Development of antibacterial Type III secretion system binding antibodies

Common agricultural crops such as wild rice and tomatoes are infected by the bacterial pathogens Xanthomonas Oryzae pv. oryzae and Pseudomonas Syringae pv. syringae respectively. These species use the same cellular structure to infect their host cells called a type three secretion system or T3SS. T3SSs are composed of multiple protein subunits used to insert effector proteins into the host cell's cytoplasm. These effectors in turn begin a cascade of events that ultimately results in invasion of the host cell. Once infected, the pathogens can take over the cell's system and ultimately kill it. Given the importance of tomatoes and rice as food sources, the spread of these infections could potentially become disastrous for the populations that depend on them. To combat this infection, biological antibiotics will be developed to bind to a critical subunit in each of the respective pathogen's T3SS, preventing proper assembly and function, and rendering the bacteria non-pathogenic. A combination of yeast surface display and directed evolution are being used to find nanobodies specific to the T3SS in Xanthomonas Oryzae pv. oryzae and Pseudomonas Syringae pv. syringae. Nanobodies are inexpensive and stable antibody fragments that hold substantial potential as antimicrobials in human and agriscience applications. To do this, an antigen mimicking that of the bacterial secretion systems must be created. The antigens herein were chosen for their relative conservation and role in T3SS pathogenicity. This antigen selection strategy is expected to both increase the applicability of the method across bacterial species, and reduce the potential for the development of antibiotic resistance. This poster outlines the selection, cloning, and purification of a T3SS antigen for directed evolution of nanobodies as T3SS-targeting antimicrobials. © FASEB.