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首页> 外文期刊>European Journal of Cell Biology: Journal of Deutsche Gesellschaft fur Elektronenmikroskopie: Journal of Deutsche Gesellschaft fur Zellbiologie >Isolation and characterization of Salmonella typhimurium and Yersiniapseudotuberculosis-containing phagosomes from infected mouse macrophages:Y-pseudotuberculosis traffics to terminal lysosomes where they aredegraded
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Isolation and characterization of Salmonella typhimurium and Yersiniapseudotuberculosis-containing phagosomes from infected mouse macrophages:Y-pseudotuberculosis traffics to terminal lysosomes where they aredegraded

机译：从感染的小鼠巨噬细胞中分离并鉴定鼠伤寒沙门氏菌和耶尔森氏假结核的吞噬体：Y假结核向贩运的最终溶酶体降解

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The interaction of Salmonella and Yersinia with macrophages is critical to the pathogenesis of these organisms. After internalization into macrophages, these bacteria reside in membrane-enclosed vacuoles, In this report, we present an approach to isolate and characterize bacteria-containing vacuoles (BCVs) to study intracellular trafficking of pathogenic bacteria within the membrane system of host cells. Using the mouse monocyte-macrophage cell line J774A.1, we found that Salmonella typhimurium replicated intracellularly to approximately 5 times its original numbers over a 9 hour infection course, while Yersinia pseudotuberculosis and Escherichia coli did not replicate inside these cells. Analysis of isolated latex bead-containing vacuoles confirmed that they trafficked normally from endosomes to lysosomes within the endocytic pathway of J774A.1 cells. We isolated BCVs free of contaminating endosomes and lysosomes using sucrose step gradients, and used quantitative immunoblotting to characterize the contents of these vacuoles at different time points after internalization. We found that the isolated BCVs contained endosomal and lysosomal marker proteins including lamp-1, mannose 6-phosphate receptor (M 6-PR), cathepsin D and cathepsin L. Further, we report on differential processing of lysosomal hydrolases (such as cathepsin I) and cathepsin L) associated with the isolated BCVs, Although there was some contamination of the S. typhimurium-containing vacuoles with endoplasmic reticulum (ER) marker protein calnexin, the Y. pseudotuberculosis-containing vacuoles were predominately free of ER contamination, The Y. pseudotuberculosis-containing vacuoles displayed properties of lysosomes, containing the M 6-PR-dependent lysosomal hydrolases cathepsin D and cathepsin L, which were shown to be processed to their mature forms incrementally over time. These results, coupled with intracellular growth and microscopic examination of infected cells over time, indicated that Y. pseudotuberculosis traffics to lysosomes where they are degraded, The described method for isolation and characterization of BCVs proved to be a valuable tool to characterize the vacuolar compartment occupied by Y. pseudotuberculosis, and has potential to be applied to other vacuole resident pathogens whose trafficking is thought to play a role in pathogenesis.
机译：沙门氏菌和耶尔森氏菌与巨噬细胞的相互作用对于这些生物的发病机制至关重要。内化成巨噬细胞后，这些细菌驻留在膜封闭的液泡中。在本报告中，我们提出了一种分离和表征含细菌的液泡（BCV）的方法，以研究宿主细胞膜系统内病原性细菌的细胞内运输。使用小鼠单核巨噬细胞细胞系J774A.1，我们发现鼠伤寒沙门氏菌在9小时的感染过程中在细胞内复制至原始数量的约5倍，而假单胞菌和大肠埃希氏菌则未在这些细胞内复制。对分离的含胶乳珠的液泡的分析证实，它们在J774A.1细胞内吞途径内从内体正常转移至溶酶体。我们使用蔗糖阶梯梯度分离了不含污染的内体和溶酶体的BCV，并使用定量免疫印迹法对内化后不同时间点上这些液泡的含量进行了表征。我们发现分离的BCV包含内体和溶酶体标记蛋白，包括lamp-1，甘露糖6-磷酸受体（M 6-PR），组织蛋白酶D和组织蛋白酶L。此外，我们报道了溶酶体水解酶（例如组织蛋白酶I）的差异加工）和组织蛋白酶L）与分离的BCV相关，尽管内质网（ER）标记蛋白钙连蛋白污染了含鼠伤寒沙门氏菌的液泡，但含假结核耶尔森氏菌的液泡主要不受ER污染，含有假结核的液泡显示了溶酶体的特性，其中包含M 6 -PR依赖性的溶酶体水解酶组织蛋白酶D和组织蛋白酶L，它们被显示为随着时间的推移逐渐加工成它们的成熟形式。这些结果，加上随着时间的推移细胞内生长和对感染细胞的显微镜检查，表明假结核耶尔森菌流向溶酶体并在其中降解。所述的分离和表征BCV的方法被证明是表征所占据的液泡隔室的有价值的工具。由假结核耶尔森氏菌（Y.pseudotuberculosis）制造，并有潜力应用于其他液泡常驻病原体，其病原体的运输被认为在发病中起作用。

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