A Single Intravenous Dose of Indole-producing Attenuated Brucella in NOD Mice: Assessment of Type 1 Diabetes Onset and Islet Immune Remodeling.

Type 1 diabetes (T1D) results from autoimmune destruction of pancreatic β cells, and current therapies fail to restore durable immune tolerance. Metabolically engineered bacteria producing immunomodulatory metabolites represent a promising single-dose therapeutic strategy. Here, this paper presents a comprehensive protocol in which a single intravenous administration of an indole-producing attenuated Brucella melitensis strain BmΔvjbR::tnaA is used to assess effects on the onset of autoimmune diabetes in prediabetic female NOD/ShiLtJ mice. The protocol begins with preparing BmΔvjbR::tnaA from frozen glycerol stocks, growing it in selective media to mid-log phase, and calculating colony-forming units based on optical density. Female NOD mice, <5-week-old are restrained without anesthesia and injected intravenously via lateral tail vein with a defined bacterial dose in phosphate-buffered saline while maintained on heating pads. Blood glucose is monitored 2x weekly after a 2 h fast for 70-105 days, with diabetes defined as glucose ≥250 mg/dL on two consecutive readings. At experimental endpoints, harvested pancreas are fixed in neutral-buffered formalin for 72 h, paraffin-embedded, and sectioned for hematoxylin and eosin staining, insulin immunohistochemistry, and total islet area quantification. Formalin-fixed paraffin-embedded sections are submitted for spatial proteomics with a 25-marker antibody panel to visualize regulatory and effector immune populations. Finally, fresh pancreatic tissue undergoes single-cell isolation, 3' library preparation, sequencing, and Seurat-based bioinformatics analysis to profile transcriptional changes across immune and stromal populations. This integrated protocol enables reproducible single-dose bacterial therapy delivery and multi-modal characterization of islet immune remodeling, providing a framework for testing metabolite-engineered microbes in autoimmune models and dissecting tissue-specific immune mechanisms at single-cell resolution.