Rickettsial Actin‐Based Motility

A bstract : Actin‐based motility (ABM) is employed by spotted fever group (SFG) rickettsiae, such as Rickettsia rickettsii , to promote cell‐to‐cell spread. Time‐lapse video microscopy revealed that ABM is not strictly confined to SFG rickettsiae as typhus group R. typhi moved at approximately the same rate as R. rickettsii (approximately 4 μm/min), but in a highly erratic fashion. A number of common behaviors were observed between ABM of R. typhi and R. rickettsii , such as entrance into plasma membrane protrusions, formation of new actin tails only on the old surface of newly formed daughter cells, and quick (within 15 sec) reassembly of the actin tail to the opposite pole upon contact with cellular structures that impede forward movement. This last behavior suggests that the rickettsial protein(s) required for ABM is uniformly localized to both poles of the bacterium and possibly throughout the rickettsial surface. Functional roles in rickettsial ABM for neuronal Wiskott‐Aldrich syndrome protein (N‐WASP) and the actin‐related protein (Arp)2/3 complex, critical regulators of ABM of other pathogens, have not been established. Domains of N‐WASP that have characterized inhibitory effects on N‐WASP or Arp2/3 complex function were expressed in HeLa cells infected with R. rickettsii . Shigella flexneri ‐infected cells were used as a control. When ectopically expressed, the VCA domain of N‐WASP (VCA) acts as a dominant/negative with respect to Arp2/3 complex function and N‐WASP missing VCA (ΔVCA) acts as a dominant/negative form of N‐WASP. Expression of VCA or ΔVCA severely inhibited S. flexneri ABM (no Shigella motility observed in the majority of expressing cells) while only moderately inhibiting ABM of R. rickettsii (approximately 35% decrease in the rate of ABM). In addition, ectopically expressed full‐length GFP‐N‐WASP was recruited by S. flexneri but not R. rickettsii , and Arp3 was detected by indirect immunofluorescence in S. flexneri actin tails but not within R. rickettsii actin tails. Collectively, these data suggest that rickettsial ABM is independent of N‐WASP and Arp2/3 complex function.