Merk Liana N, Shur Andrey S, Jena Smrutiti, Munoz Javier, Brubaker Douglas K, Murray Richard M, Green Leopold N
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, United States.
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, United States.
ACS Synth Biol. 2024 Dec 20;13(12):3885-3896. doi: 10.1021/acssynbio.3c00668. Epub 2024 Nov 28.
Bacteria genetically engineered to execute defined therapeutic and diagnostic functions in physiological settings can be applied to colonize the human microbiome, providing in situ surveillance and conditional disease modulation. However, many engineered microbes can only respond to single-input environmental factors, limiting their tunability, precision, and effectiveness as living diagnostic and therapeutic systems. For engineering microbes to improve complex chronic disorders such as inflammatory bowel disease, the bacteria must respond to combinations of stimuli in the proper context and time. This work implements a previously characterized split activator AND logic gate in the probiotic strain Nissle 1917 (EcN). Our system can respond to two input signals: the inflammatory biomarker tetrathionate and a second input signal, anhydrotetracycline (aTc), for manual control. We report 4-6 fold induction with a minimal leak when the two chemical signals are present. We model the AND gate dynamics using chemical reaction networks and tune parameters in silico to identify critical perturbations that affect our circuit's selectivity. Finally, we engineer the optimized AND gate to secrete a therapeutic anti-inflammatory cytokine IL-22 using the hemolysin secretion pathway in the probiotic strain. We used a germ-free transwell model of the human gut epithelium to show that our engineering bacteria produce similar host cytokine responses compared to recombinant cytokine. Our study presents a scalable workflow to engineer cytokine-secreting microbes driven by logical signal processing. It demonstrates the feasibility of IL-22 derived from probiotic EcN with minimal off-target effects in a gut epithelial context.
经过基因工程改造以在生理环境中执行特定治疗和诊断功能的细菌可用于定殖人类微生物群，提供原位监测和条件性疾病调节。然而，许多工程微生物只能对单一输入环境因素做出反应，这限制了它们作为活体诊断和治疗系统的可调性、精确性和有效性。为了改造微生物以改善诸如炎症性肠病等复杂慢性疾病，细菌必须在适当的背景和时间对多种刺激组合做出反应。这项工作在益生菌菌株Nissle 1917（EcN）中实现了先前表征的分裂激活剂与门逻辑门。我们的系统可以对两个输入信号做出反应：炎症生物标志物连四硫酸盐和用于人工控制的第二个输入信号脱水四环素（aTc）。我们报告说，当存在这两种化学信号时，诱导倍数为4至6倍，且泄漏最小。我们使用化学反应网络对与门动力学进行建模，并在计算机上调整参数，以识别影响我们电路选择性的关键扰动。最后，我们对优化后的与门进行改造，使其利用益生菌菌株中的溶血素分泌途径分泌治疗性抗炎细胞因子IL-22。我们使用人肠道上皮的无菌Transwell模型表明，与重组细胞因子相比，我们的工程细菌产生相似的宿主细胞因子反应。我们的研究提出了一种可扩展的工作流程，用于改造由逻辑信号处理驱动的分泌细胞因子的微生物。它证明了在肠道上皮环境中，源自益生菌EcN的IL-22具有最小脱靶效应的可行性。
