Role of c-Cbl in Invasion of Mammalian Cells by Rickettsia Conorii

Specific AimsRickettsia conorii is an intracellular bacterium that causes Mediterranean spotted fever. R. conorii is transmitted from ticks to humans and invades vascular endothelial cells. A recent publication (Martinez et al., 2005) has identified Ku70 as a host cell receptor that binds to rOmpB, an R. conorii surface ligand. The engagement of Ku70 by rOmpB leads to a rapid ubiquitination of Ku70 by c-Cbl, followed by the entry of R. conorii into the host cell. However, the role of c-Cbl in ubiquitinating Ku70 and communicating with the invading bacterium remain to be clarified. Based on the report of Martinez et al and other groups on the roles played by R. conorii surface ligands rOmpA and rOmpB, we propose the following hypothesis: Hypothesisc-Cbl ubiquitinates Ku70 in response to signals from a Rickettsia conorii cell surface protein called rOmpA. This ubiquitination event enables Ku70 and the attached bacteria to be endocytosed into the cell. Specific Aims1. To analyse the effect of c-Cbl - mediated ubiquitination on Ku70 localisation. We will investigate if ubiquitination of Ku70 by c-Cbl is necessary for Ku70 to localize to the bacterial entry foci. We will culture c-Cbl deficient cells and observe the effects of R. conorii infection on them.2. To map the putative signal(s) involved in the communication between R. conorii rOmpA outside the cell and c-Cbl inside the cell. We will identify the putative rOmpA receptor on host cells and protein(s) that may act as bridges of communication between rOmpA and c-Cbl.3. To investigate the mechanism of how ubiquitination of Ku70 enables R. conorii to enter a host cell. We will determine if any c-Cbl interacting proteins and/or endocytic proteins are recruited to the cell surface by ubiquitinated Ku70 to form a phagosome. If endocytic proteins are recruited, we will proceed.