The Rickettsia Ankyrin Repeat Protein 2 is a type IV secreted effector and co-localizes with markers of the endoplasmic reticulum in mammalian cells

University of Maryland, Baltimore. Molecular Microbiology and Immunology. Ph.D. 2018 ; Spotted fever and typhus group Rickettsia species are neglected zoonotic, vector-borne bacteria that cause fatal infections and are distributed worldwide. These pathogens efficiently invade host endothelial cells, rapidly escape the phagosome, and establish an "intracytosolic niche," where they grow to high numbers before cell lysis. Despite more than two decades of research, the mechanisms of rickettsial pathogenesis and cytosolic survival remain poorly understood. A candidate virulence factor, Rickettsia ankyrin repeat protein 2 (RARP-2), is largely conserved in pathogenic strains, absent in non-pathogenic strains, and shows increased expression in the mammalian stage of infection. Based on these observations, we hypothesized that RARP-2 functions to enhance pathogenesis during rickettsial infection in mammals. This study demonstrates that RARP-2 is a cytosolically localized effector that is secreted by the type IV secretion system (T4SS) of Rickettsia species from both the typhus and spotted fever groups in order to modulate host pathways. Analysis of homolog distribution and domains of RARP-2 in 43 Rickettsia spp. allowed for the detailed characterization of the C-terminal ankyrin repeats, an N-terminal putative cysteine protease motif, and a C-terminal secretion signal characterized by disorder and flexibility. R. typhi RARP-2 is maximally expressed during the cytosolic phase of infection, is secreted via the T4SS, and overexpression results in the formation of cytosolic vesicular structures. The virulent R. rickettsii str. Sheila Smith (SS) RARP-2 homolog was observed to have 7 more Ank repeats than the avirulent str. Iowa-RARP-2. While both SS- and Iowa-RARP-2 homologs are secreted by the T4SS, only expression of SS-RARP-2 in the avirulent Iowa strain induced a lytic plaque phenotype characteristic of virulent strains. SS-RARP-2 also induced the formation of vesicular like structures in the host cytoplasm that were ...