A novel method of Francisella infection of epithelial cells using HeLa cells expressing fc gamma receptor.

Background: 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.
Methods: 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.
Results: 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.
Conclusions: 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.
(© 2024. The Author(s).)