Fuller James R, Craven Robin R, Hall Joshua D, Kijek Todd M, Taft-Benz Sharon, Kawula Thomas H
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7290, USA.
Infect Immun. 2008 Nov;76(11):4934-43. doi: 10.1128/IAI.00475-08. Epub 2008 Sep 2.
Francisella tularensis is a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of the F. tularensis live vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequenced Francisella species yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele in trans. Based on the deletion mutant phenotype, FTL_1914 was termed ripA (required for intracellular proliferation, factor A). Following uptake by J774.A1 cells, F. tularensis LVS Delta ripA colocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of the F. tularensis LVS Delta ripA mutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. The F. tularensis LVS Delta ripA mutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved among Francisella species that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence.
土拉弗朗西斯菌是一种剧毒的细菌病原体，可在包括巨噬细胞和上皮细胞在内的多种宿主细胞类型中侵入并复制。为了更好地理解这一过程，我们筛选了土拉弗朗西斯菌活疫苗株（LVS）的转座子插入文库，寻找在肺泡上皮细胞系中能侵入但无法复制的突变株。从该筛选中分离出的一个这样的菌株在基因FTL_1914中存在插入，该基因在所有已测序的弗朗西斯菌物种中保守，但与任何已知功能的基因缺乏显著同源性。构建了缺失FTL_1914的缺失菌株。该菌株在上皮细胞或巨噬细胞样细胞中均不复制，野生型等位基因反式互补可恢复细胞内复制。基于缺失突变体表型，FTL_1914被命名为ripA（细胞内增殖所需因子A）。被J774.A1细胞摄取后，土拉弗朗西斯菌LVS ΔripA与溶酶体相关膜蛋白1（LAMP-1）共定位，然后以与野生型LVS感染细胞相同的速率和频率逃离吞噬体。土拉弗朗西斯菌LVS ΔripA突变体的电子显微镜图像显示，突变细菌以不依赖复制的过程重新进入自噬体特征性的双膜空泡。在肺鼠疫小鼠模型中，土拉弗朗西斯菌LVS ΔripA突变体在肺中持续存在的能力以及在肝脏和脾脏中扩散和定殖的能力显著受损。RipA蛋白在实验室培养基中生长期间表达，并定位于细胞质膜。因此，RipA是弗朗西斯菌物种中保守的细胞质膜蛋白，是宿主细胞质内细胞内复制以及疾病进展、扩散和毒力所必需的。
