Dynamics of Actin-Based Movement by Rickettsia rickettsii in Vero Cells

Abstract

ABSTRACT Actin-based motility (ABM) is a virulence mechanism exploited by invasive bacterial pathogens in the genera Listeria , Shigella , and Rickettsia . Due to experimental constraints imposed by the lack of genetic tools and their obligate intracellular nature, little is known about rickettsial ABM relative to Listeria and Shigella ABM systems. In this study, we directly compared the dynamics and behavior of ABM of Rickettsia rickettsii and Listeria monocytogenes . A time-lapse video of moving intracellular bacteria was obtained by laser-scanning confocal microscopy of infected Vero cells synthesizing β-actin coupled to green fluorescent protein (GFP). Analysis of time-lapse images demonstrated that R. rickettsii organisms move through the cell cytoplasm at an average rate of 4.8 ± 0.6 μm/min (mean ± standard deviation). This speed was 2.5 times slower than that of L. monocytogenes , which moved at an average rate of 12.0 ± 3.1 μm/min. Although rickettsiae moved more slowly, the actin filaments comprising the actin comet tail were significantly more stable, with an average half-life approximately three times that of L. monocytogenes (100.6 ± 19.2 s versus 33.0 ± 7.6 s, respectively). The actin tail associated with intracytoplasmic rickettsiae remained stationary in the cytoplasm as the organism moved forward. In contrast, actin tails of rickettsiae trapped within the nucleus displayed dramatic movements. The observed phenotypic differences between the ABM of Listeria and Rickettsia may indicate fundamental differences in the mechanisms of actin recruitment and polymerization.