Motility Characteristics Are Altered for Rickettsia bellii Transformed To Overexpress a Heterologous rickA Gene

Abstract

The rickettsial protein RickA activates host cell factors associated with the eukaryotic actin cytoskeleton and is likely involved with rickettsial host cell binding and infection and the actin-based motility of spotted fever group rickettsiae. The rickA gene sequence and protein vary substantially between Rickettsia species, as do observed motility-associated phenotypes. To help elucidate the function of RickA and determine the effects of species-specific RickA variations, we compared extracellular binding, intracellular motility, and intercellular spread phenotypes of three Rickettsia bellii variants. These included two shuttle vectortransformed R. bellii strains and the wild-type isolate from which they were derived, R. bellii RML 369C. Both plasmid shuttle vectors carried spectinomycin resistance and a GFPuv reporter; one contained Rickettsia monacensis-derived rickA, and the other lacked the rickA gene. Rickettsia bellii transformed to express R. monacensis rickA highly overexpressed this transcript in comparison to its native rickA. These rickettsiae also moved at higher velocities and followed a more curved path than the negative-control transformants. A lower proportion of R. monacensis rickA-expressing bacteria ever became motile, however, and they formed smaller plaques. Rickettsiae are obligate intracellular Alphaproteobacteria in the order Rickettsiales. Most, but not all, species of rickettsiae coopt host cell-produced actin to induce bacterial movement in and between host cells, a behavior termed actin-based motility. Rickettsia bellii 369C is one such motile species; it was isolated from ticks of the genera Amblyomma and Dermacentor and is considered nonpathogenic in that it does not obviously negatively affect its host ticks and has never been associated with disease in vertebrates. Most previous studies examining rickettsial motility have done so by using pathogenic species, such as Rickettsia rickettsii (1, 2), or the milder pathogen, Rickettsia parkeri (3, 4). As a nonpathogen also capable of actin-based motility and rapid cell invasion, R. bellii provides a contrast that may help illuminate virulence factors of the pathogenic species. Additionally, because of its capability of infecting both tick and mammal-derived cell lines and its tractability to genetic manipulation, R. bellii was a preferred species for this study.In Listeria and Shigella, two facultatively intracellular bacterial genera that also utilize actin-based motility, branched actin tails are polymerized by activating the host cell ARP2/3 protein complex (5, 6). The ARP2/3 complex is activated by nucleation-promoting factors, proteins that are naturally produced within eukaryotic cells, including the Wiskott-Aldrich syndrome protein (WASP) family (7), but can be mimicked by intracellular bacteria to hijack the actin polymerization process for the manufacture of a branched actin tail. In Listeria, the nucleation-promoting factor mimic is the protein ActA (8, 9). A similar rickettsial WASP-like protein, RickA, has been considered ...

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