Oh Y K, Straubinger R M
Department of Pharmaceutics, State University of New York at Buffalo, Amherst 14260, USA.
Infect Immun. 1996 Jan;64(1):319-25. doi: 10.1128/iai.64.1.319-325.1996.
Mycobacterium avium is a facultative intracellular pathogen that can survive and replicate within macrophages. We tested the hypotheses that survival mechanisms may include alteration of phagosomal pH or inhibition of phagosome-lysosome fusion. M. avium was surface labeled with N-hydroxysuccinimidyl esters of carboxyfluorescein (CF) and rhodamine (Rho) to enable measurement of the pH of individual M. avium-containing phagosomes and the interactions of bacterium-containing phagosomes with labeled secondary lysosomes. CF fluorescence is pH sensitive, whereas Rho is pH insensitive; pH can be calculated from their fluorescence ratios. Surface labeling of M. avium did not affect viability in broth cultures or within J774, a murine macrophage-like cell line. By fluorescence spectroscopy, live M. avium was exposed to an environmental pH of approximately 5.7 at 6 h after phagocytosis, whereas similarly labeled Salmonella typhimurium, zymosan A, or heat-killed M. avium encountered an environmental pH of < 5.0. Video fluorescence and laser scanning confocal microscopy gave consistent pH results and demonstrated the heterogeneity of intracellular fate early in infection. pH became more homogeneous 6 h after infection. M. avium cells were coated with immunoglobulin G (IgG) or opsonized to investigate whether phagocytosis by the corresponding receptors would alter intracellular fate. Opsonized, unopsonized, and IgG-coated M. avium cells entered compartments of similar pH. Finally, the spatial distribution of intracellular bacteria and secondary lysosomes was compared. Only 18% of live fluorescent M. avium cells colocalized with fluorescent lysosomes, while 98% of heat-killed bacteria colocalized. Thus, both inhibition of phagosome-lysosome fusion and alteration of phagosomal pH may contribute to the intracellular survival of M. avium.
鸟分枝杆菌是一种兼性细胞内病原体，可在巨噬细胞内存活和复制。我们检验了以下假说：其生存机制可能包括吞噬体pH值的改变或吞噬体-溶酶体融合的抑制。用羧基荧光素（CF）和罗丹明（Rho）的N-羟基琥珀酰亚胺酯对鸟分枝杆菌进行表面标记，以便测量单个含鸟分枝杆菌吞噬体的pH值以及含细菌吞噬体与标记的次级溶酶体的相互作用。CF荧光对pH敏感，而Rho对pH不敏感；pH值可根据它们的荧光比率计算得出。鸟分枝杆菌的表面标记不影响其在肉汤培养物中或在鼠巨噬细胞样细胞系J774内的活力。通过荧光光谱法，吞噬后6小时，活的鸟分枝杆菌暴露于约5.7的环境pH值中，而同样标记的鼠伤寒沙门氏菌、酵母聚糖A或热灭活的鸟分枝杆菌遇到的环境pH值小于5.0。视频荧光和激光扫描共聚焦显微镜给出了一致的pH结果，并证明了感染早期细胞内命运的异质性。感染后6小时pH值变得更加均匀。用免疫球蛋白G（IgG）包被鸟分枝杆菌细胞或使其调理，以研究通过相应受体的吞噬作用是否会改变细胞内命运。调理的、未调理的和IgG包被的鸟分枝杆菌细胞进入pH值相似的区室。最后，比较了细胞内细菌和次级溶酶体的空间分布。只有18%的活荧光鸟分枝杆菌细胞与荧光溶酶体共定位，而98%的热灭活细菌共定位。因此，吞噬体-溶酶体融合的抑制和吞噬体pH值的改变都可能有助于鸟分枝杆菌在细胞内的存活。
