Gayle Petoria, Freitag Nancy E, Strbo Natasa, Schesser Kurt
Department of Microbiology & Immunology, University of Miami Miller School of Medicine.
Department of Microbiology & Immunology, University of Illinois College of Medicine.
J Vis Exp. 2019 Feb 22(144). doi: 10.3791/58775.
There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.
细菌病原体一旦进入真核细胞后，会采用多种策略来生存和增殖。所谓的“胞质”病原体（单核细胞增生李斯特菌、福氏志贺菌、类鼻疽伯克霍尔德菌、土拉弗朗西斯菌和立克次氏体属）通过物理和酶促方式降解初级液泡膜进入被感染细胞的胞质溶胶。一旦进入胞质溶胶，这些病原体既能增殖，又能产生足够的机械力穿透宿主细胞的质膜以感染新细胞。在此，我们展示了如何通过集落形成单位测定法和流式细胞术对单核细胞增生李斯特菌（Lm）细胞感染周期的这一终末步骤进行定量，并举例说明病原体编码因子和宿主编码因子如何影响这一过程。我们还展示了体外感染的培养细胞与体内感染小鼠来源的肝细胞的Lm感染动态之间的密切对应关系。这些基于功能的测定相对简单，并且可以很容易地扩大规模，用于基于发现的真核细胞功能调节剂高通量筛选。
