AbstractsBiology & Animal Science

Characterization of PfFNT – a lactate transporter in Plasmodium falciparum

by Janis Rambow

Institution: Christian-Albrechts-Universität zu Kiel
Department: Mathematisch-Naturwissenschaftliche Fakultät
Degree: PhD
Year: 2015
Record ID: 1102650
Full text PDF: http://macau.uni-kiel.de/receive/dissertation_diss_00016527


A distinctive character of the mature intraerythrocytic form of the malaria parasite, Plasmodium falciparum, is a high glycolytic flow rate to fulfill its energetic requirements. This action produces two mole of lactic acid per mole of glucose as the anaerobic end product resulting in large quantities that need to be removed from the parasite cytosol. On its way out into the bloodstream lactate has to cross three different phospholipid bilayers, the parasite plasma membrane, the parasitophorous vacuole membrane and the red blood cell membrane. Although transport characteristics have been described for lactate in P. falciparum the molecular identity of the underlying permease(s) is still unknown. Here the discovery of a gene, PfFNT, responsible for the peptide that facilitates lactate transport over the parasite plasma membrane is described. It is a member of the formate nitrite transporter family (FNT) with high sequence similarities to microbial FNTs. For characterization of the protein a Saccharomyces cerevisiae knock out strain was employed that has lost the ability to transport monocarboxylates. Using this system PfFNT lactate/proton symport was found. This was confirmed by a direct proportionality of L-lactate transport to the prevailing pH gradient. Moreover when this gradient was abolished by proton decouplers, i.e. carbonylcyanide-3-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP), transport ceased. The PfFNT facilitated substrate pattern fits microbial FNTs, with acetate exhibiting the highest permeability followed by formate, L-lactate, D-lactate and pyruvate in decreasing order. The dicarboxylate malonate was excluded showing selectivity of monovalent anions over multivalent anions which is also a common feature shared by all FNTs discovered so far. The non-charged molecule glycerol, which is similar in size to lactate, was also excluded. Moreover facilitation features, such as transport rates and inhibition profile, match earlier findings from measurements performed in isolated living parasites in vitro hinting at a central position of PfFNT in parasite metabolism. For this the antiplasmodial compounds phloretin, furosemide, and cinnamate derivatives where tested revealing IC50 values around 1 mM. Inhibition requires a negative moiety though, since the uncharged cinnamamide had no effect. Notably the organomercurial p-chloromercuribenzene sulfonate (pCMBS), an inhibitor of human lactate transport, did not alter transport rates of PfFNT. This, taken together with the fact that there are no FNT homologs apparent in the human genome indicates PfFNT as a novel promising antimalarial drug target. Additionally PfFNT is the only transporter of the plasmodial glycolytic pathway for which structure information is available from crystals of homologous proteins predisposing it to further design of high affinity inhibitors. Ein herausragendes Merkmal des intraerythrozytären Malariaerregers, Plasmodium falciparum, ist eine hohe glykolytische Flussrate um seinen Energiebedarf zu decken. Bei diesem Vorgang…