Beatty W L, Ullrich H J, Russell D G
Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO/USA.
Eur J Cell Biol. 2001 Jan;80(1):31-40. doi: 10.1078/0171-9335-00131.
Bacterial cell wall constituents are released from mycobacterial phagosomes and actively traffic within infected macrophages. Colocalization of fluorescently tagged bacterial moieties with endocytic tracers revealed the dynamic movement of released mycobacterial constituents into the endocytic network with accumulation in tubular lysosomal-like compartments. The released bacterial constituents not only penetrated the infected host cell but were also present in an extracellular microvesicular fraction. To identify the intracellular source of these exocytic compartments, released vesicular material was isolated from culture supernatants by differential ultracentrifugation and characterized by Western blot and electron microscopy analyses. The presence of lysosomal membrane proteins and lysosomal proteases suggested that labeled mycobacterial cell wall constituents access a constitutive lysosomal exocytic pathway. An abundance of multilamellar extracellular compartments morphologically reminiscent of MHC class II-enriched compartments (MIIC) implicated a MHC class II transport pathway in the extracellular release of bacterial constituents. Increases in intracellular free calcium have previously been shown to trigger lysosomal exocytosis by inducing fusion of lysosomes with the plasma membrane. To test if an increase in calcium would stimulate exocytosis with release of mycobacterial constituents, infected macrophages were exposed to the calcium ionophore A23187. The ionophore triggered the release of a microvesicular fraction containing labeled bacterial moieties, implicating calcium-regulated lysosomal exocytosis as a trafficking pathway by which mycobacterial products are released from infected macrophages.
细菌细胞壁成分从分枝杆菌吞噬体中释放出来，并在受感染的巨噬细胞内活跃运输。荧光标记的细菌部分与内吞示踪剂的共定位揭示了释放的分枝杆菌成分向内吞网络的动态移动，并在管状溶酶体样区室中积累。释放的细菌成分不仅穿透了受感染的宿主细胞，还存在于细胞外微泡部分。为了确定这些胞吐区室的细胞内来源，通过差速超速离心从培养上清液中分离出释放的囊泡物质，并通过蛋白质免疫印迹和电子显微镜分析进行表征。溶酶体膜蛋白和溶酶体蛋白酶的存在表明，标记的分枝杆菌细胞壁成分进入了组成型溶酶体胞吐途径。大量形态上类似于富含MHC II类分子的区室（MIIC）的多膜细胞外区室表明，MHC II类运输途径参与了细菌成分的细胞外释放。先前已表明，细胞内游离钙的增加会通过诱导溶酶体与质膜融合来触发溶酶体胞吐作用。为了测试钙的增加是否会刺激胞吐作用并释放分枝杆菌成分，将受感染的巨噬细胞暴露于钙离子载体A23187。该离子载体触发了含有标记细菌部分的微泡部分的释放，这表明钙调节的溶酶体胞吐作用是分枝杆菌产物从受感染巨噬细胞中释放的一种运输途径。
