Mitochondrial Pyruvate Carrier Subunits Are Essential for Pyruvate-Driven Respiration, Infectivity, and Intracellular Replication of Trypanosoma cruzi

Author(s)

Negreiros, Raquel S. ^[1] ; Lander, Noelia ^{[2, 3, 4]} ; Chiurillo, Miguel A. ^{[2, 3, 4]} ; Vercesi, Anibal E. ^[1] ; Docampo, Roberto ^{[2, 3]}

Affiliation

^[1] Univ Estadual Campinas, Dept Clin Pathol, Campinas, SP - Brazil

^[2] Univ Georgia, Ctr Trop & Emerging Global Dis, Athens, GA 30602 - USA

^[3] Univ Georgia, Dept Cellular Biol, Athens, GA 30602 - USA

^[4] Univ Cincinnati, Dept Biol Sci, Cincinnati, OH - USA

• Document type: Journal article
• Source: MBIO; v. 12, n. 2 MAR-APR 2021.
• Web of Science Citations: 1

Abstract

Pyruvate is the final metabolite of glycolysis and can be converted into acetyl coenzyme A (acetyl-CoA) in mitochondria, where it is used as the substrate for the tricarboxylic acid cycle. Pyruvate availability in mitochondria depends on its active transport through the heterocomplex formed by the mitochondrial pyruvate carriers and 2 (MPC1/MPC2). We report here studies on MPC1/MPC2 of Trypanosoma cruzi, the etiologic agent of Chagas disease. Endogenous tagging of T. cruzi MPC1 (TcMPC1) and TcMPC2 with 3xc-Myc showed that both encoded proteins colocalize with MitoTracker to the mitochondria of epimastigotes. Individual knockout (KO) of TcMPC1 and TcMPC2 genes using CRISPR/Cas9 was confirmed by PCR and Southern blot analyses. Digitonin-permeabilized TcMPC1-KO and TcMPC2-KO epimastigotes showed reduced O2 consump-tion rates when pyruvate, but not succinate, was used as the mitochondrial substrate, while a-ketoglutarate increased their O2 consumption rates due to an increase in a-keto-glutarate dehydrogenase activity. Defective mitochondrial pyruvate import resulted decreased Ca21 uptake. The inhibitors UK5099 and malonate impaired pyruvate-driven oxygen consumption in permeabilized control cells. Inhibition of succinate dehydrogen-ase by malonate indicated that pyruvate needs to be converted into succinate increase respiration. TcMPC1-KO and TcMPC2-KO epimastigotes showed little growth dif-ferences in standard or low-glucose culture medium. However, the ability of trypomasti-gotes to infect tissue culture cells and replicate as intracellular amastigotes was decreased in TcMPC-KOs. Overall, T. cruzi MPC1 and MPC2 are essential for cellular respi-ration in the presence of pyruvate, invasion of host cells, and replication of amastigotes. IMPORTANCE Trypanosoma cruzi is the causative agent of Chagas disease. Pyruvate the end product of glycolysis, and its transport into the mitochondrion is mediated by the mitochondrial pyruvate carrier (MPC) subunits. Using the CRISPR/Cas9 tech-nique, we generated individual T. cruzi MPC1 (TcMPC1) and TcMPC2 knockouts and demonstrated that they are essential for pyruvate-driven respiration. Interestingly, although glycolysis was reported as not an important source of energy for the infec-tive stages, MPC was essential for normal host cell invasion and intracellular replication. <comment>Superscript/Subscript Available</comment (AU)

• FAPESP's process: 17/25084-3 - Pyruvate metabolism function identification in Trypanosoma cruzi
• Grantee: Raquel Silva de Negreiros
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 13/50624-0 - Calcium signaling in trypanosomatids
• Grantee: Roberto Docampo
• Support Opportunities: Research Projects - SPEC Program