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

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 1 and 2 (MPC1/MPC2). We report here studies on MPC1/MPC2 of Trypanosoma cruzi, the etiologic agent of Chagas disease. Endogenous tagging of T. cruziMPC1 (TcMPC1) and TcMPC2 with 3×c-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 consumption rates when pyruvate, but not succinate, was used as the mitochondrial substrate, while α-ketoglutarate increased their O2 consumption rates due to an increase in α-ketoglutarate dehydrogenase activity. Defective mitochondrial pyruvate import resulted in decreased Ca2+ uptake. The inhibitors UK5099 and malonate impaired pyruvate-driven oxygen consumption in permeabilized control cells. Inhibition of succinate dehydrogenase by malonate indicated that pyruvate needs to be converted into succinate to increase respiration. TcMPC1-KO and TcMPC2-KO epimastigotes showed little growth differences in standard or low-glucose culture medium. However, the ability of trypomastigotes 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 respiration in the presence of pyruvate, invasion of host cells, and replication of amastigotes.IMPORTANCETrypanosoma cruzi is the causative agent of Chagas disease. Pyruvate is the end product of glycolysis, and its transport into the mitochondrion is mediated by the mitochondrial pyruvate carrier (MPC) subunits. Using the CRISPR/Cas9 technique, we generated individual T. cruziMPC1 (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 infective stages, MPC was essential for normal host cell invasion and intracellular replication.

Inorganic polyphosphate interacts with nucleolar and glycosomal proteins in trypanosomatids.

Inorganic polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high-energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over-representation of nucleolar and glycosomal proteins. Using the polyphosphate-binding domain (PPBD) of Escherichia coli exopolyphosphatase (PPX), we localized long-chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre-incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration-dependent and chain length-dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress.

The major leucyl aminopeptidase of Trypanosoma cruzi (LAPTc) assembles into a homohexamer and belongs to the M17 family of metallopeptidases.

BACKGROUND: Pathogens depend on peptidase activities to accomplish many physiological processes, including interaction with their hosts, highlighting parasitic peptidases as potential drug targets. In this study, a major leucyl aminopeptidolytic activity was identified in Trypanosoma cruzi, the aetiological agent of Chagas disease. RESULTS: The enzyme was isolated from epimastigote forms of the parasite by a two-step chromatographic procedure and associated with a single 330-kDa homohexameric protein as determined by sedimentation velocity and light scattering experiments. Peptide mass fingerprinting identified the enzyme as the predicted T. cruzi aminopeptidase EAN97960. Molecular and enzymatic analysis indicated that this leucyl aminopeptidase of T. cruzi (LAPTc) belongs to the peptidase family M17 or leucyl aminopeptidase family. LAPTc has a strong dependence on neutral pH, is mesophilic and retains its oligomeric form up to 80°C. Conversely, its recombinant form is thermophilic and requires alkaline pH. CONCLUSIONS: LAPTc is a 330-kDa homohexameric metalloaminopeptidase expressed by all T. cruzi forms and mediates the major parasite leucyl aminopeptidolytic activity. Since biosynthetic pathways for essential amino acids, including leucine, are lacking in T. cruzi, LAPTc could have a function in nutritional supply.