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<p>Two of the major goals of the AAES and US agriculture are to ensure a safe food supply and improve food production. The long-term goal of this project is to develop a multifaceted approach to improve control of agriculturally important pathogens. These goals are a consolidation and continuation of the research that has been ongoing in my laboratory for the past few years to improve food safety and food production. Our hypothesis is that through early detection of pathogens and development of novel antimicrobials, we can better prevent and combat foodborne illnesses and improve food production. The primary objectives of this study include</p><p> 1) development of multiplex magnetoelastic nanoparticle biosensors for rapid detection of foodborne bacterial pathogens including pathogenic serovars of Salmonella enterica, Escherichia coli O157:H7, and Shigella species;</p><p>2) characterization of novel proteinaceous antimicrobials from subterranean termite Reticulotermes flavipes;</p><p>3) genetic characterization of a novel antifungal agent 7, 10, 12-trihydroxy-8(E)-octadecenoic acid produced by Pseudomonas aeruginosa; and</p><p>4) continuation of metabolic engineering of P. aeruginosa for overproduction of rhamnolipids, a biosurfactant with heat stable antifungal activity. Major expected outcomes from the proposed research include the ability of farmers and food industry personnel to easily detect contaminated foods, the development of effective antimicrobial therapy for pathogens that are resistant to existing drugs, and improved production of crops that are susceptible to fungal infections. The students working on this project will be trained in an integrated multidisciplinary approach, including microbiology, molecular biology, and biochemistry, to improve food safety and food production.</p>
<p>Research Procedures.Specific Aim 1: Development of multiplex magnetoelastic nanoparticle biosensors for rapiddetection of foodborne bacterial pathogens. In the past few years, we have optimized isolation ofhighly selective oligopeptide probes displayed on gpIII of a Ff class filamentous phage fordetection of pathogens. We currently have three manuscripts in preparation describing theseprocedures and isolation of highly selective S. Typhimurium probes. We are also in the processof improving our probes for S. Enteritidis, other pathogenic serovars of S. enterica, Shigellasonnei, and S. flexneri via limited mutagenesis to form the consensus for each target pathogen. Inaddition, we will isolate and characterize selective probes for E. coli O157:H7, S. aureus, andClostridium species. We have developed genetic approaches to construct combinatorial probesand chimeric phage probes to increase the selectivity for a given pathogen. Furthermore, we havedeveloped and demonstrated a genetic approach for stably immobilizing phage displayed probeson the magnetoelastic sensor platforms in self-assembling monolayer (SAM) to improve avidityof the sensor. We are preparing to file for a patent for this method in the near future. In order tosimultaneously detect multiple pathogens on a multiplex biosensor, each probe set for a targetorganism will be attached to a specific and different MEP of designed resonant frequency for itsdetection. MEPs ranging from 1 μm to 1000 μm in length may be used, depending upon targetagent size and mass. The corresponding characteristic resonant frequency range is from 2 GHz to2 MHz. For a MEP 50 μm long x 2 μm wide x 1 μm thick (which has a characteristic resonantfrequency of about 44 MHz), the attachment of one S. Typhimurium bacterium (mass of 1bacterium = 2 picogram) would result in a resonant frequency shift of 56 KHz, or 0.125 %. Inour previous experiments, we easily identified a 0.01% shift in resonant frequency (100 Hz outof 1 MHz) using standard instrumentation. The dimensions of the MEPs will also be adjustedbased on the properties of the target agents (principally mass and size) and the associatedfrequency change when the MEP biosensor is bound to the target. Finally, our biosensor can beeasily adapted for detection of other pathogens by coupling the MEPs with oligopeptide probesthat are specific for the infectious agents of interest. This aim will be pursued in collaborationwith Bryan Chin (Co-PI). Chin's group will be responsible for synthesis of MEPs as well as thebuilding and optimization of detection instruments. The Suh group will be responsible forisolation and development of phage probes for various bacterial pathogens. Both groups willparticipate in optimization of biosensors for detection of pathogens.Specific</p><p>Aim 2: Characterization of novel proteinaceous antimicrobials from subterraneantermite Reticulitermes flavipes. We will purify and characterize each protein with antimicrobialactivity and determine its biological range especially towards multidrug resistant (MDR)bacterial pathogens. Our preliminary data indicate that R. flavipes produces agents that areeffective against several MDR pathogens. The proteins will be purified by standard approachesusing liquid chromatography. Proteins will be identified via MALDI-TOF. We are alsointerested in elucidating the changes in the antimicrobial profile of the termite when it is exposedto different pathogens. Termites only have innate immunity and yet the antimicrobial profilessuggest a specific response upon exposure to a bacterium. Once we identify the proteins, we willperform reverse genetics to identify the termite genes that encode for the antimicrobials andstudy their expressions. It is our hope that by understanding the termite immune system, we candevelop approaches to combat this important pest in the future. This aim will be pursued incollaboration with Xing Ping Hu (Co-PI).Specific</p><p>Aim 3: Genetic characterization of a novel antifungal agent 7, 10, 12-trihydroxy-8(E)-octadecenoic acid produced by Pseudomonas aeruginosa. We have previously isolated severalmutants of P. aeruginosa that are defective for TOD production. We will characterize thesegenes to understand the biosynthesis of TOD in P. aeruginosa. In addition, we will determinethe biological range of TOD against a multitude of pathogens including bacteria and fungi.Finally, if successful in understanding the biosynthetic pathway of TOD, we will construct aTOD overproducing P. aeruginosa via metabolic engineering.Specific Aim 4: Continue with metabolic engineering of P. aeruginosa for overproduction ofrhamnolipids, a biosurfactant with heat stable antifungal activity. Our current metabolicallyengineered P. aeruginosa strain produces fifteen-fold more rhamnolipid than the parent strain.We will further improve the production by knocking out several more metabolic pathways thatcompete with rhamnolipid for the same precursors. In addition, we will reduce the cost ofproduction by eliminating utilization of antibiotic for maintenance of a recombinant plasmid andcostly IPTG for induction of rhamnolipid biosynthetic genes. We have developed a novel geneticapproach for maintaining plasmids without the antibiotic selection and we will implement thistechnique for this strain. In addition, we will substitute the usage of IPTG with much cheaperlactose by improving P. aeruginosa's import of the sugar and converting lactose to allolactose,the inducer of the Lac repressor. These approaches will significantly reduce the cost ofoverproducing rhamnolipids. In the future, we will use the same approach for overproduction ofTOD.</p><p>