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

Naemat, Abida; Sinjab, Faris; McDonald, Alison; Downes, Andy; Elfick, Alistair; Elsheikha, Hany M.; Notingher, Ioan

Abstract

Improved 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 micro-spectroscopy (RMS) and coherent anti-Stokes Raman spectroscopy (CARS) to visualise the time-dependent molecular interactions between the protozoan Acanthamoeba castellanii and host human cells. Human retinal pigment epithelial (ARPE-19) cells were prelabelled with deuterated Phe (L-Phe(D8)) and the uptake of the host derived L-Phe(D8) by A. castellanii trophozoites was measured by RMS for up to 48 hours 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 3h, 24h and 48h hpi. CARS 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.

• Journal Article Type: Article
• Acceptance Date: Oct 19, 2017
• Online Publication Date: Nov 23, 2017
• Publication Date: Mar 2, 2018
• Deposit Date: Oct 20, 2017
• Publicly Available Date: Nov 24, 2018
• Journal: Journal of Raman Spectroscopy
• Print ISSN: 0377-0486
• Electronic ISSN: 1097-4555
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 49
• Issue: 3
• Pages: 412-423
• DOI: https://doi.org/10.1002/jrs.5296
• Keywords: Acanthamoeba castellanii; Coherent anti-Stokes Raman scattering; Host-pathogen interaction; Isotope labelling; ; Raman Spectroscopy
• Public URL: https://nottingham-repository.worktribe.com/output/917718
• Publisher URL: http://onlinelibrary.wiley.com/doi/10.1002/jrs.5296/full
• Additional Information: This is the peer reviewed version of the following article: Naemat A, Sinjab F, McDonald A, et al. Visualizing the interaction of Acanthamoeba castellanii with human retinal epithelial cells by spontaneous Raman and CARS imaging. J Raman Spectrosc. 2018;49:412–423. https://doi.org/10.1002/jrs.5296 which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1002/jrs.5296/full This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.