Camilli A, Paynton C R, Portnoy D A
Department of Microbiology, University of Pennsylvania, School of Medicine, Philadelphia 19104-6076.
Proc Natl Acad Sci U S A. 1989 Jul;86(14):5522-6. doi: 10.1073/pnas.86.14.5522.
To dissect the determinants of Listeria monocytogenes that are required for pathogenicity, we designed an intracellular selection protocol based on penicillin selection to isolate mutants defective for intracellular growth. Eight independent mutants obtained by insertion of Tn916 were isolated that were resistant to methicillin treatment following internalization by the J774 macrophage-like cell line. Seven mutants were absolutely defective for intracellular growth, whereas one showed abortive intracellular growth. The majority of the mutants were nonhemolytic and lacked a secreted 58-kDa polypeptide thought to be the L. monocytogenes hemolysin, listeriolysin O. Southern blot analysis indicated that one mutant contained a Tn916 insertion in hlyA, the listeriolysin O structural gene, which resulted in a truncated listeriolysin O polypeptide, whereas another mutant contained an insertion immediately upstream of hlyA, which resulted in reduced expression of listeriolysin O. The other mutants contained Tn916 insertions in genes other than hlyA, although all but one were nonhemolytic. Revertants isolated by their ability to grow within tissue culture cells regained hemolytic activity. These data show that intracellular methicillin selection facilitates isolation of mutations in genes required for intracellular growth and strengthens the premise that listeriolysin O is essential for intracellular growth.
为了剖析致病性单核细胞增生李斯特菌所需的决定因素，我们设计了一种基于青霉素选择的细胞内筛选方案，以分离细胞内生长缺陷型突变体。通过插入Tn916获得了八个独立的突变体，这些突变体在被J774巨噬细胞样细胞系内化后对甲氧西林治疗具有抗性。七个突变体在细胞内生长方面完全缺陷，而一个显示出细胞内生长失败。大多数突变体不溶血，并且缺乏一种被认为是单核细胞增生李斯特菌溶血素（李斯特菌溶血素O）的58 kDa分泌多肽。Southern印迹分析表明，一个突变体在溶血素O结构基因hlyA中含有Tn916插入，这导致了截短的李斯特菌溶血素O多肽，而另一个突变体在hlyA上游紧邻处含有插入，这导致了李斯特菌溶血素O的表达降低。其他突变体在hlyA以外的基因中含有Tn916插入，尽管除一个外所有突变体都不溶血。通过其在组织培养细胞内生长的能力分离出的回复突变体恢复了溶血活性。这些数据表明，细胞内甲氧西林选择有助于分离细胞内生长所需基因中的突变，并强化了李斯特菌溶血素O对细胞内生长至关重要的前提。
