Characterization of the apicomplexan amino acid transporter (ApiAT) family inToxoplasma gondii

Abstract

Apicomplexan parasites are auxotrophic for a range of amino acids which must be salvaged from their host cells, either through direct uptake or degradation of host proteins. Here, we describe a family of plasma membrane-localized amino acid transporters, termed the Apicomplexan Amino acid Transporters (ApiATs), that are ubiquitous in apicomplexan parasites. Functional characterization of the ApiATs of Toxoplasma gondii indicate that several of these transporters are important for intracellular growth of the tachyzoite stage of the parasite, which is responsible for acute infections. We demonstrate that the ApiAT protein TgApiAT5-3 is an exchanger for aromatic and large neutral amino acids, with particular importance for L-tyrosine scavenging and amino acid homeostasis, and that TgApiAT5-3 is critical for parasite virulence. Our data indicate that T. gondii expresses additional proteins involved in the uptake of aromatic amino acids, and we present a model for the uptake and homeostasis of these amino acids. Our findings identify a family of amino acid transporters in apicomplexans, and highlight the importance of amino acid scavenging for the biology of this important phylum of intracellular parasites.Author SummaryThe Apicomplexa comprise a large number of parasitic protozoa that have obligate intracellular lifestyles and cause significant human and animal diseases, including malaria, cryptosporidiosis, toxoplasmosis, coccidiosis in poultry, and various cattle fevers. Apicomplexans must scavenge essential nutrients from their hosts in order to proliferate and cause disease, including a range of amino acids. The direct uptake of these nutrients is presumed to be mediated by transporter proteins located in the plasma membrane of intracellular stages, although the identities of these proteins are poorly defined. Using a combination of bioinformatic, genetic, cell biological, and physiological approaches, we have characterized a family of plasma membrane-localized transporter proteins that we have called the Apicomplexan Amino acid Transporters (ApiATs). The family is found in apicomplexans and their closest free-living relatives. We show that TgApiAT5-3, a member of the family in the apicomplexan Toxoplasma gondii, is an exchanger for aromatic and large neutral amino acids. In particular, it is critical for uptake of tyrosine, and for parasite virulence in a mouse infection model. We conclude that ApiATs are a family of plasma membrane transporters that play crucial roles in amino acid scavenging by apicomplexan parasites.

Characterisation of the Toxoplasma gondii tyrosine transporter and its phosphorylation by the calcium‐dependent protein kinase 3

“…A). Measuring unidirectional influx, we observed a significant (4.0‐fold) increase in the uptake of 14 C‐tyrosine into ApiAT5‐3‐expressing oocytes compared to either water‐injected or uninjected control oocytes under the conditions tested, consistent with results from (Parker et al ., ). We also observed moderate ApiAT5‐3‐dependent phenylalanine influx, but not for the BCAA valine (Fig.…”

“…To test this, we expressed apiAT5‐3 in the heterologous expression system, X. laevis oocytes. Concurrently with our study, data were presented that ApiAT5‐3 may be a tyrosine transporter (Giel van Dooren, personal communication and pre‐published in BioRx [Parker et al ., ]). We, therefore, tested BCAA import and replicated the tyrosine uptake capacity of ApiAT5‐3 in oocytes expressing WT ApiAT5‐3 (Fig.…”

“…These results differ from those in Parker et al . whereby growth can, at least partially, be rescued in high tyrosine medium (Parker et al ., ). One mechanism to counter low tyrosine levels that most organisms possess, is the ability to convert phenylalanine into tyrosine, via the enzyme phenylalanine‐4‐hydroxylase (AAH).…”

“…Transport measurements were performed at room temperature on groups of 10 oocytes in ND96 medium (96 mM NaCl, 2 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 and 5 mM HEPES) containing 1 µM radiolabelled U‐ 14 C‐L‐tyrosine (specific activity of 486 mCi/mmol; Perkin Elmer), U‐ 14 C‐L‐phenylalanine (specific activity of 508 mCi/mmol; Perkin Elmer) or U‐ 14 C‐L‐valine (specific activity of 271 mCi/mmol; Perkin Elmer). Transport was measured at 10 min, over which time uptake of L‐tyrosine is linear (Parker et al ., ). Each result was confirmed by at least three independent experiments.…”

“…The phosphorylation site that appeared to have one of the most marked reductions in phosphorylation state in Tg CDPK3 mutants compared to WT parasites (Treeck et al ., ), is situated within a putative transporter of the MFS family (TGGT1_257530, named ApiAT5‐3 as per Parker et al ., ) that has moderate homology to a BCAA transporter. Given the additional evidence from the phosphoproteomic dataset that Tg CDPK3 putatively regulates BCAA catabolism via BCKDH, we hypothesised that Tg CDPK3 might be involved in BCAA uptake in addition to regulating motility.…”