06/08/2022
By Kwok Fan Chow

The Kennedy College of Science, Department of Chemistry, invites you to attend a dissertation defense by Sajani Liyanage entitled “Carbohydrate Mediated Bacteria Interactions and Imaging.” The defense will be held in Olney 518 (North campus) on Tuesday, June 21 at 9 a.m.

Committee Chair: Prof. Mingdi Yan, Department of Chemistry, University of Massachusetts Lowell

Committee Members:

  • Prof. Olof Ramström, Department of Chemistry, University of Massachusetts Lowell
  • Prof. Matthew Gage, Department of Chemistry, University of Massachusetts Lowell
  • Prof. James Whitten, Department of Chemistry, University of Massachusetts Lowell

Abstract:

The first part of the dissertation focuses on developing fluorescent turn-on trehalose conjugated perfluorophenyl azide (PFPA)–functionalized carbazole (Tre-Cz) to image mycobacteria. As mycobacteria have a vital relationship with trehalose, we employed trehalose as the targeting agent for mycobacteria. Trehalose conjugated PFPA-carbazole was synthesized, and its imaging and detection of mycobacteria were studied. Fluorescence turn-on is achieved by photoactivation of the azide, generating a highly fluorescent product through an efficient intramolecular C-H insertion reaction. The fluorescent turn-on of Tre-Cz was successful, showing 90 times higher than Tre-Cz. Photoconversion quantum yield was 0.14, comparable to literature photoactivable azides. Labeling mycobacteria only requires 1 h incubation time with a relatively low concentration (100 µM) of Tre-Cz. Fluorescence turn-on is fast which only needs 1 min UV irradiation with a handheld UV lamp. Tre-Cz showed enhanced uptake by mycobacteria compared to E. coli, S. epidermidis and P. aeruginosa while maltose conjugated PFPA-carbazole (Mal-Cz) displayed minimal uptake. The fluorescent product of Tre-Cz showed 26 times lower fluorescence than in the case of the Tre-Cz due to photobleaching of the fluorescent product. Rapid detection with naked eyes showed that 10^3 CFU/mL and above concentrations could be detected, while LOD was calculated to be 2 CFU/mL when bacteria was in PBS. Tre-Cz could detect mycobacteria in bacteria-spiked sputum samples but the sensitivity was significantly dropped compared to bacteria in PBS. Studies using heat-killed, stationary phase and isoniazid-treated mycobacteria showed that metabolically active bacteria are required for the uptake of Tre-CBZ. The uptake decreased in the presence of trehalose in a concentration dependent fashion, indicating that Tre-Cz hijacks the trehalose uptake pathways. Mechanistic studies demonstrated that the uptake was impacted by both the Ag85 complex and the LpqY-SugABC transporter. While the uptake of the probe was reduced by ~20% in the Ag85C mutant, the uptake was mostly diminished in the mutants of LpqY, SugA and SugBC.

The second part of the dissertation focuses on the synthesis of a maltose-based fluorescence turn-on imaging probe (Mal-Cz), perfluorophenyl azide-functionalized carbazole, to detect E. coli and S. epidermidis in milk samples. Fluorescence turn-on is achieved by photoactivation of the azide, similar to the Tre-Cz probe. The photophysical and photochemical parameters of the probe and the product were characterized, showing a relatively high photo-conversion quantum yield. Confocal fluorescence microscopy demonstrated the successful uptake of the probe by E. coli and S. epidermidis and the subsequent detection of the bacteria by photoactivation. The probe could specifically image E. coli and S. epidermidis in the presence of other Gram-negative and Gram-positive bacteria. The limit of detection was determined to be 6 and 4 CFU/mL by UV-visible spectrometry, and ~10^3 CFU/mL by naked eyes using a handheld UV lamp for E. coli and S. epidermidis, respectively. Heat kill assay, competition with free maltose, growth phase dependency and sodium azide inhibition showed that the probe was metabolically incorporated into E. coli. The uptake was significantly reduced in the maltodextrin transporter mutants of E. coli.

In the third part, the impact of mechanical property (stiffness) of carbohydrate-conjugated polyacrylamide substrate on bacteria binding was explored. Trehalose and mannose containing monomers were synthesized and copolymerized to prepare polyacrylamide hydrogels by radical polymerization. Carbohydrates containing hydrogels of three different stiffness were prepared and interacted with SYTO stained bacteria. Stiffness was changed by changing the acrylamide monomer concentration and degree of cross-linking. Gel preparation was done in 96 well plates or in PDMS molds. Gels prepared in PDMS molds were more even and uniform than the gels prepared in the 96 well plate. Trehalose presenting hydrogels interacted with M. smegmatis while E. coli interacted with mannose presenting hydrogels. Interactions were recorded by the plate reader, microarray scanner and bacteria plating. Interactions were specific; minimal interactions were observed when M. smegmatis and E. coli interacted with mannose and trehalose presenting hydrogels, respectively. Results showed that the lower the stiffness of the substrate, the higher the interactions. The impact of incubation time, carbohydrate, and bacteria concentration was changed during the experiment and the interactions were measured. M. smegmatis interacted with trehalose hydrogels for up to 12 hours and plateaued thereafter due to the saturation of sugar-binding sites on the hydrogel surface. For E. coli, saturation was obtained after 8 hours of incubation. When bacteria interacted with hydrogels of increasing carbohydrate concentrations, interactions also increased, and the effect was less pronounced in stiffer hydrogels. At increasing bacteria concentrations, interactions were higher but less significant for more rigid gels. It was clear that the hydrogels prepared by PDMS molds showed better interaction correlations with bacteria than those prepared in 96 well plates. Altogether these results suggest that bacteria can mechanosense the substrate before adhering, and substrate stiffness impacts bacteria binding.

All interested students and faculty members are invited to attend.