Trypanosoma brucei causes human African trypanosomiasis (HAT) and nagana in cattle. During infection of a vertebrate, endocytosis of host transferrin (Tf) is important for viability of the parasite. The majority of proteins involved in trypanosome endocytosis of Tf are unknown. Here we identify pseudokinase NRP1 (Tb427tmp.160.4770) as a regulator of Tf endocytosis. Genetic knockdown of NRP1 inhibited endocytosis of Tf without blocking uptake of bovine serum albumin. Binding of Tf to the flagellar pocket was not affected by knockdown of NRP1. However the quantity of Tf per endosome dropped significantly, consistent with NRP1 promoting robust capture and/or retention of Tf in vesicles. NRP1 is involved in motility of Tf-laden vesicles since distances between endosomes and the kinetoplast were reduced after knockdown of the gene. In search of possible mediators of NRP1 modulation of Tf endocytosis, the gene was knocked down and the phosphoproteome analyzed. Phosphorylation of protein kinases forkhead, NEK6, and MAPK10 was altered, in addition to EpsinR, synaptobrevin and other vesicle-associated proteins predicted to be involved in endocytosis. These candidate proteins may link NRP1 functionally either to protein kinases or to vesicle-associated proteins.
SQ109 is an anti-tubercular drug candidate that has completed Phase IIb/III clinical trials for tuberculosis and has also been shown to exhibit potent in vitro efficacy against protozoan parasites including Leishmania and Trypanosoma cruzi spp. However, its in vivo efficacy against protozoa has not been reported. Here, we evaluated the activity of SQ109 in mouse models of Leishmania, Trypanosoma spp. as well as Toxoplasma infection. In the T. cruzi mouse model, 80% of SQ109-treated mice survived at 40 days post-infection. Even though SQ109 did not cure all mice, these results are of interest since they provide a basis for future testing of combination therapies with the azole posaconazole, which acts synergistically with SQ109 in vitro. We also found that SQ109 inhibited the growth of Toxoplasma gondii in vitro with an IC50 of 1.82 µM and there was an 80% survival in mice treated with SQ109, whereas all untreated animals died 10 days post-infection. Results with Trypanosoma brucei and Leishmania donovani infected mice were not promising with only moderate efficacy. Since SQ109 is known to be extensively metabolized in animals, we investigated the activity in vitro of SQ109 metabolites. Among 16 metabolites, six mono-oxygenated forms were found active across the tested protozoan parasites, and there was a ~6× average decrease in activity of the metabolites as compared to SQ109 which is smaller than the ~25× found with mycobacteria.
Acidocalcisomes are electron-dense organelles rich in polyphosphate and inorganic and organic cations that are acidified by proton pumps, and possess several channels, pumps and transporters. They are present in bacteria and eukaryotes and have been studied in greater detail in trypanosomatids. Biogenesis studies of trypanosomatid acidocalcisomes found that they share properties with lysosome-related organelles of animal cells. In addition to their described roles in autophagy, cation and phosphorus storage, osmoregulation, pH homeostasis, and pathogenesis, recent studies have defined the role of these organelles in phosphate utilization, calcium ion (Ca2+ ) signaling, and bioenergetics, and will be the main subject of this review.
Human African trypanosomiasis (HAT) is a deadly neglected tropical disease caused by the protozoan parasite Trypanosoma brucei. During the course of screening a collection of diverse nitrogenous heterocycles, we discovered two novel compounds that contain the tetracyclic core of the Yohimbine and Corynanthe alkaloids, were potent inhibitors of T. brucei proliferation and T. brucei methionyl-tRNA synthetase (TbMetRS) activity. Inspired by these key findings, we prepared several novel series of hydroxyalkyl δ-lactam, δ-lactam, and piperidine analogs and tested their anti-trypanosomal activity. A number of inhibitors are more potent against T. brucei than these initial hits with one hydroxyalkyl δ-lactam derivative being 25-fold more effective in our assay. Surprisingly, most of these active compounds failed to inhibit TbMetRS. This work underscores the importance of verifying, irrespective of close structural similarities, that new compounds designed from a lead with a known biological target engage the putative binding site.
Acidocalcisomes are membrane-bounded, electron-dense, acidic organelles, rich in calcium and polyphosphate. These organelles were first described in trypanosomatids and later found from bacteria to human cells. Some of the functions of the acidocalcisome are the storage of cations and phosphorus, participation in pyrophosphate (PPi) and polyphosphate (polyP) metabolism, calcium signaling, maintenance of intracellular pH homeostasis, autophagy, and osmoregulation. Isolation of acidocalcisomes is an important technique for understanding their composition and function. Here, we provide detailed subcellular fractionation protocols using iodixanol gradient centrifugations to isolate high-quality acidocalcisomes from Trypanosoma brucei, which are subsequently validated by electron microscopy, and enzymatic and immunoblot assays with organellar markers.
Mitochondrial Ca2+ transport mediated by the uniporter complex (MCUC) plays a key role in the regulation of cell bioenergetics in both trypanosomes and mammals. Here we report that Trypanosoma brucei MCU (TbMCU) subunits interact with subunit c of the mitochondrial ATP synthase (ATPc), as determined by coimmunoprecipitation and split-ubiquitin membrane-based yeast two-hybrid (MYTH) assays. Mutagenesis analysis in combination with MYTH assays suggested that transmembrane helices (TMHs) are determinants of this specific interaction. In situ tagging, followed by immunoprecipitation and immunofluorescence microscopy, revealed that T. brucei ATPc (TbATPc) coimmunoprecipitates with TbMCUC subunits and colocalizes with them to the mitochondria. Blue native PAGE and immunodetection analyses indicated that the TbMCUC is present together with the ATP synthase in a large protein complex with a molecular weight of approximately 900 kDa. Ablation of the TbMCUC subunits by RNA interference (RNAi) significantly increased the AMP/ATP ratio, revealing the downregulation of ATP production in the cells. Interestingly, the direct physical MCU-ATPc interaction is conserved in Trypanosoma cruzi and human cells. Specific interaction between human MCU (HsMCU) and human ATPc (HsATPc) was confirmed in vitro by mutagenesis and MYTH assays and in vivo by coimmunoprecipitation. In summary, our study has identified that MCU complex physically interacts with mitochondrial ATP synthase, possibly forming an MCUC-ATP megacomplex that couples ADP and Pi transport with ATP synthesis, a process that is stimulated by Ca2+ in trypanosomes and human cells.
IMPORTANCE The mitochondrial calcium uniporter (MCU) is essential for the regulation of oxidative phosphorylation in mammalian cells, and we have shown that in Trypanosoma brucei, the etiologic agent of sleeping sickness, this channel is essential for its survival and infectivity. Here we reveal that that Trypanosoma brucei MCU subunits interact with subunit c of the mitochondrial ATP synthase (ATPc). Interestingly, the direct physical MCU-ATPc interaction is conserved in T. cruzi and human cells.
Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3′ extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3′ adenylation, uridine insertion/deletion editing, and 3′ A/U-tailing. The rRNAs and gRNAs are 3′ uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover.
Inna Aphasizheva, Juan Alfonzo, Jason Carnes, Igor Cestari, Jorge Cruz-Reyes, H. Ulrich Göringer, Stephen Hajduk, Julius Lukeš, Susan Madison-Antenucci, Dmitri A. Maslov, Suzanne M. McDermott, Torsten Ochsenreiter, Laurie K. Read, Reza Salavati, Achim Schnaufer, André Schneider, Larry Simpson, Kenneth Stuart, Vyacheslav Yurchenko, Z. Hong Zhou, Alena Zíková, Liye Zhang, Sara Zimmer, Ruslan Aphasizhev. Trends Parasitol. 2020 Apr;36(4):337-355. doi: 10.1016/j.pt.2020.01.006. Epub 2020 Feb 28.
- Kinetoplasts (mitochondrial genome nucleoids) are important in bloodstream trypanosomes for the establishment of mitochondrial membrane potential.
- Many proteins involved in segregation of kinetoplasts have been identified.
- A region between a kinetoplast and basal bodies is described as a tripartite attachment complex (TAC).
- A set of TAC-associated proteins (TACAPs) has been proposed as the machinery for kinetoplast segregation.
- Subcomplexes of TACAPs that form in vivo have been described.
- Several proteins that do not associate with TAC are involved in the maintenance of the kinetoplast.
- New kinetoplast-associated proteins have been identified.
- We are approaching an exciting period in the field when a molecular understanding of how all aspects of kinetoplast biogenesis are executed seems achievable.
We recently reported the medicinal chemistry re-optimization of a series of compounds derived from the human tyrosine kinase inhibitor, lapatinib, for activity against Plasmodium falciparum. From this same library of compounds, we now report potent compounds against Trypanosoma brucei brucei (which causes human African trypanosomiasis), T. cruzi (the pathogen that causes Chagas disease), and Leishmania spp. (which cause leishmaniasis). In addition, sub-micromolar compounds were identified that inhibit proliferation of the parasites that cause African animal trypanosomiasis, T. congolense and T. vivax. We have found that this set of compounds display acceptable physicochemical properties and represent progress towards identification of lead compounds to combat several neglected tropical diseases.
Lori Ferrins, Amrita Sharma, Sarah M. Thomas, Naimee Mehta, Jessey Erath, Scott Tanghe, Susan E. Leed, Ana Rodriguez, Kojo Mensa-Wilmot, Richard J. Sciotti, Kirsten Gillingwater, Michael P. Pollastri. 2018. PLOS Neglected Tropical Diseases. https://doi.org/10.1371/journal.pntd.0006834