Engineered Escherichia coli Nissle 1917 carrying D-lactate dehydrogenase to suppress glioblastoma malignancy via gut-brain axis reprogramming.

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Background: Glioblastoma (GBM) is the most aggressive primary brain tumor with limited therapeutic options and poor prognosis. Metabolic reprogramming is a hallmark of GBM, yet the role of D-lactate, an understudied stereoisomer of lactate, remains elusive. While L-lactate has been extensively characterized, we identified D-lactate as a systemically circulating oncometabolite that disrupts intestinal homeostasis via the gut-brain axis, representing an unexplored therapeutic vulnerability in GBM. Methods: Through untargeted metabolomics of patient-derived GBM stem-like cells and brain organoid co-culture models, we characterized D-lactate as a key tumor-secreted metabolite. We screened human and murine gut microbiota to isolate D-lactate-metabolizing bacteria, cloned six highly active D-lactate dehydrogenases (LDHDs), and engineered probiotic Escherichia coli Nissle 1917 (EcN) to express optimal variants. Lipid-coated therapeutic bacteria (Lipo-EcN-LDHD) were developed to enhance gastric stability and intestinal colonization. Therapeutic efficacy was evaluated in orthotopic GBM xenograft models using bioluminescence imaging, survival analysis, histopathology, and metagenomic sequencing. Results: GBM-bearing mice exhibited ~4-fold elevation in serum D-lactate (~150 μM) compared to controls, accompanied by intestinal shortening and dysbiosis. Natural D-lactate-metabolizing bacteria significantly suppressed tumor growth and prolonged survival. Enzymatic characterization identified MeLDHD3 as the most catalytically efficient variant. Oral administration of Lipo-EcN-MeLDHD3 achieved robust intestinal colonization, reduced systemic D-lactate levels, significantly inhibited orthotopic tumor growth ( P <0.01), and prolonged survival ( P <0.05). Treatment restored gut microbiota homeostasis, reduced tumor Ki67 proliferation index, and attenuated D-lactate-mediated immunosuppressive microenvironment. Conclusions: This study establishes D-lactate as a novel metabolic target in GBM and demonstrates the first synthetic biology-inspired "gut-heals-brain" therapeutic strategy. Engineered probiotic EcN expressing LDHD enables non-invasive, sustained systemic clearance of tumor-derived D-lactate, offering a safe, precision metabolic intervention for GBM. This platform validates the gut-brain axis as a druggable conduit for brain tumor therapy and provides a transformative paradigm for treating metabolically driven malignancies.