Role of VirD4 in early stages of protein and DNA secretion

Type IV coupling proteins (T4CPs) are phylogenetically conserved regulators of medically and economically relevant bacterial type IV secretion systems (T4SS). T4CPs act as substrate receptors, that regulate the uptake of the substrate to the secretion channel and energize the secretion process. Elucidating their functions is crucial for our fundamental understanding of how T4SSs initiate transfer of genes and virulence factors into the cytosol of other bacterial or eukaryotic host cells. This project focuses on VirD4, the coupling protein of the T4SS of Agrobacterium tumefaciens, which is one of the best studied paradigms. The Agrobacterium VirB/D4 T4SS leads to the oncogenic transformation of plant cells and serves as an important tool for the genetic engineering of plants. Our extensive knowledge about this system, including information about its architecture and about VirD4 interaction partners, makes it the ideal model to elucidate the complex, mutual modulation circuit within which type IV coupling proteins are embedded. In this project I will identify key residues and functional domains of VirD4 that are required at multiple points of this intricate interaction network. Therefore I will randomly mutagenize VirD4 and exploit a combination of different transfer and genetic assays to dissect the impact on function or transfer by the introduced mutation. The results from this study will not only increase our knowledge of Agrobacterium mediated macromolecular transfer, but will also have an immediate impact on our functional understanding of other T4SSs involved in bacterial pathogenesis or antibiotic resistance dissemination. Furthermore, this study will provide the basis for follow-up structure-function analysis that will help to modify type IV secretion systems for further use as novel tools for genetic engineering of mammalian cells or in delivering antimicrobial agents to other bacteria to combat infections. In the long run this would allow us to benefit from the high transfer efficiency of these naturally occurring macromolecule secretion systems to establish new gene therapeutic approaches or turn bacteria against bacteria to provide alternatives to current antimicrobial agents.

Bacteria use specialized secretion processes mediated by type IV secretion systems (T4SS) to deliver protein and DNA molecules to other bacterial, plant or animal cells. As a result, T4SSs contribute significantly to the dissemination of antibiotic resistances, bacterial adherence, and the virulence of several prominent pathogens. Type IV coupling proteins (T4CPs) are essential parts of these T4SSs. They act as substrate receptors that regulate the uptake of substrates to the secretion channel and energize the secretion process. This project focused on VirD4Atu, the coupling protein of the T4SS of Agrobacterium tumefaciens, which is one of the best studied T4SS paradigm and an ideal model to elucidate the complex, mutual modulation circuit within which T4CPs are embedded. A strategy that employed random and specialized mutagenesis approaches with a combination of different transfer and genetic assays identified essential residues on VirD4Atu that are involved in the productive entry of macromolecular secretion substrates to the transport channel. To verify the importance of these findings, regions on TraGRP4, the coupling protein of conjugative plasmid RP4, were replaced with key domains of VirD4Atu and the ability of these hybrid proteins to transfer homo- and heterologous substrates was investigated. The fact, that no transfer events could be detected verified the importance of the identified VirD4Atu domains, but also underscores the complexity of the intricate network of protein interactions, that T4CPs have to fulfill. In summary, the results of this study increased our knowledge of Agrobacterium mediated macromolecular transfer and our functional understanding of T4SSs in general. Furthermore, this study provided the basis for follow-up structure-function analysis that in the long run will allow us to modify type IV secretion systems for the use as novel tools for genetic engineering of plant and mammalian cells or in delivering antimicrobial agents to other bacteria to combat infections.