Dilucca Marisa, Broz Petr
Department of Immunobiology, University of Lausanne, Epalinges, Switzerland.
Methods Mol Biol. 2023;2641:49-65. doi: 10.1007/978-1-0716-3040-2_5.
The non-canonical inflammasome pathway functions as the primary cytosolic innate immune detection mechanism for Gram-negative bacterial lipopolysaccharide (LPS) in human and mouse cells and controls the proteolytic activation of the cell death executor gasdermin D (GSDMD). The main effectors of this pathways are the inflammatory proteases caspase-11 in mice and caspase-4/caspase-5 in humans. These caspases have been shown to bind LPS directly; however, the interaction between LPS and caspase-4/caspase-11 requires a set of interferon (IFN)-inducible GTPases, known as guanylate-binding proteins (GBPs). These GBPs assemble to form coatomers on cytosolic Gram-negative bacteria, which function as recruitment and activation platforms for caspase-11/caspase-4. Here we describe an assay to monitor caspase-4 activation in human cells by immunoblotting and its recruitment to intracellular bacteria using the model pathogen Burkholderia thailandensis.
非经典炎性小体途径作为人和小鼠细胞中革兰氏阴性菌脂多糖（LPS）的主要胞质内固有免疫检测机制，并控制细胞死亡执行蛋白gasdermin D（GSDMD）的蛋白水解激活。该途径的主要效应分子是小鼠中的炎性蛋白酶caspase-11和人类中的caspase-4/caspase-5。这些半胱天冬酶已被证明可直接结合LPS；然而，LPS与caspase-4/caspase-11之间的相互作用需要一组干扰素（IFN）诱导的GTP酶，即鸟苷酸结合蛋白（GBP）。这些GBP组装形成胞质革兰氏阴性菌上的包被蛋白，作为caspase-11/caspase-4的募集和激活平台。在这里，我们描述了一种通过免疫印迹监测人细胞中caspase-4激活及其使用模式病原体泰国伯克霍尔德菌募集到细胞内细菌的检测方法。
