical illness by leveraging lactic acidosis for the production of therapeutic byproducts. Given the vast array of disease states associated with impaired lactate and pyruvate metabolism, this platform has broad potential applications beyond sepsis. As a result, completion of this work will result in a potential low risk, scalable, and convenient therapeutic option for a wide range of pathologies. Project Number: 1F32AI194752-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Noah Hutchinson | Institution: UNIVERSITY OF MICHIGAN AT ANN ARBOR, ANN ARBOR, MI | Award Amount: $75,520 | Activity Code: F32 | Study Section: Special Emphasis Panel[ZRG1 F07A-W (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1F32AI19475201