Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2023

In vitro labelling of Acanthamoeba trophozoites using ligand conjugated fluorescein isothiocyanate (FITC). (#177)

Hari Kumar Peguda 1 , Nicole Carnt 1 , Rajesh Kuppusamy 1 2 , Zi Gu 3 , Mark Willcox 1
  1. School of Optometry and Vision Science, The University of New South Wales., Sydney, New South Wales, Australia
  2. School of Chemistry, The university of New South Wales, Sydney, New South Wales, Australia
  3. School of Chemical Engineering, The university of New South Wales, Sydney, New South Wales, Australia

Purpose: Acanthamoeba causes devastating eye infections but can be very difficult to diagnose. Therefore, the aim of the current investigation was to determine whether mannose ligates to fluorescein could be used to label trophozoites of Acanthamoeba, and the specificity of the labelling.

Methods: Acanthamoeba castellanii (ATCC 30868) was cultured axenically for 7 days at 320C. Trophozoites were collected and allowed to attach to the surface of microtiter plate. Amine groups of Mannosamine (MA) or glucosamine (GA) was attached to fluorescein isothiocyanate (FITC) through isothiocyanate groups and dissolved in β-cyclodextrin solution. Acanthamoeba trophozoites were incubated with MA-FITC or Glucose-FITC or β-cyclodextrin (control) at 1000µM for 2 hours. After incubation trophozoites were washed to remove the loosely bound compounds. Trophozoites were observed for the fluorescence under an inverted microscope using FITC (515-550nm) filter. Fluorescence intensity was further quantified using ImageJ software.

Results: Trophozoites incubated with 1000µM MA-FITC showed brighter fluorescence than GA-FITC and β-cyclodextrin. The MA-FITC appeared to be taken up into vacuoles of the trophozoites. The addition of free mannose before incubation reduced binding/uptake of MA-FITC but had no effect on glucose-FITC. This suggest mannose binding receptors are playing role in the uptake of fluorescence.

Conclusion: Ma-FITC dissolved in β-cyclodextrin was specifically bound and taken up by trophozoites. This suggest that MA-FITC may be developed as a specific stain to help diagnose Acanthamoeba ocular infections.