Characterization of Rickettsia parkeri Sca2 N-terminal Domain and its Role in Actin Polymerization

Rickettsia parkeri is an obligate intracellular Gram-negative bacterium that causes spotted fever disease. Following invasion of host cells, R. parkeri polymerizes host actin filaments to form actin comet tails for movement within and between cells. We are studying the interactions of R. parkeri with actin to help further our understanding of bacterial pathogenesis and host cell biology. Other pathogens utilize the host cell’s Arp2/3 complex to make actin tails consisting of short branched filaments. However, R. parkeri makes tails composed of long straight filaments using the bacterial protein Sca2. Sca2 is thought to have similar properties to host formins, a family of proteins that promote nucleation and elongation of actin filaments. Although full length Sca2 protein was shown to exhibit formin-like activity, the functions of individual domains are not yet defined. The N terminal domain (NTD) of Sca2 is thought to be similar to the FH2 domain of formins based on its predicted structure and involvement in actin nucleation. Previous attempts at purifying a stable NTD were limited in success. I am working to purify four smaller truncations to define a minimal structural domain required for activity. To determine if each truncation has a formin FH2-like nucleation function, I will use pyrene-actin assembly assays to obtain quantitative data of each truncation’s polymerization activity. Preliminary results indicate that even the shortest truncation retains activity. These experiments will reveal information about the role of the NTD in Sca2 activity in vitro and function in Rickettsia pathogens actin based motility.