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 develop methods for the extraction, recovery and purification of fimbriae/pili from various pathogenic bacteria (E. coli; Salmonella). Virtually all bacteria possess one or more fimbriae or pili, hair-like structures extending from the cell surface. Fimbriae/pili associated with pathogens possess adhesins 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. However, fimbriae/pili also facilitate attachment to various inert surfaces (harvesting/processing equipment) and to plant tissues. Once purified, we intend to develop monoclonal antibodies against these fimbriae in order to evaluate the effect of different environmental and growth conditions on fimbrial expression. In addition, assays using glycoprotein micelles will be developed to identify adhesions. Meetings are conducted weekly to discuss results and plan future experiments.