Abstract
This study investigates the temporal and spatial interchange of the aromatic amino acid phenylalanine (Phe) between human retinal pigment epithelial cell line (ARPE-19) and tachyzoites of the apicomplexan protozoan parasite Toxoplasma gondii (T. gondii). Stable isotope labelling by amino acids in cell culture (SILAC) is combined with Raman micro-spectroscopy to selectively monitor the incorporation of deuterium-labelled Phe into proteins in individual live tachyzoites. Our results show a very rapid uptake of l-Phe(D8) by the intracellular growing parasite. T. gondii tachyzoites are capable of extracting l-Phe(D8) from host cells as soon as it invades the cell. l-Phe(D8) from the host cell completely replaces the l-Phe within T. gondii tachyzoites 7–9 hours after infection. A quantitative model based on Raman spectra allowed an estimation of the exchange rate of Phe as 0.5–1.6 × 104 molecules/s. On the other hand, extracellular tachyzoites were not able to consume l-Phe(D8) after 24 hours of infection. These findings further our understanding of the amino acid trafficking between host cells and this strictly intracellular parasite. In particular, this study highlights new aspects of the metabolism of amino acid Phe operative during the interaction between T. gondii and its host cell.
Highlights
Toxoplasma gondii is the most prevalent protozoal infection of the Central Nervous System
Even though T. gondii can synthesise some amino acids (AA) de novo, this parasite lacks synthetic pathways for some AA, which must be retrieved from the host cells[6,7,8,9]
We investigated the rate at which l-Phe(D8) tracer replaces l-Phe within the host cells
Summary
Toxoplasma gondii is the most prevalent protozoal infection of the Central Nervous System. Little is known regarding the transport of Phe from mammalian cell hosts to the parasite, the formation of precursor pool, protein synthesis, or protein turnover within the parasite Unravelling these complex processes requires novel approaches that are capable of monitoring the shuttling of AA from or toward the host during T. gondii infection. Various isotope labelling approaches exist for studying protein dynamics and turnover These methods include stable isotope labelling by amino acids in cell culture (SILAC), chemical derivatization (GC-MS, ICAT, iTRAQ, TMT), and enzymatically catalyzed incorporation (18O labelling). These approaches are invasive, costand/or time-prohibitive, and might not be amenable to the biological system of interest. These techniques alone cannot quantitatively describe the complex dynamic interactions between engaging host and parasite systems
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