Lasa I, David V, Gouin E, Marchand J B, Cossart P
Unité des Interactions Bactéries-Cellules, CNRS URA 1300, Institut Pasteur, Paris, France.
Mol Microbiol. 1995 Nov;18(3):425-36. doi: 10.1111/j.1365-2958.1995.mmi_18030425.x.
The intracellular bacterial pathogen Listeria monocytogenes moves inside the host-cell cytoplasm propelled by continuous actin assembly at one pole of the bacterium. This process requires expression of the bacterial surface protein ActA. Recently, in order to identify the regions of ActA which are required for actin assembly, we and others have expressed different domains of ActA by transfection in eukaryotic cells. As this type of approach cannot address the role of ActA in the actin-driven bacterial propulsion, we have now generated several L. monocytogenes strains expressing different domains of ActA and analysed the ability of the different domains to trigger actin assembly and bacterial movement in both infected cells and cytoplasmic extracts. We show here that the amino-terminal part is critical for F-actin assembly and movement. The internal proline-rich repeats and the carboxy-terminal domains are not essential. However, in vitro motility assays have demonstrated that mutants lacking the proline-rich repeats domain of ActA moved two times slower (6+/-2 micrometers min(-1)) than the wild type (13 +/-3 micrometers min(-1)). In addition, phosphatase treatment of protein extracts of cells infected with the L. monocytogenes strains expressing the ActA variants suggested that phosphorylation may not be essential for ActA activity.
细胞内细菌病原体单核细胞增生李斯特菌在宿主细胞质内移动，由细菌一端持续的肌动蛋白组装推动。这一过程需要细菌表面蛋白ActA的表达。最近，为了确定ActA中肌动蛋白组装所需的区域，我们和其他人通过在真核细胞中进行转染来表达ActA的不同结构域。由于这种方法无法解决ActA在肌动蛋白驱动的细菌推进中的作用，我们现在构建了几种表达ActA不同结构域的单核细胞增生李斯特菌菌株，并分析了不同结构域在感染细胞和细胞质提取物中触发肌动蛋白组装和细菌移动的能力。我们在此表明，氨基末端部分对于F-肌动蛋白组装和移动至关重要。富含脯氨酸的内部重复序列和羧基末端结构域并非必需。然而，体外运动分析表明，缺失ActA富含脯氨酸重复序列结构域的突变体移动速度比野生型慢两倍（6±2微米/分钟）（野生型为13±3微米/分钟）。此外，用表达ActA变体的单核细胞增生李斯特菌菌株感染的细胞的蛋白质提取物进行磷酸酶处理表明，磷酸化对于ActA活性可能并非必需。
