Joint Faculty of Veterinary Medicine, Laboratory of Veterinary Public Health, Yamaguchi University, 1677-1 Yoshida, Yamaguchi, 753-8515, Japan.
School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, 192-0392, Japan.
BMC Infect Dis. 2024 Oct 17;24(1):1171. doi: 10.1186/s12879-024-10083-y.
Francisella tularensis, the causative agent of tularemia, is a facultative intracellular bacterium. Although the life cycle of this bacterium inside phagocytic cells (e.g., macrophages, neutrophils) has been well analyzed, the difficulty of gene silencing and editing genes in phagocytic cells makes it difficult to analyze host factors important for the infection. On the other hand, epithelial cell lines, such as HeLa, have been established as cell lines that are easy to perform gene editing. However, the infection efficiency of Francisella into these epithelial cells is extremely low.
In order to facilitate the molecular biological analysis of Francisella infection using epithelial cells, we constructed an efficient infection model of F. tularensis subsp. novicida (F. novicida) in HeLa cells expressing mouse FcγRII (HeLa-FcγRII), and the system was applied to evaluate the role of host GLS1 on Francisella infection.
As a result of colony forming unit count, HeLa-FcγRII cells uptake F. novicida in a serum-dependent manner and demonstrated an approximately 100-fold increase in intracellular bacterial infection compared to parental HeLa cells. Furthermore, taking advantage of the gene silencing capability of HeLa-FcγRII cells, we developed GLS1, a gene encoding glutaminase, knockdown cells using lentiviral sh RNA vector and assessed the impact of GLS1 on F. novicida infection. LDH assay revealed that GLS1-knockdown HeLa-FcγRII cells exhibited increased cytotoxicity during infection with F. novicida compared with control HeLa-FcγRII cells. Furthermore, the cell death was inhibited by the addition of ammonia, the metabolite produced through glutaminase activity. These results suggest that ammonia plays an important role in the proliferation of F. novicida.
In this report, we proposed a new cell-based infection system for Francisella infection using HeLa-FcγRII cells and demonstrated its effectiveness. This system has the potential to accelerate cell-based infection assays, such as large-scale genetic screening, and to provide new insights into Francisella infection in epithelial cells, which has been difficult to analyze in phagocytic cells.
土拉弗朗西斯菌是造成兔热病的病原体，是一种兼性细胞内细菌。尽管该细菌在吞噬细胞（如巨噬细胞、中性粒细胞）内的生命周期已得到充分分析，但由于基因沉默和编辑吞噬细胞中基因的难度，使得分析对感染很重要的宿主因素变得困难。另一方面，上皮细胞系，如 HeLa 细胞，已被建立为易于进行基因编辑的细胞系。然而，弗朗西斯菌感染这些上皮细胞的效率极低。
为了便于使用上皮细胞进行弗朗西斯菌感染的分子生物学分析，我们构建了一种在表达小鼠 FcγRII 的 HeLa 细胞（HeLa-FcγRII）中高效感染土拉弗朗西斯菌亚种 novicida（F. novicida）的模型，该系统被用于评估宿主 GLS1 在弗朗西斯菌感染中的作用。
通过菌落形成单位计数，HeLa-FcγRII 细胞以血清依赖性方式摄取 F. novicida，与亲本 HeLa 细胞相比，细胞内细菌感染增加了约 100 倍。此外，利用 HeLa-FcγRII 细胞的基因沉默能力，我们使用慢病毒 shRNA 载体构建了编码谷氨酰胺酶的 GLS1 基因敲低细胞，并评估了 GLS1 对 F. novicida 感染的影响。LDH 测定显示，与对照 HeLa-FcγRII 细胞相比，GLS1 敲低的 HeLa-FcγRII 细胞在感染 F. novicida 时表现出更高的细胞毒性。此外，加入氨（通过谷氨酰胺酶活性产生的代谢物）可抑制细胞死亡。这些结果表明，氨在 F. novicida 的增殖中发挥重要作用。
本研究报告提出了一种使用 HeLa-FcγRII 细胞进行弗朗西斯菌感染的新型细胞感染系统，并证明了其有效性。该系统有望加速基于细胞的感染测定，如大规模基因筛选，并为上皮细胞中的弗朗西斯菌感染提供新的见解，这在吞噬细胞中很难分析。
