Working Group: Cellular Interactions of Bacterial Pathogens, Centre for Biological Threats and Special Pathogens, Highly Pathogenic Microorganisms (ZBS 2), Robert Koch Institute, Berlin, Germany.
Department of Chemistry, Chair of Biochemistry, Technische Universität München, 85748 Garching, Germany.
Int J Med Microbiol. 2019 Sep;309(6):151341. doi: 10.1016/j.ijmm.2019.151341. Epub 2019 Aug 19.
Francisella tularensis is the causative agent of the human disease referred to as tularemia. Other Francisella species are known but less is understood about their virulence factors. The role of environmental amoebae in the life-cycle of Francisella is still under discussion. Francisella sp. strain W12-1067 (F-W12) is an environmental Francisella isolate recently identified in Germany which is negative for the Francisella pathogenicity island, but exhibits a putative alternative type VI secretion system. Putative virulence factors have been identified in silico in the genome of F-W12. In this work, we established a "scatter screen", used earlier for pathogenic Legionella, to verify experimentally and identify candidate fitness factors using a transposon mutant bank of F-W12 and Acanthamoeba lenticulata as host organism. In these experiments, we identified 79 scatter clones (amoeba sensitive), which were further analyzed by an infection assay identifying 9 known virulence factors, but also candidate fitness factors of F-W12 not yet described as fitness factors in Francisella. The majority of the identified genes encoded proteins involved in the synthesis or maintenance of the cell envelope (LPS, outer membrane, capsule) or in the metabolism (glycolysis, gluconeogenesis, pentose phosphate pathway). Further C-flux analysis of the Tn5 glucokinase mutant strain revealed that the identified gene indeed encodes the sole active glucokinase in F-W12. In conclusion, candidate fitness factors of the new Francisella species F-W12 were identified using the scatter screen method which might also be usable for other Francisella species.
土拉弗朗西斯菌是导致土拉热的病原体。其他弗朗西斯菌也已被发现，但对其毒力因子的了解较少。环境阿米巴在弗朗西斯菌生命周期中的作用仍在讨论中。弗朗西斯菌 W12-1067 株（F-W12）是一种最近在德国发现的环境弗朗西斯菌分离株，它对弗朗西斯菌致病性岛呈阴性，但表现出一种假定的替代型 VI 型分泌系统。在 F-W12 的基因组中，通过计算机预测鉴定了潜在的毒力因子。在这项工作中，我们建立了一个“分散筛选”，该筛选之前用于致病性军团菌，使用 F-W12 和 Acanthamoeba lenticulata 的转座子突变体库进行实验验证和鉴定候选适应因子。在这些实验中，我们鉴定了 79 个分散克隆（对阿米巴敏感），通过感染实验进一步分析，鉴定了 9 个已知的毒力因子，但也鉴定了 F-W12 中尚未描述为适应因子的候选适应因子。大多数鉴定的基因编码参与细胞包膜（LPS、外膜、荚膜）合成或维持或代谢（糖酵解、糖异生、戊糖磷酸途径）的蛋白质。进一步的 Tn5 葡萄糖激酶突变株 C 通量分析表明，所鉴定的基因确实编码 F-W12 中唯一的活性葡萄糖激酶。总之，使用分散筛选方法鉴定了新的弗朗西斯菌 F-W12 的候选适应因子，该方法也可能适用于其他弗朗西斯菌。
