Utilization of Double-Immunofluorescence Microscopy to determine the role of FTL₁199 in erythrocyte invasion by Francisella tularensis.

Francisella tularensis is an intracellular bacterium that is 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 transcriptional regulator, FTL₁199 was determined to be modulated in the presence of erythrocytes. We hypothesized that this gene may therefore play a role in erythrocyte invasion. Deletion of FTL₁199 resulted in a significant reduction in the invasion of rabbit erythrocytes, and complementation of FTL₁199 restored this strain’s ability to acquire the cytoplasmic space of erythrocytes. These findings were determined by a gentamicin protection assay to quantify erythrocyte invasion. To validate these findings, we conducted double-immunofluorescence microscopy. F. tularensis strains incubated with rabbit erythrocytes to allow for invasion. Cells were probed with anti-F. tularensis antibodies followed by red fluorescent secondary antibodies. Cells were washed and erythrocytes were permeabilized. Bacteria were probed a second time utilizing a green fluorescent secondary antibody. Therefore, intracellular bacteria were only labeled with a singular secondary antibody, while extracellular bacteria were tagged with two secondary antibodies. Intracellular bacterial enumeration showed that deletion of FTL₁199 resulted in a significant reduction of invasion (p = 0. 0253), while FTL₁199 complementation to the deletion mutant restored invasion to that of wild type (p = 0. 3258). These data demonstrate that FTL₁199 plays an important role in erythrocyte invasion. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence, R15HL14735 from NHLBI, and funds from the NASA West Virginia Space Grant Consortium).