DESIGN OF CHIMERIC ANTI-MICROBIAL PROTEINS FOR RAPID CLEARANCE OF XYLELLA

• Author(s): Dandekar, Abhaya; Gupta, Goutam; Hong-Geller, Elizabeth; McDonald, Karen; Bruening, George; Civerolo, Ed; Shiflett, Patrick; Unkefer, Cliff; Unkefer, Pat;
• Abstract: Xylella fastidiosa (Xf), is a gram-negative xylem-limited bacterium and causative agent of Pierces disease (PD) in California grapevines. During very early stages of Xf infection, specific carbohydrates/lipids/proteins on the outer membrane of Xf interact with plant cells and are important for virulence (3). Design of a protein inhibitor that interrupts this step of the plant- Xf interaction will be useful in anti-microbial therapy and controlling PD. Traditionally, antibiotics are prescribed as a preferred therapy; however, a pathogen often develops antibiotic resistance and escapes their anti-microbial action (4). In this UC/LANL project, we propose a novel protein-based therapy that circumvents the shortcomings of an antibiotic. We have designed a chimeric anti-microbial protein with two functional domains. One domain (called the surface recognition domain or SRD) will specifically target the bacterium outer-membrane whereas the other will lyse the membrane and kill Xf. In this chimera, Elastase is the SRD that recognizes mopB, the newly discovered Xf outer membrane protein (5). The second domain is Cecropin B, a lytic peptide that targets and lyses gram-negative bacteria. We have successfully tested each of these components individually and demonstrated that they each (Elastase and Cecropin B) display activity against Xf, which is increased when both proteins are combined. We have tested Elastase against purified mopB and intact Xf cells and found that mopB is degraded in both cases, suggesting that it is potentially a target for Elastase. The HNE-GSTA-Cecropin B chimera gene has been synthesized and is currently being cloned into vectors for overexpression in insect and grapevine cells in order to test its activity against Xf in vitro. We have also initiated transgenic grapevine cultures expressing a pear polygalacturonase inhibiting protein that is secreted into the medium using a CELLline 350 bioreactor. In the future, we plan to use this system to test secretion and anti-Xf of the chimeric protein.
• Publication Date: Dec 2004
• Journal: 2004 Pierce's Disease Research Symposium