Ultrastructure of Rickettsia rickettsii actin tails and localization of cytoskeletal proteins

Actin-based motility (ABM) is a mechanism for intercellular spread that is utilized by vaccinia virus and the invasive bacteria within the genera Rick ettsia, Listeria, and Shigella. Within the Rickettsia, ABM is confined to m embers 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 ho st cytosol and into cell membrane protrusions that can be engulfed by neigh boring cells, initiating a new infectious cycle. Little is known about the structure and morphogenesis of the Rickettsia rickettsii actin tail relativ e to Shigella and Listeria actin tails. In this study we examined the ultra structure of the rickettsial actin tail by confocal, scanning electron, and transmission electron microscopy. Confocal microscopy of rhodamine phalloi din-stained infected Vero cells revealed the typhus group rickettsiae, Rick ettsia prowazekii and Rickettsia typhi, to have no actin tails and short (s imilar to 1- to 3-mu m) straight or hooked actin tails, respectively. The S FG rickettsia, R. rickettsii, displayed long actin tails (>10 mu m) that we re frequently comprised of multiple, distinct actin bundles, wrapping aroun d each other in a helical fashion. Transmission electron microscopy, in con junction with myosin S1 subfragment decoration, revealed that the individua l actin filaments of R. rickettsii tails are >1 mu m long, arranged roughly parallel to one another, and oriented with the fast-growing barbed end tow ards the rickettsial pole. Scanning electron microscopy of intracellular ri ckettsiae demonstrated R. rickettsii to have polar associations of cytoskel etal material and R. prowazekii to be devoid of cytoskeletal interactions. By indirect immunofluorescence, both R. rickettsii and Listeria monocytogen es actin tails were shown to contain the cytoskeletal proteins vasodilator- stimulated phosphoprotein profilin, vinculin, and filamin. However, rickett sial tails lacked ezrin, paxillin, and tropomyosin, proteins that were asso ciated with actin tails of cytosolic or protrusion-bound Listeria. The uniq ue ultrastructural and compositional characteristics of the R. rickettsii a ctin tail suggest that rickettsial ABM is mechanistically different from pr eviously described microbial ABM systems.