Flaherty Rebecca A, Lee Shaun W
Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame.
Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame;
J Vis Exp. 2016 Aug 19(114):54406. doi: 10.3791/54406.
Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.
许多细菌病原体分泌强效毒素，以帮助破坏宿主组织、引发宿主细胞的信号变化或在感染过程中操纵免疫系统反应。尽管已经开发出方法来成功纯化和生产许多这些重要的毒力因子，但仍有许多细菌毒素因其独特的结构或广泛的翻译后修饰而难以在体外系统中纯化和研究。此外，即使能够获得纯毒素，在相关生理条件下研究毒素的特定作用仍存在许多挑战。大多数旨在评估分泌型细菌毒素对宿主细胞影响的体外细胞培养模型，都涉及用一次性剂量的毒素孵育宿主细胞。这种方法很难模拟宿主细胞在感染期间实际经历的情况，即细菌细胞持续产生毒素，并在感染过程中逐渐积累。本方案描述了一种基于可渗透膜插入物的细菌感染系统的设计，用于研究A组链球菌产生的强效毒素链球菌溶素S对人上皮角质形成细胞的影响。与将纯毒素或细菌上清液直接应用于宿主细胞的方法相比，该系统更接近感染期间的自然生理环境。重要的是，该方法还消除了由于细菌与宿主细胞直接接触而导致的宿主反应偏差。该系统已被用于有效评估链球菌溶素S（SLS）对宿主膜完整性、细胞活力和细胞信号反应的影响。这种技术可以很容易地应用于研究其他分泌型毒力因子对多种哺乳动物宿主细胞类型的影响，以研究分泌型细菌因子在感染过程中的特定作用。
