Visualizing the interaction of Acanthamoeba castellanii with human retinal epithelial cells by spontaneous Raman and CARS imaging

Abida Naemat,Hany M Elsheikha,Faris Sinjab,Ioan Notingher,Alistair Elfick,Alison Mcdonald,Andy Downes

doi:10.1002/jrs.5296

Abstract

AbstractImproved understanding of the mechanism of nutrient's uptake can enable targeted manipulation of nutrient sensing pathways in medically important pathogens to a greater degree than is currently possible. In this context, we present the use of spontaneous Raman microspectroscopy and coherent anti‐Stokes Raman spectroscopy to visualize the time‐dependent molecular interactions between the protozoan Acanthamoeba castellanii and host human cells. Human retinal pigment epithelial (ARPE‐19) cells were pre‐labelled with deuterated Phe (L‐Phe[D8]) and the uptake of the host derived L‐Phe(D8) by A. castellanii trophozoites was measured by Raman microspectroscopy for up to 48 hr post infection (hpi). This approach revealed a time‐dependent uptake pattern of this essential amino acid by A. castellanii trophozoites during the first 24 hpi with complete enrichment with L‐Phe(D8) detected in trophozoites at 48 hpi. In contrast, cell free A. castellanii trophozoites showed a modest uptake of only 16–18% L‐Phe(D8) from L‐Phe(D8)–supplemented culture medium after 3, 24, and 48 hr hpi. Coherent anti‐Stokes Raman spectroscopy microscopy was successfully used to monitor the reprogramming of lipids within the trophozoites as they engaged with host cells. The methodology presented here provides new advances in the ability to analyze the kinetic of amino acid acquisition by A. castellanii from host cell and extracellular environment, and to visualize lipid reprogramming within the trophozoite.

Highlights

Acanthamoeba species are single cell eukaryotic protozoa that can survive in a variety of environmental niches, including soil and water, 1 swimming pools, 2 eye wash stations, 3 and noses and throats of asymptomatic individuals.[4]
With the use of high-resolution Coherent anti-Stokes Raman scattering (CARS), we investigated whether the interaction of A. castellanii trophozoites with host cells triggers reprogramming of lipid contents within trophozoites
A. castellanii trophozoites were grown in normal medium conditions containing L-Phe and were subsequently incubated in media supplemented with L-Phe(D8)

Summary

Introduction

Acanthamoeba species are single cell eukaryotic protozoa that can survive in a variety of environmental niches, including soil and water, 1 swimming pools, 2 eye wash stations, 3 and noses and throats of asymptomatic individuals.[4] Acanthamoeba exists in two different life cycle forms: trophozoite and cyst. Under favourable conditions, such as abundant food supply, appropriate temperature, osmolarity and neutral pH, Acanthamoeba exists in the vegetative, infective and replicative trophozoite stage. Considered as freeliving ubiquitous protozoans, several species of Acanthamoeba can cause severe infections in humans, mainly localized in immune privileged sites, such as the brain 8 and the eyes.[9]

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