Sun A N, Camilli A, Portnoy D A
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6076.
Infect Immun. 1990 Nov;58(11):3770-8. doi: 10.1128/iai.58.11.3770-3778.1990.
To dissect the regulatory and structural requirements for Listeria monocytogenes intracellular growth and cell-to-cell spread, we designed a protocol based on transposon mutagenesis and the isolation of mutants which form small plaques in monolayers of mouse L2 cell fibroblasts. Two different transposable elements were used to generate libraries of insertion mutants: Tn916 and a derivative of Tn917-lac, Tn917-LTV3. Ten classes of mutants were isolated and evaluated for growth and cell-to-cell spread in J774 mouse macrophagelike cells, Henle 407 human epithelial cells, and mouse bone marrow-derived macrophages. Mutants were also evaluated for secretion of hemolysin and phospholipase (assayed by egg yolk opacity) and association with F-actin in the cytoplasm of cells, using NBD-phallacidin staining. The ten classes of mutants included (i) mutants showing abortive intracellular and extracellular growth; (ii) mutants showing abortive intracellular growth; (iii) rough mutants; (iv) mutants showing greatly reduced hemolysin and phospholipase secretion but showing normal growth in cells and little or no association with F-actin; (v) mutants with mutations mapping to an open reading frame (ORF) adjacent to hlyA and referred to as ORF U, lacking phospholipase activity, and with 50% normal hemolysin activity; (vi) mutants with reduced secretion of both hemolysin and phospholipase; (vii) nonhemolytic mutants with mutations mapping to the structural gene, hlyA; (viii) mutants with 25% normal hemolysin secretion and absolutely no association with F-actin; (ix) mutants with mutations mapping to ORF U, lacking phospholipase activity, and with normal hemolysin activity; and (x) mutants showing a mixed-plaque morphology but normal for all other parameters.
为剖析单核细胞增生李斯特菌细胞内生长及细胞间传播的调控和结构要求，我们设计了一种基于转座子诱变和分离在小鼠L2细胞成纤维细胞单层中形成小噬斑的突变体的方案。使用两种不同的转座元件来生成插入突变体文库：Tn916和Tn917-lac的衍生物Tn917-LTV3。分离出十类突变体，并在J774小鼠巨噬样细胞、Henle 407人上皮细胞和小鼠骨髓来源的巨噬细胞中评估其生长和细胞间传播情况。还使用NBD-鬼笔环肽染色评估突变体的溶血素和磷脂酶分泌（通过蛋黄不透明度测定）以及与细胞胞质中F-肌动蛋白的关联。这十类突变体包括：（i）细胞内和细胞外生长失败的突变体；（ii）细胞内生长失败的突变体；（iii）粗糙型突变体；（iv）溶血素和磷脂酶分泌大幅减少但在细胞中生长正常且与F-肌动蛋白几乎无关联或无关联的突变体；（v）突变定位到与hlyA相邻的开放阅读框（ORF）且称为ORF U的突变体，缺乏磷脂酶活性，溶血素活性为正常的50%；（vi）溶血素和磷脂酶分泌均减少的突变体；（vii）突变定位到结构基因hlyA的非溶血突变体；（viii）溶血素分泌为正常的25%且与F-肌动蛋白绝对无关联的突变体；（ix）突变定位到ORF U、缺乏磷脂酶活性且溶血素活性正常的突变体；（x）呈现混合噬斑形态但所有其他参数正常的突变体。
