Sturgill-Koszycki S, Haddix P L, Russell D G
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Electrophoresis. 1997 Dec;18(14):2558-65. doi: 10.1002/elps.1150181411.
The intramacrophage pathogen Mycobacterium avium resides in a vacuole which displays unusual fusion characteristics, expressed as both a failure to mature into phagolysosomes and a continued access to the early recycling pathway. In contrast, compartments containing inert IgG-opsonized latex beads mature to phagolysosomes. Techniques were developed for the isolation of these particle-containing phagosomes from macrophages to facilitate analysis of phagosomal constituents by electrophoresis and autoradiography. Metabolic labeling of macrophages followed by phagosome isolation and two-dimensional polyacrylamide gel electrophoresis revealed only minor differences in the protein profiles between the M. avium and IgG-bead phagosomes despite the marked differences in the fusigenicity of the respective vacuoles. Pulse-chase labeling experiments revealed greater differences in the accessibility of Mycobacterium avium and IgG-bead phagosomes to newly synthesized proteins. These phagosome isolation techniques were extended to analyze the protein synthesis profile of intracellular M. avium for comparison with bacteria that were metabolically labeled in broth culture. Not surprisingly, the majority of polypeptides in the bacilli were common to both growth conditions. However, despite these similarities, intracellular M. avium express several unique proteins, most notably one abundant protein with a molecular weight of 51 kDa. In addition, the bacteria manifest a restricted set of proteins expressed while in stasis shortly after infection.
巨噬细胞内病原体鸟分枝杆菌寄居于一个具有异常融合特性的液泡中，表现为既不能成熟为吞噬溶酶体，又持续进入早期再循环途径。相比之下，含有惰性免疫球蛋白G调理的乳胶珠的区室则成熟为吞噬溶酶体。已开发出从巨噬细胞中分离这些含颗粒吞噬体的技术，以利于通过电泳和放射自显影分析吞噬体成分。对巨噬细胞进行代谢标记，随后分离吞噬体并进行二维聚丙烯酰胺凝胶电泳，结果显示，尽管各自液泡的融合性存在显著差异，但鸟分枝杆菌吞噬体和免疫球蛋白G珠吞噬体之间的蛋白质谱仅有微小差异。脉冲追踪标记实验显示，鸟分枝杆菌吞噬体和免疫球蛋白G珠吞噬体对新合成蛋白质的可及性存在更大差异。这些吞噬体分离技术被扩展用于分析细胞内鸟分枝杆菌的蛋白质合成谱，以便与在肉汤培养中进行代谢标记的细菌进行比较。不出所料，两种生长条件下杆菌中的大多数多肽是相同的。然而，尽管存在这些相似之处，细胞内鸟分枝杆菌表达几种独特的蛋白质，最显著的是一种分子量为51 kDa的丰富蛋白质。此外，细菌在感染后不久处于停滞状态时表现出一组受限的蛋白质表达。
