The Rickettsia Late Motility Factor Sca2 Exhibits Species Differences in its Actin Assembly Mechanism by Julie Eunkyoung Choe A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Molecular and Cell Biology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Matthew D. Welch, Chair Professor Eva Nogales Professor David Drubin Professor Kathleen Ryan Fall 2015 The Rickettsia Late Motility Factor Sca2 Exhibits Species Differences in its Actin Assembly Mechanism © 2015 by Julie Eunkyoung Choe Abstract The Rickettsia Late Motility Factor Sca2 Exhibits Species Differences in its Actin Assembly Mechanism by Julie Eunkyoung Choe Doctor of Philosophy in Molecular and Cell Biology University of California, Berkeley Professor Matthew D. Welch, Chair Intracellular pathogens commonly subvert the host cell actin cytoskeleton during various points of their infection cycles. My work has focused on the exploitation of actin by pathogens that grow within the cytosol. Many such pathogens assemble actin filaments at their surface to power motility within the cytoplasm, facilitating cell-cell spread during infection. Diverse bacterial species have independently evolved this strategy, and each uses a distinct mechanisms to intercept or mimic different host proteins involved in actin polymerization. Rickettsia are one genus of bacterial pathogens that exploit actin for intracellular motility. These are Gram-negative, obligate intracellular pathogens that include the causative agents of various types of spotted fever disease and typhus. Species within the genus Rickettsia can express up to two bacterial factors that power motility at either early or late times during infection. My work has focused on orthologs of the Rickettsia late motility factor Sca2 (surface cell antigen 2). Interestingly, I have found that these exhibit significant sequence differences between species. I participated in the discovery that Sca2 from the spotted fever group (SFG) Rickettsia species R. parkeri mimics eukaryotic formins in its ability to nucleate and processively elongate actin filaments, resulting in the assembly of an actin comet tail consisting of long and bundled filaments. Furthermore, for R. parkeri Sca2, I identified a minimal truncation that retains nucleation function, and showed that it can bind to three molecules of profilin, also similar to the behavior of formins. In contrast, Sca2 from typhus group (TG) and ancestral group (AG) Rickettsia species lack the formin-mimicking domain, and the organization of the actin filaments in their comet tails is poorly defined. I found that Sca2 from the TG species R. typhi nucleates actin, though its specific actin assembly mechanism remains undetermined. I further found that Sca2 from the AG species R. bellii nucleates actin efficiently when dimerized, and binds to a single actin monomer through a WASP-homology 2 (WH2) motif. These results suggest that R. bellii Sca2 may assemble actin via a mechanism that mimics eukaryotic tandem- WH2 nucleators, and its activity may be enhanced by dimerization or oligomerization of Sca2 on the bacterial surface. Additionally, I found that during infection, R. bellii move more slowly, in more curved paths, and more frequently than R. parkeri, and generate narrower actin tails consisting of bundled actin filaments. Nevertheless, R. bellii and R. parkeri use a similar set of host actin cytoskeletal proteins for efficient motility. Together, these data support the conclusion that R. bellii Sca2 utilizes a distinct mechanism to assemble actin filaments, and yet harness a similar set of host proteins to promote a motility that occurs with distinct parameters when compared with other Rickettsia species. This work reveals that even related bacterial species exhibit a surprising diversity of actin assembly mechanisms to subvert host actin for intracellular movement. 1 Table of Contents Abstract ........................................................................................................................................... 1 Table of Contents .............................................................................................................................. i List of Figures ................................................................................................................................... ii List of Abbreviations ....................................................................................................................... iii Acknowledgments........................................................................................................................... iv Chapter 1 – Introduction: An evolutionary perspective on the role of actin-based motility in virulence of intracellular bacterial pathogens ................................................................................ 1 Roles of actin-based motility in survival and virulence ...................................................... 4 Bacterial motility factors mimic eukaryotic host actin nucleators ..................................... 6 Actin motility proteins – orthologs comparison and evolution .......................................... 7 Conclusion and future directions ...................................................................................... 11 References ........................................................................................................................ 13 Chapter 2 – Actin assembly by the late actin-based motility factor Sca2 in spotted fever group and typhus group Rickettsia species ............................................................................................. 18 Introduction ...................................................................................................................... 19 Results ............................................................................................................................... 21 Discussion.......................................................................................................................... 27 Materials & Methods ........................................................................................................ 31 References ........................................................................................................................ 34 Chapter 3 – The Sca2 protein from the ancestral group Rickettsia species Rickettsia bellii employs a distinctive mechanism of actin assembly .................................................................... 37 Introduction ...................................................................................................................... 38 Results ............................................................................................................................... 40 Discussion.......................................................................................................................... 50 Materials & Methods ........................................................................................................ 55 References ........................................................................................................................ 59 Chapter 4 – Future Directions & Remaining Questions ................................................................ 63 Sca2 Ortholog Mechanisms .............................................................................................. 64 Host Factor Requirements ................................................................................................ 65 Expression Patterns and Regulation of Sca2 Orthologs.................................................... 66 Genetic Manipulation of Rickettsia .................................................................................. 67 References ........................................................................................................................ 69 i List of Figures Figure 1.1 – Bacteria intercept actin pathways for several purposes during infection ................. 3 Figure 1.2 – Eukaryotic actin nucleators and their bacterial mimics.............................................. 5 Figure 1.3 – Mechanisms of ABM of select bacterial species ......................................................... 8 Figure 2.1 – R. parkeri Sca2 domain organization and biochemical characterization of a minimal active fragment ........................................................................................... 20 Figure 2.2 – The proline-rich region of RpSca2 binds profilin ...................................................... 23 Figure 2.3 – RtSca2 passenger is largely insoluble and prone to aggregation. RtSca2-785 conditionally enhances actin assembly .................................................................... 25 Figure 3.1 – Rickettsia phylogeny and domain organization of Sca2 orthologs ........................... 39 Figure 3.2 – R. bellii actin-based motility parameters differ from those of R. parkeri ................. 41 Figure 3.3 – A similar set of host cytoskeleton proteins is important for R. bellii and R. parkeri motility ...................................................................................................... 43 Figure 3.4 – Sca2 has a polar localization more often in R. bellii than in R. parkeri .................... 45 Figure 3.5 – The R. bellii Sca2 passenger domain nucleates actin when dimerized .................... 46 Figure 3.6 – R.