SOLUBLE FORMS OF AN ANTI-XYLELLA ANTIBODY AND STRAINS OF ALCALIGENES XYLOSOXIDANS DENITRIFICANS CAPABLE OF SECRETING THEM Project Leader:

Several methods were used to create soluble forms of a single chain antibody (scFv S1) that binds to the surface of the grape strains of Xylella fastidiosa (Xf). S1 fused to a pelB leader and secreted from E. coli. These forms were not secreted correctly and could not bind Xf in an ELISA. Maltose binding protein fusions of S1 were soluble and could be used to detect Xf in an ELISA. We also successfully secreted S1 from Alcaligenes xylosoxidans denitrificans (Axd) using a leader sequence that directed S1 to the periplasmic space. Strains of Axd that secrete anti-Xylella factors are being developed for use in a strategy to prevent the spread of Xf. INTRODUCTION The glassy-winged sharpshooter (GWSS) is the principal vector of the xylem-limited bacterium Xylella fastidiosa (Xf), which causes Pierce’s disease (PD) in grapes. Limiting the spread of this pathogen by rendering GWSS incapable of pathogen transmission (paratransgenesis) is a promising method of pathogen control. Paratransgenesis seeks to modify the phenotype of an organism indirectly by modifying its symbiotic bacteria to confer vector-incompetence Paratransgenic approaches to disrupt pathogen infection of humans are being developed by several groups. These include interference with the ability of triatomid bugs to transmit pathogens causing Chagas’ disease (Beard et al., 2001), interference with HIV attachment to its target cells in the reproductive tracts of humans (Chang et al., 2003; Rao et al., 2005), and the elimination of persistent Candida infections from biofilms in chronically infected patients (Beninati et al., 2000). Paratransgenesis has also been applied to deliver cytokines mammalian guts to relieve colitis (Steidler et al., 2000; Steidler, 2001). Thus, the method has wide applicability. Alcaligenes xylosoxidans denitrificans (Axd) is Gram negative, beta proteobacterial species that can colonize the GWSS foregut and cibarium, as well as various plant tissues, including xylem. It is non-pathogenic in insects, plants and healthy humans. Given these characteristics, Axd has become the focus of our paratransgenesis efforts to control PD in grapes. Over the past several years we developed the technology to stably modify Axd by inserting genes into its chromosome, have developed methods to suppress horizontal gene transfer, and have isolated a single chain antibody (scFv) that recognized an epitope on the surface of the PD strain of Xf. (Bextine et al., 2004). We are currently engaged in combining these systems in order to produce strains of Axd that are suitable for environmental release in a practical strategy symbiotic control strategy for PD. We report here the evaluation of various S1 constructs for solubility and the construction of a prototype Axd strain capable of secreting S1. OBJECTIVES 1. To create soluble and functional forms of the S1 single chain antibody. 2. To construct strains of Axd capable of secreting scFvs. RESULTS Objective 1: Soluble forms of the S1 scF. We expressed a soluble form of the S1 scFv in two ways. S1 was expressed from a construct carrying a pelB leader sequence which targets the protein to the periplasm of the cell, from which it can “leak” out into the growth medium and be collected. Several strains of E. coli were used for this test. We also fused the S1 sequence to E. coli maltose binding protein and purified the fusion protein using affinity chromatography. S1 proteins expressed in these two ways were assayed in a western blot to see if they could be detected at all and were also used in an ELISA to determine whether or not they could still bind to the surface of Xf. The results of these assays are shown in Table 1.