Weber Brent S, Ly Pek Man, Feldman Mario F
Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO, 63110, USA.
Methods Mol Biol. 2017;1615:465-472. doi: 10.1007/978-1-4939-7033-9_32.
The bacterial type VI secretion system (T6SS) is a secretory apparatus encoded by many Gram-negative bacteria. The T6SS facilitates the secretion and injection of toxic effector proteins into host cells, providing a competitive advantage to bacteria encoding this machinery. The activity of the T6SS can be monitored by probing for the conserved tubule component Hcp, which is secreted to the supernatants by the T6SS. Detection of Hcp in culture supernatants is indicative of an active T6SS, but this secretion system is often tightly regulated or inactive under laboratory conditions and different bacterial strains display differing Hcp secretion phenotypes. Herein, we describe an enzyme-linked immunosorbent assay (ELISA) and colony blot methods to facilitate large-scale screening of isolates for Hcp secretion and, thus, T6SS activity.
细菌VI型分泌系统（T6SS）是一种由许多革兰氏阴性菌编码的分泌装置。T6SS有助于将有毒效应蛋白分泌并注射到宿主细胞中，为编码该机制的细菌提供竞争优势。T6SS的活性可以通过探测保守的小管成分Hcp来监测，Hcp由T6SS分泌到上清液中。在培养上清液中检测到Hcp表明T6SS处于活跃状态，但该分泌系统在实验室条件下通常受到严格调控或处于非活性状态，并且不同的细菌菌株表现出不同的Hcp分泌表型。在此，我们描述了一种酶联免疫吸附测定（ELISA）和菌落印迹方法，以促进对分离株进行大规模筛选，检测其Hcp分泌情况，从而评估T6SS活性。
