Garcia-del Portillo F, Finlay B B
Biotechnology Laboratory, University of British Columbia, Vancouver, Canada.
J Cell Biol. 1995 Apr;129(1):81-97. doi: 10.1083/jcb.129.1.81.
Salmonella typhimurium is an intracellular bacterial pathogen that remains enclosed in vacuoles (SCV) upon entry into the host cell. In this study we have examined the intracellular trafficking route of S. typhimurium within epithelial cells. Indirect immunofluorescence analysis showed that bacteria initiated fusion with lysosomal membrane glycoprotein (lgp)-containing compartments approximately 15 min after bacterial internalization. This process was completed approximately 75 min later and did not require microtubules. Cation-independent (CI)- or cation-dependent (CD)-mannose 6-phosphate receptors (M6PRs) were not observed at detectable levels in SCV. Lysosomal enzymes showed a different distribution in SCV: lysosomal-acid phosphatase (LAP) was incorporated into these vacuoles with the same kinetics as lgps, while cathepsin D was present in a low proportion (approximately 30%) of SCV. Uptake experiments with fluid endocytic tracers such as fluorescein-dextran sulphate (F-DX) or horseradish-peroxidase (HRP) showed that after 2 h of uptake, F-DX was present in approximately 75% of lgp-containing vesicles in uninfected cells, while only approximately 15% of SCV contained small amounts of the tracer during the same uptake period. SCV also showed only partial fusion with HRP-preloaded secondary lysosomes, with approximately 30% of SCV having detectable amounts of HRP at 6 h after infection. These results indicate that SCV show limited accessibility to fluid endocytic tracers and mature lysosomes, and are therefore functionally separated from the endocytic route. Moreover, the unusual intracellular trafficking route of S. typhimurium inside epithelial cells has allowed us to establish the existence of two different lgp-containing vesicles in Salmonella-infected cells: one population is separated from the endocytic route, fusogenic with incoming SCV and may arise from a secretory pathway, while the second involves the classical secondary or mature lysosomes.
鼠伤寒沙门氏菌是一种细胞内细菌病原体，进入宿主细胞后会被包裹在液泡（SCV）中。在本研究中，我们检测了鼠伤寒沙门氏菌在上皮细胞内的细胞内运输途径。间接免疫荧光分析表明，细菌内化后约15分钟开始与含有溶酶体膜糖蛋白（lgp）的区室融合。这个过程大约在75分钟后完成，且不需要微管。在SCV中未检测到可检测水平的不依赖阳离子（CI）或依赖阳离子（CD）的甘露糖6-磷酸受体（M6PRs）。溶酶体酶在SCV中的分布不同：溶酶体酸性磷酸酶（LAP）以与lgps相同的动力学被纳入这些液泡，而组织蛋白酶D仅存在于约30%的SCV中。用荧光素硫酸葡聚糖（F-DX）或辣根过氧化物酶（HRP）等流体胞吞示踪剂进行的摄取实验表明，摄取2小时后，F-DX存在于未感染细胞中约75%的含lgp囊泡中，而在相同摄取期内，只有约15%的SCV含有少量示踪剂。SCV也仅与预加载HRP的次级溶酶体部分融合，感染后6小时约30%的SCV含有可检测量的HRP。这些结果表明，SCV对流体胞吞示踪剂和成熟溶酶体的可及性有限，因此在功能上与胞吞途径分离。此外，鼠伤寒沙门氏菌在上皮细胞内异常的细胞内运输途径使我们能够确定在沙门氏菌感染的细胞中存在两种不同的含lgp囊泡：一种群体与胞吞途径分离，与进入的SCV融合，可能起源于分泌途径，而另一种涉及经典的次级或成熟溶酶体。
