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The overall aim of this three-year project is to develop a second generation paratransgenic approach to delivery of engineered Alcaligenes xylosoxidans denitrificans (AXD) to Homalodisca vitripennis, the arthropod vector of Xylella fastidiosa, causative agent of Pierce's disease of grapevines. This proposal will focus on strategies to contain unwanted environmental spread of recombinant bacteria and potential horizontal gene transfer (HGT) of foreign DNA. <P>
Specific Aim 1: To develop a synthetic alginate-chitosan microsphere for encapsulation and field delivery of engineered AXD to H. vitripennis <P>
Specific Aim 2: To establish efficacy of a synthetic alginate-chitosan microsphere in preventing environmental escape of recombinant AXD and horizontal gene transfer of foreign DNA to environmental bacteria <P>
Specific Aim 3: To establish in closed-cage settings the efficacy of a Resin-Microsphere System (RMS) in delivery of engineered AXD to the cibarial region of H. vitripennis <P>
TIMELINE, DELIVERABLES: <BR> Month 6 Formulation of Alginate-Chitosan Microsphere; Bacterial stability; pH-gated release of bacteria/re-design with new polymers as needed ALC Microsphere with desired characteristics <BR>Month 18 Containment of R-AXD by ALC Microsphere Prevention of HGT and transfer within earthworm; ALC Microsphere with containment properties <BR>Month 24 Development of Resin Microsphere System Microsphere stability, water impermeability and coating of plants/ re-design as needed RMS that permits microsphere viability and water impermeability <BR>Month 36 Simulated field trial with H. vitripennis and grape plants Delivery of R-AXD via RMS; colonization of cibarium; no release into rhizosphere; Proof-of-concept in simulated conditions of targeted release of R-AXD <BR><BR>Expected Outcomes and Alternative Approaches: We expect the alginate microspheres to provide partial protection of the R-AXD. Whereas, freezing temperatures will cause death of most control AXD, we expect a statistically significant increase in survival of the microsphere-encased population. Similar results are expected for the trials involving extremes of high UV light and aridity. We expect the alginate matrix to prevent HGT in either direction. Comparison of recombinant events between the experimental (alginate microspheres) and control (liquid co-incubation) groups should reveal a statistically significant difference that validates the hypothesis. <BR><BR>Again, we expect that ALC microspheres will prevent release of R-AXD into the gut of the earthworm and secondary sequelae such as HGT within gut microbial consortia. There should be a very significant difference in free R-AXD in the gut lumen of experimental versus control worms. In the absence of high fluid flux, ingested ALC microspheres are expected to remain closed with no leakage of R-AXD into the gut of the worm. If the RMS performs in a similar fashion to CRUZIGARD, we expect stability of the ALC microspheres and water impermeability of the matrix itself. Since the R-AXD bacteria are stabilized within the microspheres, it is possible that they will remain viable for the entire duration of 6 months.
NON-TECHNICAL SUMMARY: Despite advances in public health, insect-transmitted diseases remain a leading cause of morbidity and mortality. Additionally, the global impact of diseases to agriculture exceeds $100 billion. Currently,the best methods for control of many insect-borne diseases involve the use of pesticides which are toxic, expensive and allow evolution of insect resistance. Evolving methods for control of vector-borne diseases rely on modification rather than elimination of insects. These strategies involve either direct transformation of an insect genome or expression of gene products in the insect via transformed symbiotic microbes (paratransgenesis). Paratransgenesis is a "Trojan Horse" approach to control of disease transmission. It employs the interactions between disease-transmitting vectors, bacterial symbionts of the vectors and transmitted pathogens. Symbiotic bacteria are isolated and genetically transformed to export molecules that interfere with pathogens.The genetically altered symbionts are then introduced into the host vector where expression of engineered molecules affects the host's ability to transmit the pathogen.Pierce's Disease is a deadly disease of grapevines causing tremendous economic loss to the wine industry of California each year. It is caused by the bacterium Xylella fastidiosa, which is spread by xylem-feeding sharpshooters. The predominant vector of this disease in the US is the Glassy Winged Sharpshooter (GWSS), Homalodisca vitripennis. Pierce's Disease is prevalent within the USA from Florida to California, and outside the USA in Central and South America. In the paratransgenic approach, a commensal bacterium of H. vitripennis, Alcaligenes xylosoxidans var. dentrificans (AXD), is modified to export molecules that disrupt the transmission of X. fastidiosa, the causative agent of Pierce's disease of grapevines. Both AXD and Xylella colonize the anterior mouthparts (cibarium) of H. vitripennis, thus assuring that exported molecules from AXD contact Xylella and interrupt transmission to plants. Broadcast of engineered Alcaligenes to field sites such as vineyards with subsequent uptake by sharpshooters would result in disruption of regional Xylella transmission. Release of genetically engineered Alcaligenes could pose environmental risks: (1) Alcaligenes species have been associated with human diseases such as pneumonia in immunocompromised persons.(2) Potential horizontal gene transfer to other microbes of the environmental consortium could pose risks above and beyond scenarios involving release of unmodified organisms. Field application of the paratransgenic strategy for control of Pierce's disease would therefore require additional measures to contain human contact with Alcaligenes and minimize gene spread in the environment. This proposal introduces the concept of second generation paratransgenics in which advanced material engineering at the nano- and micro-scale is used to target release of engineered microbes and restrict gene transcription to highly specific sites of pathogen residence within the arthropod itself, with the aim of greatly reducing the risk of foreign gene release into the environment.
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APPROACH: We will develop an alginate-chitosan microsphere for encapsulation of genetically modified AXD stabilized at an acidic pH of 3.7 and gated to open under high fluid flux and pH change (greater than 7.5).The associated flow of fluid at a neutral pH through the mouthparts and anterior gut of the insect will serve as the gating mechanism that causes swelling of the alginate microspheres and release of recombinant AXD (R-AXD). Experiment 1: Synthesis and characterization of Alginate-Chitosan (ALC) microspheres.Alginate microcapsules will be synthesized and loaded. Experiment 2: Containment of AXD within ALC microspheres R-AXD will be encapsulated. Containment of R-AXD will be verified by 3 methods: (1) Fluorescence microscopy (2)Serial washes of ALC microspheres 3)Washed spheres will be immersed in fluid that approximates xylem (pH =7.5) Experiment 3: Microsphere function under extreme environmental conditions Alginate microspheres containing R-AXD will be subjected to simulated conditions of extreme environmental stress: heat, light, aridity. Experiment 4: Containment of bacteria and genetic material in the setting of microbial consortia.Populations of R-AXD contained within alginate microspheres will be exposed microbes commonly found in soil consortia to determine the extent of horizontal gene transfer.2)We will evaluate possible HGT from donor bacteria of the rhizosphere to recipient AXD contained in alginate microspheres. Experiment 5: Containment of recombinant AXD upon ingestion of microspheres by the earthworm, L. terristris. We will evaluate the barrier functions of the alginate-chitosan microsphere in the gut of L. terristris. Experiment 6: Development of a water impermeable Resin Microsphere System (RMS) for field application. In this set of experiments, we will design a RMS for containment of ALC microspheres that will (1) provide a barrier against rain, (2) permit stability of ALC microspheres, (3) permit coating of shoots of grape vines where H. vitripennis is likely to feed, and (4) permit probing and release of ALC microspheres during the initial phase of penetration of the shoot by H. vitripennis. Experiment 7: Delivery of R-AXD to the cibarium of H. vitripennis For this experiment we will use adult H. vitripennis collected from citrus orchards at the Agricultural Operations at UC Riverside. This 3-year program will develop new tools for the delivery of engineered symbiotic bacteria in a paratransgenic system. Robust methods for containment of genetically engineered microorganisms are possible given recent developments in nano-scale material engineering and controlled release. We propose to adapt these technologies to the prevention of arthropod-borne infectious diseases while minimizing risk of transgene delivery. Several other potential applications are already under development in the Durvasula lab related to delivery of engineered symbiotic bacteria to triatomine bugs and larval stages of phlebotomine sand flies. We expect to develop these strategies toward control of other arthropod-borne agricultural diseases and continue to expand the armamentarium against these devastating global scourges.