The goal of this proposal is to develop novel biochemical methods and devices for the attomolar detection of all seven serotypes and subtypes of botulinum neurotoxin (BoNT). Based on our preliminary data, we hypothesize that it is feasible to generate both fluorescent and bioluminescent substrates that are resistant to non-BoNT proteolytic cleavage, but can rapidly be activated by BoNTs.
The specific aims of the proposed research are: 1) Development of novel fluoroaenic substrates for BoNT serotypes A to G. These substrates will be designed through peptide libraries with proteinogenic and non-proteinogenic amino acids and will be selected for resistance to non-BoNT proteases and highly efficient cleavage by BoNTs. We will test ALISSAs with those novel substrates in various BoNT-spiked samples of serum, solid organ extracts, enema and stool specimens as well as a variety of foods. 2) Development of a luminogenic protein substrate for botulinum toxin. We are proposing to genetically engineer variants of recombinant luciferase proteins that become activated by specific cleavage reactions mediated by the metalloprotease activity of botulinum toxin. Luminescent substrates will be created for all seven serotypes (A to G) of botulinum toxin. We will also bioluminescent assays that emit light at multiple wavelenght for multiplexed simultaneous detection of more than one serotype per sample. 3) Application of existing and novel BoNT ALISSAs to measure toxin distribution in animal sera and organs. In collaboration with Dr. Luisa Cheng at the U.S. Department for Agriculture in Albany, CA, we will utilize BoNT ALISSA to measure the systemic content and tissue distribution in sub-lethally BoNT-intoxicated mice. Our goal is to determine pharmacokinetics and serotypedependent toxin distributions in organs following parenteral or oral intoxication. Furthermore, in collaboration with Dr. Nedelkov at Intrinsic Bioprobes and with Dr. Chris Myatt at Precision Photonics we will develop and test fully automated devices that will utilize our novel methodology for the quantitative detection of BoNTs at attomolar sensitivity. The BoNT ALISSA technology will be adapted into into a monolithic microcolumn format to be utilized by common liquid handling robots. The resulting benchtop instrumentation will also incorporate a highly sensitive, and inexpensive wave-guide-based optical readout system. Our assay system will be tested and validated with complex biological matrices such as serum, solid organ and food extracts as well as clinical samples (at Dr. Stephen Arnon's laboratory at the California Department for Public Health).
|Tsai, Wen-Yang; Youn, Han Ha; Tyson, Jasmine et al. (2018) Use of Urea Wash ELISA to Distinguish Zika and Dengue Virus Infections. Emerg Infect Dis 24:1355-1359|
|Thongsripong, Panpim; Chandler, James Angus; Green, Amy B et al. (2018) Mosquito vector-associated microbiota: Metabarcoding bacteria and eukaryotic symbionts across habitat types in Thailand endemic for dengue and other arthropod-borne diseases. Ecol Evol 8:1352-1368|
|Katzelnick, Leah C; Ben-Shachar, Rotem; Mercado, Juan Carlos et al. (2018) Dynamics and determinants of the force of infection of dengue virus from 1994 to 2015 in Managua, Nicaragua. Proc Natl Acad Sci U S A 115:10762-10767|
|Clemens, Daniel L; Lee, Bai-Yu; Horwitz, Marcus A (2018) The Francisella Type VI Secretion System. Front Cell Infect Microbiol 8:121|
|Huwyler, Camille; Heiniger, Nadja; Chomel, Bruno B et al. (2017) Dynamics of Co-Infection with Bartonella henselae Genotypes I and II in Naturally Infected Cats: Implications for Feline Vaccine Development. Microb Ecol 74:474-484|
|Norris, Michael H; Heacock-Kang, Yun; Zarzycki-Siek, Jan et al. (2017) Burkholderia pseudomallei natural competency and DNA catabolism: Identification and characterization of relevant genes from a constructed fosmid library. PLoS One 12:e0189018|
|Marques, Adriana R; Yang, Xiuli; Smith, Alexis A et al. (2017) Citrate Anticoagulant Improves the Sensitivity of Borreliella (Borrelia) burgdorferi Plasma Culture. J Clin Microbiol 55:3297-3299|
|Nualnoi, Teerapat; Norris, Michael H; Tuanyok, Apichai et al. (2017) Development of Immunoassays for Burkholderia pseudomallei Typical and Atypical Lipopolysaccharide Strain Typing. Am J Trop Med Hyg 96:358-367|
|Parameswaran, Poornima; Wang, Chunling; Trivedi, Surbhi Bharat et al. (2017) Intrahost Selection Pressures Drive Rapid Dengue Virus Microevolution in Acute Human Infections. Cell Host Microbe 22:400-410.e5|
|Bortell, Nikki; Flynn, Claudia; Conti, Bruno et al. (2017) Osteopontin Impacts West Nile virus Pathogenesis and Resistance by Regulating Inflammasome Components and Cell Death in the Central Nervous System at Early Time Points. Mediators Inflamm 2017:7582437|
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