Laboratory of Functional Biology of Protists
Current research projects
MitoSignal: Determining signaling mechanisms that drive cellular differentiation of the highly diverged eukaryote, Trypanosoma brucei
Mitochondria were primarily classified as biosynthetic and bioenergetic organelles, until it was recently revealed that they also transmit signaling molecules that can decide cell fate. To remove layers of complexity, we will utilize the unicellular pathogen, Trypanosoma brucei, to decipher the underlying mechanisms of the singular mitochondrion to drive cell differentiation under normal physiological conditions. Since the parasite must adapt to the various nutrient restrictions of the diverse environments during its complex life cycle, the mitochondrion is drastically remodeled both structurally and metabolically. Our data suggest that during the parasite´s insect form differentiation, increased levels of ROS and TCA cycle intermediates may act as signaling molecules. We propose to screen for proteins that are post-translationally modified by oxidation or acetylation and to utilize 3D electron microscopy to map remodeling of the mitochondrion architecture. Our results will provide novel mechanistic insights into mitochondria-dependent signaling pathways driving differentiation.
The role of ATP synthase structure in the biogenesis and bioenergetics of the unique Trypanosoma brucei mitochondrion
Mitochondrial (mt) cristae are inner membrane convolutions where protein factories responsible for bioenergy conversion reside. The cristae exhibit extremely large variability in their ultrastructure, except for one common attribute - the presence of ATP synthase dimer rows at the crista ridges. Little is known about the role of these arrays in cristae structure and mt bioenergetics. However, Trypanosoma brucei is an excellent model system as the singular mitochondrion of the digenetic parasite is drastically remodeled structurally and metabolically as it progresses through a complex life cycle. Notably, the highly branched, cristae-containing and ATP-producing mitochondrion transitions to a streamlined tubular, cristae-lacking and ATP-consuming organelle. Combining traditional biochemical methods with state-of-art structural approaches (e.g. cryo-EM), we will solve the ATP synthase dimer structure, identify dimer-specific subunits and explore their role in cristae shaping, mt bioenergetics and biogenesis in two major life stages of this parasite.
A paradigm shift for Trypanosoma brucei ATP production: Rewiring of mitochondrial metabolism allows for the infection of various host environments
For the last 50 years, the bioenergetics dogma of the infectious stage of the human pathogen, Trypanosoma brucei, states that the mitochondrion is an ATP consuming organelle and glycolysis is the only source of cellular ATP. Therefore, the ATP/ADP carrier (AAC) transports cytosolic ATP into the mitochondrion, where it is hydrolyzed by FoF1-ATPase to maintain the essential mitochondrial (mt) membrane potential (Δψm). However, we have generated AACDKO parasites that remain virulent in a mouse model. Furthermore, the Δψm in these mutants is still maintained by FoF1-ATPase and functional assays do not indicate the presence of another mt ATP carrier. This suggests that substrate-level phosphorylation by succinyl coenzyme A synthetase (SCoAS) produces mt ATP. Indeed, SCoASDKO parasites are less virulent in a mouse and unable to grow on glycerol. Therefore, we will apply metabolomics approaches along with bioenergetics assays to define the mt physiology of the bloodstream parasites. We will also determine if mt ATP production allows the parasite to establish newly discovered host resevoirs.
Acyclic nucleaoside phosphonates as potential inhibitors of adenine phosphoribosyltransfereases in human trypanosomatid parasites
The trypanosomatid parasites (e.g. Trypanosoma brucei, Leishmania spp.) do not possess the de novo purine synthesis pathway and thus they fully rely on the purine salvage pathway (PSP) for their nucleotide generation. 6-oxopurine phosphoribosyltransfereases (PRTs), central enzymes of the versatile PSP, are inhibited by 6-oxopurine acyclic nucleoside phosphonates (ANPs). Since adenine-bearing ANPs also exerted strong cytotoxic effects towards T. brucei, we propose to examine the parasite’s adenine PRT (APRT) as a possible drug target. Using our experience with 6-oxopurine PRTs and the APRT solved crystal structure, we will design and synthesize new selective ANP-based inhibitors and evaluate their activity in vitro. We will also synthesize suitable prodrugs of the best inhibitors and test them in cell-based assays using wildtype L. donovani and T. brucei cells, as well as genetically modified T. brucei cells that silence the expression of 6-oxo- or 6-aminopurine PRTs. We aim to use the developed inhibitors in combination with 6-oxopurine PRT inhibitors to strongly impede the essential PSP.