Leung K Y, Finlay B B
Department of Biochemistry, University of British Columbia, Vancouver, Canada.
Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11470-4. doi: 10.1073/pnas.88.24.11470.
Salmonella typhimurium is a facultative intracellular parasite, capable of penetrating, surviving, and multiplying within diverse eukaryotic cell types, including epithelial and phagocytic cells. We have been studying intracellular replication of S. typhimurium and found that it is essential in the pathogenesis of this bacterium. A total of 45,000 independent mini-Mu MudJ transposon mutants in S. typhimurium SL1344 were screened in Madin-Darby canine kidney (MDCK) epithelial cells with a beta-lactam, cefotaxime, to enrich for mutants defective for intracellular replication. Ten different auxotrophic (purine, pyrimidine, purine/methionine, and valine/isoleucine) and three prototrophic replication-defective mutants (Rep-) were identified. All Rep- mutants showed no differences in aerobic and anaerobic growth patterns, motility, serum sensitivity, mouse macrophage survival, iron uptake, and phosphate requirements. All Rep- mutants were unable to multiply inside MDCK, HeLa, and Caco-2 epithelial cells. When required nutrients for various auxotrophs were supplemented, auxotrophs then replicated inside MDCK cells. Although the parental strain multiplies in large vacuoles inside MDCK cells that distort the host cells, MDCK cells infected with the Rep- mutants appeared relatively normal and few bacteria were seen inside vacuoles. The purine auxotrophs and the three prototrophic Rep- mutants were highly attenuated in mice, and oral and intraperitoneal LD50 levels were 3 to 4 orders of magnitude higher than the wild type level. The three prototrophs were invasive and persisted in the murine organs such as livers and spleens for at least 3 weeks. Therefore, these prototrophic genes are needed for intracellular replication and are essential to the virulence of S. typhimurium.
鼠伤寒沙门氏菌是一种兼性细胞内寄生虫,能够在多种真核细胞类型(包括上皮细胞和吞噬细胞)内穿透、存活并繁殖。我们一直在研究鼠伤寒沙门氏菌的细胞内复制情况,并发现这在该细菌的发病机制中至关重要。在Madin-Darby犬肾(MDCK)上皮细胞中,使用β-内酰胺类抗生素头孢噻肟对鼠伤寒沙门氏菌SL1344中的45000个独立的mini-Mu MudJ转座子突变体进行筛选,以富集细胞内复制缺陷的突变体。鉴定出了10种不同的营养缺陷型(嘌呤、嘧啶、嘌呤/蛋氨酸和缬氨酸/异亮氨酸)和3种原养型复制缺陷突变体(Rep-)。所有Rep-突变体在需氧和厌氧生长模式、运动性、血清敏感性、小鼠巨噬细胞存活、铁摄取和磷酸盐需求方面均无差异。所有Rep-突变体都无法在MDCK、HeLa和Caco-2上皮细胞内繁殖。当为各种营养缺陷型补充所需营养时,营养缺陷型随后在MDCK细胞内复制。尽管亲本菌株在MDCK细胞内的大液泡中繁殖,这些液泡会使宿主细胞变形,但感染了Rep-突变体的MDCK细胞看起来相对正常,液泡内可见的细菌很少。嘌呤营养缺陷型和3种原养型Rep-突变体在小鼠中高度减毒,口服和腹腔注射的半数致死剂量(LD50)水平比野生型水平高3至4个数量级。这3种原养型具有侵袭性,并在肝脏和脾脏等小鼠器官中持续存在至少3周。因此,这些原养型基因是细胞内复制所必需的,对鼠伤寒沙门氏菌的毒力至关重要。