Identifying the major lactate transporter of Toxoplasma gondii tachyzoites

Abstract

Toxoplasma gondii and Plasmodium falciparum parasites both extrude l-lactate, a byproduct of glycolysis. The P. falciparum Formate Nitrite Transporter, PfFNT, mediates l-lactate transport across the plasma membrane of P. falciparum parasites and has been validated as a drug target. The T. gondii genome encodes three FNTs that have been shown to transport l-lactate, and which are proposed to be the targets of several inhibitors of T. gondii proliferation. Here, we show that each of the TgFNTs localize to the T. gondii plasma membrane and are capable of transporting l-lactate across it, with TgFNT1 making the primary contribution to l-lactate transport during the disease-causing lytic cycle of the parasite. We use the Xenopus oocyte expression system to provide direct measurements of l-lactate transport via TgFNT1. We undertake a genetic analysis of the importance of the tgfnt genes for parasite proliferation, and demonstrate that all three tgfnt genes can be disrupted individually and together without affecting the lytic cycle under in vitro culture conditions. Together, our experiments identify the major lactate transporter in the disease causing stage of T. gondii, and reveal that this transporter is not required for parasite proliferation, indicating that TgFNTs are unlikely to be targets for anti-Toxoplasma drugs.

Toxoplasma gondii excretion of glycolytic products is associated with acidification of the parasitophorous vacuole during parasite egress

“…Knockdown of an uncharacterized sodium/H+ exchanger, NHE4, also showed no effect on PV pH. Several studies have identified formate-nitrite transporters (FNTs) as transporters of monocarboxylate metabolites, such as formate and lactate (27)(28)(29). FNT transport of substrates depends on the cotransport of H + , thus export of lactate or other substrates is intrinsically linked to acidification of the environment.…”

“…FNT transport of substrates depends on the cotransport of H + , thus export of lactate or other substrates is intrinsically linked to acidification of the environment. Previous work suggested that FNT1 is the major FNT in tachyzoites (27)(28)(29). To determine the role of FNT1 and potentially FNT2 in acidification of the PV during egress, we endogenously tagged FNT1 and FNT2 with an mNG-AIDTy cassette.…”

“…Regardless, taken together our findings suggest that FNT1 and FNT2 both contribute to the release of lactate and pyruvate (and cotransport of H + ) during egress, but that other transporters can also export H + for residual acidification of the PV in the absence of FNT1 and FNT2. It was unexpected that the iFNT2 parasites showed reduced lactate and pyruvate release given the recent finding that FNT2 contributes little to lactate transport in WT extracellular parasites (29). One potential explanation is that FNT2 plays a more prominent role in export of lactate, as tested in our study, versus uptake of lactate as examined in Zeng et al A common finding between the two studies is that the lack of expression of all three FNTs had no effect on replication of T. gondii under in vitro culture conditions.…”

“…More recent gene knockout studies reported that FNT1 plays a prominent role in lactate export (28,29).…”

“…Release of Lactic acid and pyruvic acid potentially contribute to acidification of the PV L-lactate is a major by-product of glycolysis. FNT1 was recently shown to be the principal lactate transporter in T. gondii tachyzoites (29). Lactate dehydrogenases (LDHs) convert the end-product of glycolysis, pyruvate, to lactate.…”