A QUANTITATIVE MODEL OF INTRACELLULAR GROWTH OF LEGIONELLA-PNEUMOPHILA IN ACANTHAMOEBA-CASTELLANII
A QUANTITATIVE MODEL OF INTRACELLULAR GROWTH OF LEGIONELLA-PNEUMOPHILA IN ACANTHAMOEBA-CASTELLANII
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DOI：
10.1128/iai.60.1.296-301.1992
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发表时间：
1992-01-01
影响因子：
3.1
通讯作者：
TOMPKINS, LS
中科院分区：
文献类型：
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作者：
MOFFAT, JF;TOMPKINS, LS
已经开发了卡氏棘阿米巴嗜肺军团菌的细胞内生长模型，并提供了进入后存活和复制的定量测量。 在此模型中，在营养限制条件下，将单层棘阿米巴与组织培养板中的细菌一起孵育。 培养后用庆大霉素杀死细胞外细菌，并在阿米巴裂解后测定细胞内细菌的数量。 在 37 摄氏度下进行测定时，观察到有毒力的嗜肺军团菌和其他野生型军团菌的细胞内生长。 在室温下，测试的军团菌菌株均未在细胞内生长，而无毒力的嗜肺军团菌菌株在该测定中在任一温度下均无法复制。 测定过程中营养限制对卡斯特拉尼阿米巴的影响阻止了阿米巴的繁殖并增加了军团菌的感染水平。 阿米巴的感染程度与细菌感染的复数成正比；将 1.03 x 10(7) 细菌接种物添加到含有 1.10 x 10(5) 阿米巴原虫的孔中（感染复数为 100），大约 4.4% 的 A.castellanii 细胞被感染。 细胞松弛素 D 减少阿米巴原虫对细菌的摄取，主要是通过使阿米巴原虫脱离培养皿，减少目标宿主的数量；甲胺也降低了初始感染水平，但这两种抑制剂都无法阻止军团菌的细胞内复制。 因此，一旦细菌进入细胞，只有降低温度才能限制复制。 这种细胞内生长模型提供了一步生长曲线，应该有助于研究宿主-寄生虫相互作用的分子基础。
A model of intracellular growth for Legionella pneumophila in Acanthamoeba castellanii has been developed and provides a quantitative measure of survival and replication after entry. In this model, Acanthamoeba monolayers were incubated with bacteria in tissue culture plates under nutrient-limiting conditions. Gentamicin was used to kill extracellular bacteria following the period of incubation, and the number of intracellular bacteria was determined following lysis of amebae. Intracellular growth of virulent L. pneumophila and other wild-type Legionella species was observed when the assay was performed at 37-degrees-C. At room temperature, none of the Legionella strains tested grew intracellularly, while an avirulent L. pneumophila strain was unable to replicate in this assay at either temperature. The effect of nutrient limitation on A. castellanii during the assay prevented multiplication of the amebae and increased the level of infection by Legionella spp. The level of infection of the amebae was directly proportional to the multiplicity of infection with bacteria; at an inoculum of 1.03 x 10(7) bacteria added to wells containing 1.10 x 10(5) amebae (multiplicity of infection of 100), approximately 4.4% of A. castellanii cells became infected. Cytochalasin D reduced the uptake of bacteria by the amebae primarily by causing amebae to lift off the culture dish, reducing the number of target hosts; methylamine also reduced the level of initial infection, yet neither inhibitor was able to prevent intracellular replication of Legionella spp. Consequently, once the bacteria entered the cell, only lowered temperature could restrict replication. This model of intracellular growth provides a one-step growth curve and should be useful to study the molecular basis of the host-parasite interaction.