Brucella suis-impaired specific recognition of phagosomes by lysosomes due to phagosomal membrane modifications
Brucella suis-impaired specific recognition of phagosomes by lysosomes due to phagosomal membrane modifications
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DOI：
10.1128/iai.69.1.486-493.2001
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发表时间：
2001-01-01
影响因子：
3.1
通讯作者：
Porte, F
中科院分区：
文献类型：
--
作者：
Naroeni, A;Jouy, N;Porte, F
布鲁氏菌属是革兰氏阴性的兼性细胞内细菌，可感染人类和动物。这些生物可以在吞噬细胞和非专业吞噬细胞的膜结合室中生存和复制。在这两种类型的细胞中，抑制吞噬体-溶酶体融合被认为是细胞内存活的机制。然而，布鲁氏菌成熟的生化机制和微生物因素仍是完全未知的。我们开发了两种不同的方法，试图进一步深入了解这些机制：(i)在存在布鲁氏菌的情况下，用荧光显微镜分析整个细胞内的一般运输；（li）用流式细胞术分析体外重构实验，显示含布鲁氏菌的吞噬体和溶酶体之间的相互作用。荧光显微镜结果显示，细胞中有活的猪布鲁氏菌存在时，含乳胶珠的吞噬体与溶酶体的融合特性没有改变。我们得出结论，融合抑制仅限于病原体吞噬体，并且宿主细胞融合机制不会因细胞中存在活布鲁氏菌而改变。通过体外重组实验，我们观察到被杀死的猪双球菌吞噬体和溶酶体之间存在特定的关联，这种关联依赖于外源提供的细胞质、能量和温度。这种关联在被杀死的细菌中观察到，而在活的细菌中没有观察到。因此，正如在体内实验中所指出的那样，这种特异性识别抑制似乎仅限于病原体吞噬体膜。
Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (li) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells. We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell. By in vitro reconstitution experiments, we observed a specific association between killed B. suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments.