Substrate-mediated regulation of the arginine transporter ofToxoplasma gondii

Abstract

14 Intracellular parasites, such as the apicomplexan Toxoplasma gondii, are adept at 15 scavenging nutrients from their host. However, there is little understanding of how 16 parasites sense and respond to the changing nutrient environments they encounter during 17 an infection. TgApiAT1, a member of the apicomplexan ApiAT family of amino acid 18 transporters, is the major uptake route for the essential amino acid L-arginine (Arg) in 19 T. gondii. Here, we show that the abundance of TgApiAT1, and hence the rate of uptake 20 of Arg, is regulated by the availability of Arg in the parasite's external environment, 21 increasing in response to decreased [Arg]. Using a luciferase-based 'biosensor' strain of 22 T. gondii, we demonstrate that parasites vary the expression of TgApiAT1 in different 23 organs within their host, indicating that parasites are able to modulate TgApiAT1-24 dependent uptake of Arg as they encounter different nutrient environments in vivo. 25 Finally, we show that Arg-dependent regulation of TgApiAT1 expression is post-26 transcriptional, mediated by an upstream open reading frame (uORF) in the TgApiAT1 27transcript, and we provide evidence that the peptide encoded by this uORF is critical for 28 mediating regulation. Together, our data reveal the mechanism by which an 29 apicomplexan parasite responds to changes in the availability of a key nutrient. 30 31The uptake of nutrients by T. gondii parasites is mediated primarily by plasma membrane 56 transporters [13]. We recently characterised a family of plasma membrane amino acid 57 transporters that are found throughout apicomplexans and have termed these the Apicomplexan 58Amino acid Transporter (ApiAT) family [14]. We have demonstrated that one member of this 59 family, TgApiAT1, is an Arg transporter that is essential for T. gondii virulence [15]. 60In this study, we have investigated the ability of T. gondii parasites to sense and respond to the 61Arg levels that they encounter in their host. We report Arg-dependent regulation of TgApiAT1 62 expression, and demonstrate that this process is mediated by an upstream open reading frame 63 (uORF) in the TgApiAT1 transcript. We also present evidence, obtained using a luciferase-64 based 'biosensor' strain of T. gondii, that parasites vary the expression of TgApiAT1 in 65 different organs within their host. Our data demonstrate how T. gondii parasites are able to 66 sense and respond to changes in the abundance of a key nutrient, as well as illustrating their 67 ability to do so within the course of an infection. 68 69 RESULTS 70 Regulation of TgApiAT1 protein abundance and parasite arginine uptake. 71 To investigate whether the abundance of TgApiAT1 is dependent upon Arg availability, we 72 introduced a haemagglutinin (HA3) epitope tag into the open reading frame of the TgApiAT1 73 genomic locus. The resultant TgApiAT1-HA3-expressing parasites were grown in modified 74 Roswell Park Memorial Institute 1640 (RPMI) medium in which [Arg] ranged from 10 µM to 75 5 mM. Western blotting revealed that the a...

Coordinated Action of Multiple Transporters in the Acquisition of Essential Cationic Amino Acids by the Intracellular Parasite Toxoplasma gondii

“…Based on our findings, and on several other recent studies into Arg uptake in T. gondii [27,31,41], we can now propose a comprehensive model for the uptake of cationic amino acids into these parasites (Fig 7). The scavenging of AA + by parasites results in a depletion of these amino acids in the host cell cytosol, causing upregulation of the host CAT1 AA + transporter [41].…”

“…The data in the present study indicate that TgApiAT6-1 functions as this alternative Arg transporter. We have recently shown that the expression of TgApiAT1 is up-regulated under Arg limiting conditions and expressed at low levels under Arg replete conditions [31]. The differential expression of TgApiAT1 may therefore allow these parasites to survive when Arg levels are limited, while TgApiAT6-1 may ensure regulated uptake of Arg and Lys under nutrient rich conditions.…”

“…in the cells of organs with high Arg catabolism such as the liver [45]; Fig 7,right), Lys uptake by TgApiAT6-1 will out-compete Arg uptake. The parasite responds by upregulating TgApiAT1, which enables sufficient Arg uptake for parasite proliferation [31]. If the ratio of Arg:Lys in the host cell is high (e.g.…”

“…All three ApiATs have been shown to be important at particular parasite life-cycle stages: the T. gondii transporters for tachyzoite proliferation [20,27], and PbApiAT8 for P. berghei gamete development in mice and transmission to Anopheles mosquitos [27,29,30]. TgApiAT1 expression is regulated by the intracellular availability of Arg, allowing parasites to exert tight control over the uptake of this amino acid [31]. The dearth of other candidate amino acid transporter homologues in apicomplexan genomes suggests that ApiATs may be the primary amino acid/amino acid metabolite transporter family in these parasites [20,31,32].…”

“…TgApiAT1 expression is regulated by the intracellular availability of Arg, allowing parasites to exert tight control over the uptake of this amino acid [31]. The dearth of other candidate amino acid transporter homologues in apicomplexan genomes suggests that ApiATs may be the primary amino acid/amino acid metabolite transporter family in these parasites [20,31,32].…”