Expression of listeriolysin O and ActA by intracellular and extracellular Listeria monocytogenes
Expression of listeriolysin O and ActA by intracellular and extracellular Listeria monocytogenes
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DOI：
10.1128/iai.67.1.131-139.1999
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发表时间：
1999-01-01
影响因子：
3.1
通讯作者：
Portnoy, DA
中科院分区：
文献类型：
--
作者：
Moors, MA;Levitt, B;Portnoy, DA
单核细胞增多性李斯特菌需要嗜酸乳杆菌溶血素O（LLO）和ActA（分别是hly和actA的产物）来建立生产性细胞内感染。LLO对于空泡溶解和进入胞质溶胶是必需的，而ActA对于细菌扩散到相邻细胞是必需的。我们已经使用了一个转录报告基因系统，比较actA和hly在细胞内生长过程中的肉汤培养物的生长过程中的表达。将hly和actA基因转录融合到大肠杆菌lacZ和短小芽孢杆菌cat-86（cat）中，并以单拷贝整合到L.单核细胞增多染色体氯霉素抗性测定表明，在Luria-Bertani（LB）肉汤中，hly融合体而不是actA融合体被显著激活，并且该发现与肉汤培养物中可检测到的LLO和ActA le，eI相关。基于β-半乳糖苷酶活性的启动子活性的定量揭示了在LB肉汤中hly融合体相对于actA融合体的高达10倍高的表达水平。相比之下，两种融合物在J774细胞的胞质溶胶中均具有活性，并且在这些条件下actA融合物的活性比hly融合物的活性高约3倍。然而，来自感染的J774细胞的ActA和LLO的定量免疫沉淀证明胞质溶胶ActA比胞质溶胶LLO高约70倍。最后，与肉汤培养物中的诱导相比，在J774细胞中，actA被高度诱导（226倍），hly被中度诱导（20倍）。总的来说，这些结果表明，actA和hly的差异调节响应于生长环境，这两个基因在细胞内生长过程中优先表达。此外，虽然发酵液中ActA的产生水平低于LLO的产生水平可以通过转录调节来解释，但与细胞内LLO的相对丰度相比，细胞内ActA的相对丰度是额外的（可能是宿主介导的）因子的函数。
Listeria monocytogenes requires listeriolysin O (LLO) and ActA, the products of hly and actA, respectively, to establish a productive intracellular infection, LLO is essential for vacuolar lysis and entry into the cytosol, while ActA is required for bacterial spread to adjacent cells. We have used a transcriptional reporter gene system to compare the expression of actA and hly during intracellular growth to that during growth in broth cultures. The hly and actA genes were transcriptionally fused to Escherichia coli lacZ and Bacillus pumilus cat-86 (cat), and the fusions were integrated in single copies into the L. monocytogenes chromosome. A chloramphenicol resistance assay indicated that the hly fusion but not the actA fusion was significantly activated in Luria-Bertani (LB) broth, and this finding correlated with LLO and ActA le,els detectable in broth cultures. Quantitation of promoter activity on the basis of beta-galactosidase activity revealed up to 10-fold-higher level of expression of the hly fusion relative to the actA fusion in LB broth. In contrast, both fusions were active in the cytosol of J774 cells, and the activity of the actA fusion was approximately 3-fold higher than that of the hly fusion under these conditions, However, quantitative immunoprecipitation of ActA and LLO from infected J774 cells demonstrated approximately 70-fold more cytosolic ActA than cytosolic LLO, Finally, in comparison to induction in broth cultures, actA was highly induced (226-fold) and hly was moderately induced (20-fold) in J774 cells. Collectively, these results indicate that actA and hly are differentially regulated in response to the growth environment and that both genes are preferentially expressed during intracellular growth. Further, while the lower level of production of ActA than of LLO in broth can be accounted for by transcriptional regulation, the relative abundance of intracellular ActA compared to that of intracellular LLO is a function of additional, possibly host-mediated, factors.