Host-pathogen interactions involved in erythrocyte invasion by Francisella tularensis

<i>Francisella tularensis</i> is a gram negative, facultative intracellular bacterium that causes the zoonotic disease tularemia. <i>F. tularensis</i> is capable of invading mammalian erythrocytes, a phenomenon that enhances colonization of ticks following a blood meal. The colonization of these blood-sucking arthropods presumably enhances transmission to mammalian hosts and increases the persistence of this bacterium within the environment. Therefore, we sought to elucidate host-pathogen interactions involved in erythrocyte invasion by <i>F. tularensis</i>. In this study, we identified the red blood cell (RBC) membrane protein, Band 3, is required for erythrocyte invasion by <i>F. tularensis.</i> Erythrocyte proteins that complex with Band 3 were also evaluated for their role in erythrocyte invasion. While Glycophorin A impedes invasion, Ankyrin-1, the peripheral membrane protein that links Band 3 to the spectrin cytoskeleton, is required for erythrocyte invasion. Furthermore, expression of the recombinant cytoplasmic domain of Band 3 in <i>F. tularensis</i> LVS was used to identify interacting bacterial proteins. Here, we identified that the <i>F. tularensis</i> Glycine Cleavage Protein T, GcvT, interacts with the cytoplasmic domain of Band 3. A mutational analysis confirmed GcvT is required for RBC invasion by <i>F. tularensis</i> validating the finding that GcvT interacts with Band 3. Lastly, we determined that the <i>F. tularensis</i> Type VI Secretion System Effector Protein, PdpC, interacts with the erythrocyte cytoskeletal protein, Spectrin, alpha chain, likely contributing to the ability of <i>F. tularensis</i> to invade erythrocytes.