The intracellular parasite Toxoplasma gondii harbors three druggable FNT-type formate and l-lactate transporters in the plasma membrane

Abstract

Edited by Karen G. FlemingToxoplasma gondii is a globally prevalent parasitic protist. It is well-known for its ability to infect almost all nucleated vertebrate cells, which is reflected by its unique metabolic architecture. Its fast-growing tachyzoite stage catabolizes glucose via glycolysis to yield L-lactate as a major by-product that must be exported from the cell to prevent toxicity; the underlying mechanism remains to be elucidated, however. Herein, we report three formate-nitrite transporter (FNT)-type monocarboxylate/proton symporters located in the plasma membrane of the T. gondii tachyzoite stage. We observed that all three proteins transport both L-lactate and formate in a pH-dependent manner and are inhibited by 2-hydroxy-chromanones (a class of small synthetic molecules). We also show that these compounds pharmacologically inhibit T. gondii growth. Using a chemical biology approach, we identified the critical residues in the substrateselectivity region of the parasite transporters that determine differential specificity and sensitivity toward both substrates and inhibitors. Our findings further indicate that substrate specificity in FNT family proteins from T. gondii has evolved such that a functional repurposing of prokaryotic-type transporters helps fulfill a critical metabolic role in a clinically important parasitic protist. In summary, we have identified and characterized the lactate transporters of T. gondii and have shown that compounds blocking the FNTs in this parasite can inhibit its growth, suggesting that these transporters could have utility as potential drug targets.

Transmembrane Facilitation of Lactate/H+ Instead of Lactic Acid Is Not a Question of Semantics but of Cell Viability

“…While AQPs act as lactic acid channels, the FNTs functionally represent lactate/H + co-transporters that are comparable in efficiency to the MCTs. Evolutionary, the FNT-type of monocarboxylate transport remained restricted to microbes [39][40][41], whereas the alternating-access mechanism of the MCTs with a more specific substrate binding site certainly constitutes a more modern principle.…”

Formate–nitrite transporters carrying nonprotonatable amide amino acids instead of a central histidine maintain pH-dependent transport

“…Initially, we attempted to produce EhFNT and BtFdhC in a well-established Saccharomyces cerevisiae yeast system (2,(7)(8)(9)12). However, the yeast cells produced fragmented (EhFNT) or little amounts of protein (BtFdhC), and transport functionality was absent.…”

“…NirC, thus, contributes to the global biogeochemical nitrogen cycle. Eukaryotic FNTs carry substrate selectivity filters that are wider in diameter permitting efficient transport of larger monocarboxylates, such as L-lactate (7,8). The viability and virulence of human-pathogenic malaria parasites depends on the swift release of L-lactate/H ϩ via its single FNT-type monocarboxylate transporter, PfFNT (2).…”

Cell-Free and Yeast-Based Production of the Malarial Lactate Transporter, PfFNT, Delivers Comparable Yield and Protein Quality

“…We reconstituted purified P. pastoris cell-based and cell-free produced PfFNT into proteoliposomes following a protocol that we established earlier (Müller-Lucks et al, 2012; von Bülow et al, 2012; Golldack et al, 2017; Erler et al, 2018; Helmstetter et al, 2019). We evaluated PfFNT transport functionality by challenging the proteoliposomes with an inward directed, osmotic lactate gradient of 200 mM.…”