Cryptococcus neoformans resides in an acidic phagolysosome of human macrophages
Cryptococcus neoformans resides in an acidic phagolysosome of human macrophages
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DOI：
10.1128/iai.67.2.885-890.1999
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发表时间：
1999-02-01
影响因子：
3.1
通讯作者：
Simons, ER
中科院分区：
文献类型：
--
作者：
Levitz, SM;Nong, SH;Simons, ER
最近，我们证明了氯喹或氯化铵处理的人单核细胞衍生的巨噬细胞（MDM）对艾滋病相关病原体新型隐球菌的抗真菌活性显着增加。这两种药物均升高溶酶体pH，这表明抗真菌活性的增加是碱化吞噬溶酶体的功能。此外，生长与C。这些数据表明，C.新形动物非常适应在酸性环境中生存。为了验证这一假设，我们进行了研究，以确定人MDM和中性粒细胞吞噬体的pH值含有C。新人类用两种对pa敏感的探针的异硫氰酸酯衍生物标记真菌：荧光素和2 '，7'-二氟荧光素（俄勒冈州绿色）。这些探针的pK（a）分别为6.4和4.7，允许在宽范围内进行灵敏的pH检测。无论是活菌还是热灭活菌，其吞噬体pH值平均约为5，并随时间保持相对恒定，这表明C.新形虫不主动调节其吞噬体的pH。加入10和100 μ M氯喹导致吞噬体pH值从基线5.1分别增加到6.5和7.3。最后，通过免疫荧光技术，共定位C。证实了新生隐球菌和MDM溶酶体膜蛋白LAMP-1的融合，建立了C.具有溶酶体隔室的装载有新型生物的吞噬体发生。因此，与许多其他细胞内病原体不同，C.新生儿不避免与巨噬细胞溶酶体区室融合，而是驻留在酸性吞噬溶酶体中并存活。
Recently, we demonstrated that human monocyte-derived macrophages (MDM) treated with chloroquine or ammonium chloride had markedly increased antifungal activity against the AIDS-related pathogen Cryptococcus neoformans. Both of these agents raise the lysosomal pH, which suggested that the increased antifungal activity was a function of alkalinizing the phagolysosome. Moreover, there was an inverse correlation between growth of C. neoformans in cell-free media and pH. These data suggested that C. neoformans was well adapted to survive within acidic compartments. To test this hypothesis, we performed studies to determine the pH of human MDM and neutrophil phagosomes containing C. neoformans. Fungi were labeled with the isothiocyanate derivatives of two pa-sensitive probes: fluorescein and 2',7'-difluorofluorescein (Oregon Green). These probes have pK(a)s of 6.4 and 4.7, respectively, allowing sensitive pH detection over a broad range. The phagosomal pH averaged approximately 5 after ingestion of either live or heat-killed fungi and remained relatively constant over time, which suggested that C. neoformans does not actively regulate the pH of its phagosome. The addition of 10 and 100 mu M chloroquine resulted in increases in the phagosomal pH from a baseline of 5.1 up to 6.5 and 7.3, respectively. Finally, by immunofluorescence, colocalizatiom of C. neoformans and the MDM lysosomal membrane protein LAMP-1 was demonstrated, establishing that fusion of C. neoformans-laden phagosomes with lysosomal compartments takes place. Thus, unlike many other intracellular pathogens, C. neoformans does not avoid fusion with macrophage lysosomal compartments but rather resides and survives in an acidic phagolysosome.