Department of Clinical Microbiology, Clinical Bacteriology, and Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden.
National Research Council of Canada, Human Health Therapeutics Portfolio, Ottawa, Ontario, Canada.
Infect Immun. 2014 Sep;82(9):3622-35. doi: 10.1128/IAI.01406-13. Epub 2014 Jun 16.
Previously, we identified a spontaneous, essentially avirulent mutant, FSC043, of the highly virulent strain SCHU S4 of Francisella tularensis subsp. tularensis. We have now characterized the phenotype of the mutant and the mechanisms of its attenuation in more detail. Genetic and proteomic analyses revealed that the pdpE gene and most of the pdpC gene were very markedly downregulated and, as previously demonstrated, that the strain expressed partially deleted and fused fupA and fupB genes. FSC043 showed minimal intracellular replication and induced no cell cytotoxicity. The mutant showed delayed phagosomal escape; at 18 h, colocalization with LAMP-1 was 80%, indicating phagosomal localization, whereas the corresponding percentages for SCHU S4 and the ΔfupA mutant were <10%. However, a small subset of the FSC043-infected cells contained up to 100 bacteria with LAMP-1 colocalization of around 30%. The unusual intracellular phenotype was similar to that of the ΔpdpC and ΔpdpC ΔpdpE mutants. Complementation of FSC043 with the intact fupA and fupB genes did not affect the phenotype, whereas complementation with the pdpC and pdpE genes restored intracellular replication and led to marked virulence. Even higher virulence was observed after complementation with both double-gene constructs. After immunization with the FSC043 strain, moderate protection against respiratory challenge with the SCHU S4 strain was observed. In summary, FSC043 showed a highly unusual intracellular phenotype, and based on our findings, we hypothesize that the mutation in the pdpC gene makes an essential contribution to the phenotype.
先前，我们鉴定了一株自发产生的、本质上无毒力的弗朗西斯菌属土拉弗朗西斯菌亚种土拉弗朗西斯菌强毒株 SCHU S4 的突变株 FSC043。现在，我们已经更详细地描述了突变株的表型和减毒机制。遗传和蛋白质组学分析显示，pdpE 基因和大部分 pdpC 基因的表达水平显著下调，正如之前所证明的，该菌株表达部分缺失和融合的 fupA 和 fupB 基因。FSC043 显示出最小的细胞内复制能力，且不会诱导细胞毒性。突变株表现出延迟的吞噬体逃逸；在 18 小时时，与 LAMP-1 的共定位率为 80%，表明吞噬体定位，而 SCHU S4 和 ΔfupA 突变株的相应百分比小于 10%。然而，一小部分感染 FSC043 的细胞中含有多达 100 个与 LAMP-1 共定位约 30%的细菌。这种不寻常的细胞内表型与 ΔpdpC 和 ΔpdpC ΔpdpE 突变株相似。用完整的 fupA 和 fupB 基因互补 FSC043 并不影响表型，而用 pdpC 和 pdpE 基因互补则恢复了细胞内复制并导致明显的毒力。用两个双基因构建体互补后观察到更高的毒力。用 FSC043 菌株免疫后，观察到对 SCHU S4 菌株呼吸道攻击的中度保护。总之，FSC043 表现出一种非常特殊的细胞内表型，根据我们的发现，我们假设 pdpC 基因的突变对表型有重要贡献。
