McDonough K A, Kress Y, Bloom B R
Howard Hughes Medical Institute, Bronx, New York 10461.
Infect Immun. 1993 Jul;61(7):2763-73. doi: 10.1128/iai.61.7.2763-2773.1993.
Central to understanding the pathogenesis of tuberculosis is the interaction between the pathogen and mononuclear phagocytes. A key question about that interaction is whether Mycobacterium tuberculosis exerts an effect on phagolysosome fusion. We have reexamined the dynamics of phagolysosome fusion and its effect on intracellular bacterial replication in M. tuberculosis-infected macrophages by performing an extensive study at the electron microscopic level. Thoria-labelled murine and human macrophages were infected with a virulent (H37Rv) or avirulent (H37Ra) strain of M. tuberculosis or with Mycobacterium bovis BCG vaccine for times ranging from 2 h to 7 days. In all cases, by 2 h postinfection, approximately 85% of the bacteria clearly resided in fused vacuoles. However, at 4 days postinfection, fusion levels for viable H37Rv and H37Ra were reduced by half, whereas the fusion profiles of BCG and of heat-killed H37Rv and H37Ra were unchanged. A comparison of the numbers of bacteria per fused and nonfused vacuoles suggests both a net transfer of bacteria out of fused vacuoles and preferential bacterial multiplication in nonfused vacuoles. H37Rv and H37Ra appeared to bud from the phagolysosomes into tightly apposed membrane vesicles that did not fuse with secondary lysosomes. In some cases, no such membrane was seen and the bacteria appeared to be free in the cytoplasm. Only viable H37Rv showed a significant increase in bacterial counts during the course of infection. Thus, both of the attenuated strains we examined differed from the virulent strain H37Rv in their abilities to replicate successfully within macrophages, but each diverged from H37Rv at a different point in the process. Viable tubercle bacilli H37Rv and H37Ra had the capacity to escape from fused vesicles as the infection progressed; BCG did not. After extrusion from the phagolysosome, H37Rv, but not H37Ra, was able to multiply. These results suggest a novel mechanism by which virulent M. tuberculosis eludes the microbicidal mechanisms of macrophages by escaping from fused phagolysosomes into nonfused vesicles or the cytoplasm.
了解结核病发病机制的核心在于病原体与单核吞噬细胞之间的相互作用。关于这种相互作用的一个关键问题是结核分枝杆菌是否对吞噬溶酶体融合产生影响。我们通过在电子显微镜水平上进行广泛研究，重新审视了吞噬溶酶体融合的动态过程及其对结核分枝杆菌感染的巨噬细胞内细菌复制的影响。用钍标记的小鼠和人类巨噬细胞分别感染毒力强的（H37Rv）或无毒力的（H37Ra）结核分枝杆菌菌株或牛分枝杆菌卡介苗，感染时间从2小时到7天不等。在所有情况下，感染后2小时，约85%的细菌明显存在于融合的液泡中。然而，感染后4天，存活的H37Rv和H37Ra的融合水平降低了一半，而卡介苗以及热灭活的H37Rv和H37Ra的融合情况没有变化。对每个融合和未融合液泡中细菌数量的比较表明，细菌有从融合液泡中净转移出来的现象，并且在未融合液泡中细菌优先繁殖。H37Rv和H37Ra似乎从吞噬溶酶体芽生到紧密相邻的膜泡中，这些膜泡不与次级溶酶体融合。在某些情况下，看不到这样的膜，细菌似乎游离在细胞质中。只有存活的H37Rv在感染过程中细菌数量显著增加。因此，我们检测的两种减毒株在巨噬细胞内成功复制的能力上与毒力强的菌株H37Rv不同，但它们在这个过程的不同点上与H37Rv有所差异。随着感染的进展，存活的结核杆菌H37Rv和H37Ra有能力从融合的囊泡中逃逸；卡介苗则不能。从吞噬溶酶体挤出后，可以繁殖，而H37Ra则不能。这些结果提示了一种新机制，即毒力强的结核分枝杆菌通过从融合的吞噬溶酶体逃逸到未融合的囊泡或细胞质中，从而逃避巨噬细胞的杀菌机制。
