FTL_1199, a gene required for erythrocyte invasion by Francisella tularensis, is not necessary for zinc or iron acquisition

Francisella tularensis is an intracellular bacterium and the causative agent of the zoonotic disease, tularemia. F. tularensis invades host erythrocytes during infection, a phenomenon that leads to increased colonization of ticks after a blood meal. To better understand the mechanism of erythrocyte invasion, an RNA-seq analysis was conducted to identify genes whose transcription was affected by the presence of erythrocytes. A putative Zinc/ferric uptake regulator gene, FTL_1199 was identified. Therefore, we hypothesized that this gene may play a role in erythrocyte invasion. Two separate methods to evaluate Intracellular bacteria (gentamicin protection assay and double immunofluorescence microscopy) both showed that deletion of FTL_1199 resulted in a significant reduction in the invasion of rabbit erythrocytes, and complementation of FTL_1199 restored this mutant strain’s ability to acquire the cytoplasmic space of erythrocytes. Interestingly, the ΔFTL_1199 strain exhibited similar intracellular growth in RAW 264.7 macrophages compared to wild-type bacteria suggesting that FTL_1199 is not required for phagosomal escape or replication in the cytosol of this host cell. Therefore, FTL_1199 is the first gene identified that is required for erythrocyte invasion but not replication within macrophages. FTL_1199 is annotated as a putative zinc/ferric uptake regulator. However, deletion of FTL_1199 did not seemingly affect the ability of F. tularensis to acquire zinc or iron when these metals were scarce suggesting a novel role for the product of this gene. RNA-seq was used comparing transcripts of F. tularensis LVS to ΔFTL_1199 to potentially unveil genes regulated by FTL_1199. This analysis confirmed the zinc/iron acquisition studies further suggesting a novel role for FTL_1199.