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

Xylella fastidiosa (Xf) is a gram-negative xylem-limited bacterium and causative agent of Pierce’s 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 (1). Design of a protein inhibitor that interrupts this step of the plant-Xf interaction will be useful in anti-microbial therapy and controlling PD. 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, human neutrophil Elastase (5-10) is the SRD that recognizes MopB, the major outer membrane protein of Xf (11). The second domain is Cecropin B, a lytic peptide that targets and lyses gram-negative bacteria. We have combined Elastase and CecropinB using a flexible linker such that both components can simultaneously bind to their respective targets. This chimeric gene was synthesized and cloned into different vectors for insect and plant transformation. Five transformed insect cell lines are being evaluated and production and processing of the protein is being optimized in in liter size preps. Plant transformation experiments have been completed and we have obtained plants of Nicotiana tabacum var benthamiana and plants of Vitis vinifera ‘Thompson Seedless ’ transformed with this gene that are currently being analyzed for gene expression and protein production. Th