Ultrastructure of Rickettsia rickettsii Actin Tails and Localization of Cytoskeletal Proteins

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Abstract
Actin-based motility (ABM) is a mechanism for intercellular spread that is utilized by vaccinia virus and the invasive bacteria within the genera Rickettsia , Listeria , and Shigella . Within the Rickettsia , ABM is confined to members of the spotted fever group (SFG), such as Rickettsia rickettsii , the agent of Rocky Mountain spotted fever. Infection by each agent induces the polymerization of host cell actin to form the typical F (filamentous)-actin comet tail. Assembly of the actin tail propels the pathogen through the host cytosol and into cell membrane protrusions that can be engulfed by neighboring cells, initiating a new infectious cycle. Little is known about the structure and morphogenesis of the Rickettsia rickettsii actin tail relative to Shigella and Listeria actin tails. In this study we examined the ultrastructure of the rickettsial actin tail by confocal, scanning electron, and transmission electron microscopy. Confocal microscopy of rhodamine phalloidin-stained infected Vero cells revealed the typhus group rickettsiae, Rickettsia prowazekii and Rickettsia typhi , to have no actin tails and short (∼1- to 3-μm) straight or hooked actin tails, respectively. The SFG rickettsia, R. rickettsii , displayed long actin tails (>10 μm) that were frequently comprised of multiple, distinct actin bundles, wrapping around each other in a helical fashion. Transmission electron microscopy, in conjunction with myosin S1 subfragment decoration, revealed that the individual actin filaments of R. rickettsii tails are >1 μm long, arranged roughly parallel to one another, and oriented with the fast-growing barbed end towards the rickettsial pole. Scanning electron microscopy of intracellular rickettsiae demonstrated R. rickettsii to have polar associations of cytoskeletal material and R. prowazekii to be devoid of cytoskeletal interactions. By indirect immunofluorescence, both R. rickettsii and Listeria monocytogenes actin tails were shown to contain the cytoskeletal proteins vasodilator-stimulated phosphoprotein profilin, vinculin, and filamin. However, rickettsial tails lacked ezrin, paxillin, and tropomyosin, proteins that were associated with actin tails of cytosolic or protrusion-bound Listeria . The unique ultrastructural and compositional characteristics of the R. rickettsii actin tail suggest that rickettsial ABM is mechanistically different from previously described microbial ABM systems.