1a. Objectives (from AD-416)
The objective of this agreement is to establish the proof-of-concept that floating glycoprotein film-coated micelles can be used to capture pathogens from aqueous matrices and stabilize them during transport and storage. Initial research conducted at The MITRE Corporation (MITRE) was patented and licensed to Quickpath Bioscience.
1b. Approach (from AD-416)
Fimbriae, hair-like structures produced by many species of bacteria, vary in the composition of the fimbrial shaft and protein adhesins found on the tips. Bacterial adhesins selectively bind to tissue-specific glycans. For example, pathogens such as Salmonella and E. coli expressing Type I fimbriae attach to tissues/micelles coated with mannose. There are about twenty-two known fimbrial types. However, reproducibility and predictably of attachment is uncertain. Little is known about how the environment influences fimbrial production. Furthermore, Type I fimbriae are distributed among many bacterial species and there can be variations in the shaft fimbrin protein while the tip adhesin protein remains the same. We have selected a model strain, uropathogenic Escherichia coli (UPEC), to which we will obtain monoclonal antibodies produced against the target fimbriae. Immunoassay studies elucidating fimbrial expression as a function of environmental parameters will be conducted with methods developed for the Signalyte spectrofluorimeter (Creativ MicroTech) and imaged by Epifluorescent and Atomic Force Microscopy (NIST). Environmental parameters to be investigated include: pH (including shifts in pH), redox, temperature, culture age, nutrient ratios (carbon:nitrogen: phosphorus), nutrient composition, ionic strength, and cell chemical signaling effects. After establishing environmental conditions that reliably produce fimbrial expression, attachment studies to lipid-filled glycoprotein micelles provided by MITRE Corporation will be performed to establish optimal binding kinetics and survival. Target fimbriae will also be screened against available glycan microarrays.
3. Progress Report
The objective of this agreement is to establish the proof-of-concept that floating glycoprotein film-coated micelles can be used to capture pathogens from aqueous matrices and stabilize them during transport and storage. Virtually all bacteria possess one or more fimbriae or pili, hair-like structures extending from the cell surface. These fimbriae/pili allow for attachment to various surfaces, including a variety of inert surfaces. Fimbriae/pili associated with pathogens possess adhesions that are responsible for specific attachment to receptor molecules found on human tissues, typically at the site of infection. For example, uropathogenic E. coli (UPEC) possess Type I fimbriae that mediate attachment to mannose-containing glycoproteins coating the urinary tract. Our research indicates that this process may be simulated using oil micelles coated with gycoproteins with appropriate receptor sugars. To date, we demonstrated that this approach can be used for the capture and concentration of uropathogenic E. coli, Salmonella and Yersinia pseudotuberculosis. In addition, assays for removing fimbriae from cell surface and purifying them have been developed. The goal is to obtain monoclonal antibodies against these fimbriae in order to evaluate the effect of different environmental and growth conditions on fimbrial expression. Meetings are conducted weekly to dicsuss results and plan future experiments.