Cytosolic Replication of Group A Streptococcus in Human Macrophages.
Cytosolic Replication of Group A Streptococcus in Human Macrophages.
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DOI：
10.1128/mbio.00020-16
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发表时间：
2016-04-12
期刊：
影响因子：
6.4
通讯作者：
Holden DW
中科院分区：
文献类型：
--
作者：
O'Neill AM;Thurston TL;Holden DW
巨噬细胞作为先天免疫防御的关键组成部分，在控制包括A群链球菌（GAS）在内的细菌病原体中至关重要。尽管如此，只有有限数量的研究分析了人类中性粒细胞和巨噬细胞中GAS的恢复。在这里，我们通过几种定量方法确定了人巨噬细胞中GAS的细胞内命运。在U937和原代人巨噬细胞中，随着时间的推移，长GAS链的出现表明，尽管GAS介导的细胞毒性，但在活的、碘化丙啶阴性的巨噬细胞中发生了复制。虽然主要毒力因子M1对细菌生长没有贡献，但缺乏链溶素O （SLO）的GAS突变株在细胞内复制能力受损。SLO促进细菌从含气液泡（GCV）逃逸到巨噬细胞细胞质中。多达一半的胞质GAS与泛素和p62共定位，表明细菌是自噬机制的目标。尽管如此，U937巨噬细胞的实时成像显示，GCV破裂后，巨噬细胞能够熟练地复制GAS，这表明从GCV中逃逸对于巨噬细胞中GAS的生长很重要。我们的研究结果表明，尽管自噬受体被细菌招募，但GAS可以在活的人巨噬细胞内复制，其中SLO促进GCV逃逸和细胞质生长。GAS通常被认为是一种细胞外病原体，可以在人上皮细胞、中性粒细胞和巨噬细胞中持续存在。一些研究表明，GAS可以调节其细胞内液泡以促进巨噬细胞的存活和复制。然而，缺乏对单细胞生存和复制动力学的深入分析。我们使用巨噬细胞样细胞系和原代巨噬细胞在群体和单细胞水平上测量GAS的细胞内生长。虽然CFU计数显示细菌总体生长没有增加，但定量荧光显微镜、流式细胞术和延时成像显示细菌在一定比例的感染巨噬细胞中复制。这项研究强调了单细胞分析的重要性，特别是在研究细胞毒性病原体的细胞内命运时，并且在细胞内杀伤和生长方面表现出异质性。据我们所知，这项研究首次提供了人类细胞内GAS复制的直接可视化。
As key components of innate immune defense, macrophages are essential in controlling bacterial pathogens, including group A Streptococcus (GAS). Despite this, only a limited number of studies have analyzed the recovery of GAS from within human neutrophils and macrophages. Here, we determined the intracellular fate of GAS in human macrophages by using several quantitative approaches. In both U937 and primary human macrophages, the appearance over time of long GAS chains revealed that despite GAS-mediated cytotoxicity, replication occurred in viable, propidium iodide-negative macrophages. Whereas the major virulence factor M1 did not contribute to bacterial growth, a GAS mutant strain deficient in streptolysin O (SLO) was impaired for intracellular replication. SLO promoted bacterial escape from the GAS-containing vacuole (GCV) into the macrophage cytosol. Up to half of the cytosolic GAS colocalized with ubiquitin and p62, suggesting that the bacteria were targeted by the autophagy machinery. Despite this, live imaging of U937 macrophages revealed proficient replication of GAS after GCV rupture, indicating that escape from the GCV is important for growth of GAS in macrophages. Our results reveal that GAS can replicate within viable human macrophages, with SLO promoting GCV escape and cytosolic growth, despite the recruitment of autophagy receptors to bacteria. Classically regarded as an extracellular pathogen, GAS can persist within human epithelial cells, as well as neutrophils and macrophages. Some studies suggest that GAS can modulate its intracellular vacuole to promote survival and perhaps replicate in macrophages. However, an in-depth single-cell analysis of the dynamics of survival and replication is lacking. We used macrophage-like cell lines and primary macrophages to measure the intracellular growth of GAS at both the population and single-cell levels. While CFU counts revealed no increase in overall bacterial growth, quantitative fluorescence microscopy, flow cytometry, and time-lapse imaging revealed bacterial replication in a proportion of infected macrophages. This study emphasizes the importance of single-cell analysis especially when studying the intracellular fate of a pathogen that is cytotoxic and displays heterogeneity in terms of intracellular killing and growth. To our knowledge, this study provides the first direct visualization of GAS replication inside human cells.