Abstract
The capacity of pathogens to acquire nutrients from their host cells is one of the most fundamental aspects of infection biology. Hence, measuring the patterns of nutrients’ uptake by pathogens is essential for understanding the interactions of pathogens with eukaryotic host cells. In this study, we optimized a technique that allows fast and non-destructive measurement of the amino acid Phenylalanine (Phe) acquired by the trophozoite stage of the protozoan Acanthamoeba castellanii (A. castellanii) as they engage with individual human retinal pigment epithelial cells (ARPE-19). ARPE-19 host cells were pre-saturated with Deuterated Phe (L-Phe(D8)) to replace the native substrate Phe (L-Phe). The uptake of L-Phe(D8) by A. castellanii trophozoites was measured by Raman microspectroscopy. This approach allowed us to characterize the uptake patterns of this essential amino acid into A. castellanii trophozoites at a single cell level. At 24 hours post infection (PI) A. castellanii trophozoites are capable of salvaging L-Phe(D8) from host cells. The uptake pattern was time-dependent during the first 24 hours of infection and complete substitution with L-Phe(D8) in all parasites was detected at 48 hours PI. On the other hand, isolated A. castellanii trachyzoites (grown without host cells) did not show significant uptake for L-Phe(D8) from the media; only achieved an uptake ratio of 16-18% of L-Phe(D8) from the culture medium after 24 hours. These findings demonstrate the potential of combining Raman microspectroscopy and stable isotope labelling approaches to elucidate the role of metabolism in mediating A. castellanii interaction with host cells.
Highlights
Acanthamoeba species, discovered in 1930, are the most prevalent free-living protozoa and inhabit a variety of environments including air, soil and water[1], swimming pools[2], eye wash stations[3], and noses and throats of asymptomatic individuals[4]
In order to investigate the intake of L-Phe(D8) from the culture medium, A. castellanii trophozoites were grown in normal medium conditions containing L-Phe and were subsequently incubated in media supplemented with L-Phe(D8)
The aim of this study was to address how A. castellanii trophozoites breach the human retinal pigment epithelial cells (ARPE-19) cells, which represent the main constituent of the blood-retinal barrier (BRB) from a metabolic perspective
Summary
Acanthamoeba species, discovered in 1930, are the most prevalent free-living protozoa and inhabit a variety of environments including air, soil and water[1], swimming pools[2], eye wash stations[3], and noses and throats of asymptomatic individuals[4]. Under favourable conditions i.e. abundant food supply, neutral pH, appropriate temperature and osmolarity, Acanthamoeba exists in the vegetative, infective and reproducible trophozoite stage. This form of the life cycle has a diameter of 10-25μm[5] and exhibits irregular shape with locomotor pseudopods and surface acanthopodia[6,7]. Under physiological stress i.e. extreme temperature, osmolarity, pH and lack of nutrients or desiccation, trophozoite transforms into a metabolically inactive cyst[8] and encloses itself within a thick wall[7] and becomes resistant to biocides, chlorination and antibiotics[9]. Cysts return to their trophozoite form with improved conditions in a process known as excystment[10]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
