Spotted fever group (SFG) rickettsiae are the etiologic agents of serious human diseases including Rocky Mountain spotted fever, caused by Rickettsia rickettsii. Because of limitations imposed by the lack of workable genetic systems and the necessity of using eucaryotic cells as a growth medium, there is a paucity of information on specific virulence mechanisms exploited by these organisms. Insight into the virulence of SFG rickettsiae has recently been achieved with the discovery that, upon entering the cytoplasmic compartment, organisms recruit and polymerize host cell actin to promote direct cell-to-cell spread. To achieve a greater understanding of rickettsial actin-based motility, the research conducted in this proposal has the following goals: 1) identify the rickettsial protein ligand(s) that mediates actin-based motility and the cognate cytosolic proteins that bind this ligand(s), and 2) fully characterize rickettsial actin tail structure and formation. A priori, the rickettsial protein involved in actin recruitment is likely surface localized. Therefore, aim 1 will focus upon the potential involvement of the two immunodominant rickettsial outer membrane proteins, rOmpA and rOmpB. Transfected Vero cells producing specific domains of rOmpA or rOmpB, and infected with R. rickettsii, will be examined to determine whether cytosolic rOmp domain expression disrupts rickettsial actin recruitment and/or host cell microfilament structure. A parallel approach will utilize microinjection of rOmpA or rOmpB-specific Mab into the cytoplasm of infected cells to determine if rickettsial-induced actin polymerization can be inhibited. Because a rickettsial surface protein other than rOmpA and rOmpB may be involved, a global screen of rickettsial small-plaque-forming mutants generated by UV irradiation will be conducted in aim 2 to identify rickettsial mutants deficient for actin mobilization. The protein profiles of mutant and wild type rickettsiae will be compared to identify and allow molecular cloning of the candidate rickettsial actin recruitment protein.
Aim 3 will identify and clone host cytosolic proteins that interact with the identified rickettsial actin recruitment protein. Interacting host proteins will initially be identified as proteins that co-immunoprecipitate with the rickettsial protein produced in Vero cells by transfection. Affinity chromatography will be performed with cytoplasmic extracts to purify the interacting host protein and allow identification and cloning of the encoding gene. A separate strategy will employ yeast-two hybrid technology. Finally, aim 4 will examine rickettsial actin tails to identify actin filament length and orientation, associated host actin-binding proteins, and the relative rate of rickettsial actin-based movement. Not only will these studies lead to a greater understanding of molecular mechanisms of rickettsial pathogenesis, but they will also provide insight into the dynamic control of actin polymerization.
| Heinzen, Robert A (2003) Rickettsial actin-based motility: behavior and involvement of cytoskeletal regulators. Ann N Y Acad Sci 990:535-47 |
| Harlander, Ronald S; Way, Michael; Ren, Qun et al. (2003) Effects of ectopically expressed neuronal Wiskott-Aldrich syndrome protein domains on Rickettsia rickettsii actin-based motility. Infect Immun 71:1551-6 |
| Van Kirk, L S; Hayes, S F; Heinzen, R A (2000) Ultrastructure of Rickettsia rickettsii actin tails and localization of cytoskeletal proteins. Infect Immun 68:4706-13 |
| Heinzen, R A; Grieshaber, S S; Van Kirk, L S et al. (1999) Dynamics of actin-based movement by Rickettsia rickettsii in vero cells. Infect Immun 67:4201-7 |
