Towards delivery of DNA into mammalian mitochondria by bacterial conjugation

We are investigating alternative methods for delivering functional DNA into mitochondria. Our approach takes advantage of the fact that mitochondria are evolved from ancient endosymbiotic bacteria, and also the fact that present-day bacteria are able to transfer genetic material among each other via conjugation, a process that is entirely donor-driven. In other words, we would like to use bacteria to deliver DNA into mitochondria by conjugation.;Since there are many ways in which such a method could fail, we first conducted a proof-of-principle experiment to show that the bacterial conjugation machinery is functional within the host cell cytoplasm, by demonstrating conjugation between intracellular bacteria. To do this, we constructed a bacterial strain that is capable of infecting mouse cells and escaping from the phagosomes into the cytoplasm. In addition, the donor bacteria carried a conjugative plasmid that is capable of self-transfer, as well as an antibiotic marker distinct from the antibiotic marker carried on recipient bacteria. We found that the conjugative plasmid was transferred into recipient bacteria within the host cell cytoplasm in a matter of hours. Control experiments using a conjugative plasmid incapable of self-transfer showed that the process was indeed conjugation and not due to transformation of recipient bacteria by DNA from dead (lysed) donor bacteria.;Besides using bacteria to deliver DNA into mitochondria by conjugation, we are also investigating a simplified approach utilizing key components of the conjugation process. For plasmid RSF1010, these components are the mobilization protein, MobA, and the origin of conjugative transfer, oriT. MobA is the DNA strand transferase that binds and nicks a specific sequence in its cognate oriT, creating a covalent bond with the DNA in order to transfer the plasmid into the recipient cell. We created a chimeric protein consisting of the portion of MobA that is responsible for DNA binding and nicking, and the mitochondrial targeting pre-sequence from the human mitochondrial DNA polymerase gamma (POLG). We showed that the chimeric protein retains both conjugative and mitochondrial targeting functions. We plan to create covalently bound DNA-protein conjugates for delivery into cells using phosphonium cations or some other delivery vehicle.;Others have shown that bacteria can conjugate to isolated mitochondria. We are currently in the process of replicating those experiments, as well as extending those findings to show that bacteria can conjugate to mitochondria within cells. Our attempts to deliver DNA into mitochondria using variations on the approach are also described and discussed. (Abstract shortened by UMI.)...