EVIDENCE THAT VESICLES CONTAINING LIVING, VIRULENT MYCOBACTERIUM-TUBERCULOSIS OR MYCOBACTERIUM-AVIUM IN CULTURED HUMAN MACROPHAGES ARE NOT ACIDIC
EVIDENCE THAT VESICLES CONTAINING LIVING, VIRULENT MYCOBACTERIUM-TUBERCULOSIS OR MYCOBACTERIUM-AVIUM IN CULTURED HUMAN MACROPHAGES ARE NOT ACIDIC
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DOI：
10.1128/iai.59.5.1823-1831.1991
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发表时间：
1991-05-01
影响因子：
3.1
通讯作者：
MAY, MH
中科院分区：
文献类型：
--
作者：
CROWLE, AJ;DAHL, R;MAY, MH
结核分枝杆菌和鸟分枝杆菌在培养的人巨噬细胞 (MP) 内的膜封闭囊泡内繁殖。 通常认为这些囊泡是酸性的。 这一假设最常被引用的证据是，吡嗪酰胺（需要酸性 pH 值才能发挥作用）是有效的，而链霉素在低 pH 值下会失去大部分活性，对结核杆菌的效果较差。 通过使用两种弱碱氯喹和 NH4Cl 来提高实验感染结核分枝杆菌或鸟分枝杆菌的 MP 中酸性囊泡的 pH 值，测试了这一假设。 MP 中酸性区域的免疫细胞化学定位器用于监测细胞内杆菌与酸度的关联。 MP被结核分枝杆菌或鸟分枝杆菌感染，并与药物和弱碱的各种组合一起孵育。 MP 中细菌的复制通过培养物计数来测量。 通过电子显微照片和使用弱碱 3-(2,4-二硝基苯胺基)-3'-氨基-N-甲基二丙胺评估分枝杆菌与酸度的细胞内关联，并用胶体金标记抗体检测。 免疫细胞化学证实氯喹和NH4Cl均可升高感染MP中酸性囊泡的pH值。 然而，吡嗪酰胺或链霉素或非 pH 依赖性药物异烟肼的抗分枝杆菌活性均未引起任何与 pH 相关的变化。 免疫化学分析表明，酸度与 MP 中杀死的分枝杆菌有关，但与活的分枝杆菌无关。 这些发现表明，活的结核分枝杆菌和鸟分枝杆菌位于非酸性囊泡中的人类 MP 中。
Mycobacterium tuberculosis and Mycobacterium avium multiply in cultured human macrophages (MP) within membrane-enclosed vesicles. These vesicles are generally assumed to be acidic. The evidence most frequently cited for this assumption is that pyrazinamide, which requires an acid pH to be effective, is effective and streptomycin, which loses most of its activity at a low pH, is poorly effective against tubercle bacilli. This assumption was tested by using the two weak bases chloroquine and NH4Cl to raise the pH of acidic vesicles in MP experimentally infected with M. tuberculosis or M. avium. An immunocytochemical locator of acidic regions in the MP was used to monitor the association of intracellular bacilli with acidity. MP were infected with M. tuberculosis or M. avium and incubated with various combinations of the drugs and the weak bases. Replication of the bacteria in the MP was measured by culture counts. Intracellular associations of the mycobacteria with acidity were assessed by electron micrographs and by using the weak base 3-(2,4-dinitroanilino)-3'-amino-N-methyl dipropylamine, which was detected with colloidal gold-labeled antibodies. It was confirmed by immunocytochemistry that both chloroquine and NH4Cl raise the pH of acidic vesicles in the infected MP. However, neither caused any pH-related change in the antimycobacterial activities of pyrazinamide or streptomycin or of the pH-independent drug isoniazid. Immunochemical analyses showed acidity to be associated with killed but not living mycobacteria in the MP. These findings suggest that living M. tuberculosis and M. avium are located in human MP in vesicles which are not acidic.