Host-pathogen interactions involved in erythrocyte invasion by Francisella tularensis

Francisella tularensis is a gram negative, facultative intracellular bacterium that causes the zoonotic disease tularemia. F. tularensis 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 F. tularensis. In this study, we identified the red blood cell (RBC) membrane protein, Band 3, is required for erythrocyte invasion by F. tularensis. 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 F. tularensis LVS was used to identify interacting bacterial proteins. Here, we identified that the F. tularensis Glycine Cleavage Protein T, GcvT, interacts with the cytoplasmic domain of Band 3. A mutational analysis confirmed GcvT is required for RBC invasion by F. tularensis validating the finding that GcvT interacts with Band 3. Lastly, we determined that the F. tularensis Type VI Secretion System Effector Protein, PdpC, interacts with the erythrocyte cytoskeletal protein, Spectrin, alpha chain, likely contributing to the ability of F. tularensis to invade erythrocytes.