A Differential Fluorescence-Based Genetic Screen Identifies Listeria monocytogenes Determinants Required for Intracellular Replication
A Differential Fluorescence-Based Genetic Screen Identifies Listeria monocytogenes Determinants Required for Intracellular Replication
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DOI：
10.1128/jb.00210-13
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发表时间：
2013-08-01
影响因子：
3.2
通讯作者：
Higgins, Darren E.
中科院分区：
文献类型：
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作者：
Perry, Kyle J.;Higgins, Darren E.
单核细胞增生李斯特菌是一种革兰氏阳性的兼性细胞内病原体，能够引起严重的侵袭性疾病，在人类中具有高死亡率。虽然先前的研究已经在很大程度上阐明了细菌和宿主细胞入侵、空泡逃逸和随后的细胞间传播所必需的机制，但在宿主细胞质的限制性环境中快速复制所需的单核增生乳杆菌因子却知之甚少。在本报告中，我们描述了一种基于差异荧光的遗传筛选，利用荧光激活细胞分选（FACS）和高通量显微镜来鉴定单核增生乳杆菌在最佳细胞内复制中存在缺陷的突变体。在所鉴定的基因menD或pepP中存在缺失的细菌在原代小鼠巨噬细胞中生长缺陷和L2成纤维细胞单层斑块形成缺陷，从而验证了筛选方法鉴定细胞内复制缺陷突变体的能力。menD和pepP缺失菌株的遗传互补挽救了体外细胞内感染缺陷。此外，menD缺失菌株表现出普遍的细胞外复制缺陷，可以通过厌氧条件下的生长来弥补，而该菌株的细胞内生长缺陷可以通过添加外源甲基萘醌来弥补。正如先前的研究表明有氧代谢在单核增生乳杆菌感染中的重要性，这些发现进一步证明了甲基萘醌和有氧代谢在单核增生乳杆菌发病机制中的重要性。最后，在小鼠体内感染过程中，menD和pepP缺失菌株均被减毒。这些发现表明，基于差异荧光的筛选方法为鉴定多种细菌系统中的细胞内复制决定因素提供了有力的工具。
Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen capable of causing severe invasive disease with high mortality rates in humans. While previous studies have largely elucidated the bacterial and host cell mechanisms necessary for invasion, vacuolar escape, and subsequent cell-to-cell spread, the L. monocytogenes factors required for rapid replication within the restrictive environment of the host cell cytosol are poorly understood. In this report, we describe a differential fluorescence-based genetic screen utilizing fluorescence-activated cell sorting (FACS) and high-throughput microscopy to identify L. monocytogenes mutants defective in optimal intracellular replication. Bacteria harboring deletions within the identified gene menD or pepP were defective for growth in primary murine macrophages and plaque formation in monolayers of L2 fibroblasts, thus validating the ability of the screening method to identify intracellular replication-defective mutants. Genetic complementation of the menD and pepP deletion strains rescued the in vitro intracellular infection defects. Furthermore, the menD deletion strain displayed a general extracellular replication defect that could be complemented by growth under anaerobic conditions, while the intracellular growth defect of this strain could be complemented by the addition of exogenous menaquinone. As prior studies have indicated the importance of aerobic metabolism for L. monocytogenes infection, these findings provide further evidence for the importance of menaquinone and aerobic metabolism for L. monocytogenes pathogenesis. Lastly, both the menD and pepP deletion strains were attenuated during in vivo infection of mice. These findings demonstrate that the differential fluorescence-based screening approach provides a powerful tool for the identification of intracellular replication determinants in multiple bacterial systems.