High resolution localization of cruzipain and Ssp4 in Trypanosoma cruzi by replica staining label fracture

Summary— The replica staining label fracture technique was used to analyse the distribution of cruzipain and Ssp4 in Trypanosoma cruzi. Intense labeling for the two proteins was seen on the E fracture face of amastigote forms. Gold particles did not co-localize with the intramembranous particles. Labeling was abolished by previous treatment of the parasites with phospholipase C from Trypanosoma brucei, which removes glycosylphosphatidyl inositol (GPI) anchored proteins. These observations suggest that cruzipain and Ssp4 are attached to the parasite surface via a GPI anchor.     

Stearoyl-CoA desaturase is an essential enzyme for the parasitic protist Trypanosoma brucei

Trypanosoma brucei, the etiologic agent of sleeping sickness, is exposed to important changes in nutrients and temperature during its life cycle. To adapt to these changes, the fluidity of its membranes plays a crucial role. This fluidity, mediated by the fatty-acid composition, is regulated by enzymes named desaturases. We have previously shown that the oleoyl desaturase is essential for Trypanosoma cruzi and T. brucei. In this work, we present experimental support for the relevance of stearoyl-CoA desaturase (SCD) for T. brucei’s survival, in both its insect or procyclic-form (PCF) and bloodstream-form (BSF) stages. We evaluated this essentiality in two different ways: by generating a SCD knocked-down parasite line using RNA interference, and by chemical inhibition of the enzyme with two compounds, Isoxyl and a thiastearate with the sulfur atom at position 10 (10-TS). The effective concentration for 50% growth inhibition (EC₅₀) of PCF was 1.0 ± 0.2 μM for Isoxyl and 5 ± 2 μM for 10-TS, whereas BSF appeared more susceptible with EC₅₀ values 0.10 ± 0.03 μM (Isoxyl) and 1.0 ± 0.6 μM (10-TS). RNA interference showed to be deleterious for both stages of the parasite. In addition, T. brucei-infected mice were fed with Isoxyl, causing a reduction of the parasitemia and an increase of the rodents’ survival.     

Structural characterization reveals a novel bilobed architecture for the ectodomains of insect stage expressed Trypanosoma brucei PSSA‐2 and Trypanosoma congolense ISA

African trypanosomiasis, caused by parasites of the genus Trypanosoma, is a complex of devastating vector‐borne diseases of humans and livestock in sub‐Saharan Africa. Central to the pathogenesis of African trypanosomes is their transmission by the arthropod vector, Glossina spp. (tsetse fly). Intriguingly, the efficiency of parasite transmission through the vector is reduced following depletion of Trypanosoma brucei Procyclic‐Specific Surface Antigen‐2 (TbPSSA‐2). To investigate the underlying molecular mechanism of TbPSSA‐2, we determined the crystal structures of its ectodomain and that of its homolog T. congolense Insect Stage Antigen (TcISA) to resolutions of 1.65 Å and 2.45 Å, respectively using single wavelength anomalous dispersion. Both proteins adopt a novel bilobed architecture with the individual lobes displaying rotational flexibility around the central tether that suggest a potential mechanism for coordinating a binding partner. In support of this hypothesis, electron density consistent with a bound peptide was observed in the inter‐lob cleft of a TcISA monomer. These first reported structures of insect stage transmembrane proteins expressed by African trypanosomes provide potentially valuable insight into the interface between parasite and tsetse vector.     

The SCP2‐thiolase‐like protein (SLP) of Trypanosoma brucei is an enzyme involved in lipid metabolism

Bioinformatics studies have shown that the genomes of trypanosomatid species each encode one SCP2‐thiolase‐like protein (SLP), which is characterized by having the YDCF thiolase sequence fingerprint of the Cβ2‐Cα2 loop. SLPs are only encoded by the genomes of these parasitic protists and not by those of mammals, including human. Deletion of the Trypanosoma brucei SLP gene (TbSLP) increases the doubling time of procyclic T. brucei and causes a 5‐fold reduction of de novo sterol biosynthesis from glucose‐ and acetate‐derived acetyl‐CoA. Fluorescence analyses of EGFP‐tagged TbSLP expressed in the parasite located the TbSLP in the mitochondrion. The crystal structure of TbSLP (refined at 1.75 Å resolution) confirms that TbSLP has the canonical dimeric thiolase fold. In addition, the structures of the TbSLP‐acetoacetyl‐CoA (1.90 Å) and TbSLP‐malonyl‐CoA (2.30 Å) complexes reveal that the two oxyanion holes of the thiolase active site are preserved. TbSLP binds malonyl‐CoA tightly (K_d 90 µM), acetoacetyl‐CoA moderately (K_d 0.9 mM) and acetyl‐CoA and CoA very weakly. TbSLP possesses low malonyl‐CoA decarboxylase activity. Altogether, the data show that TbSLP is a mitochondrial enzyme involved in lipid metabolism. Proteins 2016; 84:1075–1096. © 2016 Wiley Periodicals, Inc.     

Crystal structure of the small GTPase Arl6/BBS3 from Trypanosoma brucei

Arl6/BBS3 is a small GTPase, mutations in which are implicated in the human ciliopathy Bardet–Biedl Syndrome (BBS). Arl6 is proposed to facilitate the recruitment of a large protein complex known as the BBSome to the base of the primary cilium, mediating specific trafficking of molecules to this important sensory organelle. Orthologues of Arl6 and the BBSome core subunits have been identified in the genomes of trypanosomes. Flagellum function and motility are crucial to the survival of Trypanosoma brucei, the causative agent of human African sleeping sickness, in the human bloodstream stage of its lifecycle and so the function of the BBSome proteins in trypanosomes warrants further study. RNAi knockdown of T. brucei Arl6 (TbArl6) has recently been shown to result in shortening of the trypanosome flagellum. Here we present the crystal structure of TbArl6 with the bound non‐hydrolysable GTP analog GppNp at 2.0 Å resolution and highlight important differences between the trypanosomal and human proteins. Analysis of the TbArl6 active site confirms that it lacks the key glutamine that activates the nucleophile during GTP hydrolysis in other small GTPases. Furthermore, the trypanosomal proteins are significantly shorter at their N‐termini suggesting a different method of membrane insertion compared to humans. Finally, analysis of sequence conservation suggests two surface patches that may be important for protein–protein interactions. Our structural analysis thus provides the basis for future biochemical characterisation of this important family of small GTPases.     

Distinct donor and acceptor specificities of Trypanosoma brucei oligosaccharyltransferases

Asparagine‐linked glycosylation is catalysed by oligosaccharyltransferase (OTase). In Trypanosoma brucei OTase activity is catalysed by single‐subunit enzymes encoded by three paralogous genes of which TbSTT3B and TbSTT3C can complement a yeast Δstt3 mutant. The two enzymes have overlapping but distinct peptide acceptor specificities, with TbSTT3C displaying an enhanced ability to glycosylate sites flanked by acidic residues. TbSTT3A and TbSTT3B, but not TbSTT3C, are transcribed in the bloodstream and procyclic life cycle stages of T. brucei. Selective knockdown and analysis of parasite protein N‐glycosylation showed that TbSTT3A selectively transfers biantennary Man₅GlcNAc₂ to specific glycosylation sites whereas TbSTT3B selectively transfers triantennary Man₉GlcNAc₂ to others. Analysis of T. brucei glycosylation site occupancy showed that TbSTT3A and TbSTT3B glycosylate sites in acidic to neutral and neutral to basic regions of polypeptide, respectively. This embodiment of distinct specificities in single‐subunit OTases may have implications for recombinant glycoprotein engineering. TbSTT3A and TbSTT3B could be knocked down individually, but not collectively, in tissue culture. However, both were independently essential for parasite growth in mice, suggesting that inhibiting protein N‐glycosylation could have therapeutic potential against trypanosomiasis.     

The Trypanosoma cruzi nucleic acid binding protein Tc38 presents changes in the intramitochondrial distribution during the cell cycle

Background  

Tc38 of Trypanosoma cruzi has been isolated as a single stranded DNA binding protein with high specifiCity for the poly [dT-dG] sequence. It is present only in Kinetoplastidae protozoa and its sequence lacks homology to known functional domains. Tc38 orthologues present in Trypanosoma brucei and Leishmania were proposed to participate in quite different cellular processes. To further understand the function of this protein in Trypanosoma cruzi, we examined its in vitro binding to biologically relevant [dT-dG] enriched sequences, its expression and subcellular localization during the cell cycle and through the parasite life stages.     

NMR structure of the calflagin Tb24 flagellar calcium binding protein of Trypanosoma brucei

Flagellar calcium binding proteins are expressed in a variety of trypanosomes and are potential drug targets for Chagas disease and African sleeping sickness. The flagellar calcium binding protein calflagin of Trypanosoma brucei (called Tb24) is a myristoylated and palmitoylated EF‐hand protein that is targeted to the inner leaflet of the flagellar membrane. The Tb24 protein may also interact with proteins on the membrane surface that may be different from those bound to flagellar calcium binding proteins (FCaBPs) in T. cruzi. We report here the NMR structure of Tb24 that contains four EF‐hand motifs bundled in a compact arrangement, similar to the overall fold of T. cruzi FCaBP (RMSD = 1.0 Å). A cluster of basic residues (K22, K25, K31, R36, and R38) located on a surface near the N‐terminal myristoyl group may be important for membrane binding. Non‐conserved residues on the surface of a hydrophobic groove formed by EF2 (P91, Q95, D103, and V108) and EF4 (C194, T198, K199, Q202, and V203) may serve as a target protein binding site and could have implications for membrane target recognition.     

Characterization of di‐, tri‐ and tetranucleotide microsatellite markers with perfect repeats for Trypanosoma brucei and related species

Trypanosoma brucei, a unicellular parasite causing human sleeping sickness and animal nagana, has a great impact on the socioeconomic environment of sub‐Saharan Africa. The dynamics of the parasite are still poorly understood. We have characterized 14 polymorphic di‐, tri‐ and tetranucleotide microsatellite loci with perfect repeats (only one motif) exhibiting between five and 16 alleles in T. brucei isolates from all over Africa and from all described subspecies. The microsatellites will be useful in addressing population genetic questions in T. brucei to better understand the population structure and spread of this important parasite.     

Trypanosoma cruzi: A kinetoplast-associated protein of the photolyase/cryptochrome family

A photolyase-like protein gene found in the Trypanosoma cruzi genome database was cloned and expressed in Escherichia coli resulting in the formation of inclusion bodies. Antibodies against this protein were used to determine expression of the protein in the different forms of the parasite. It was visualized in the epimastigote form but not in amastigote or trypomastigote forms obtained from culture in Vero cells. In epimastigotes, this protein is located at the level of the mitochondrion associated to both sides of the kinetoplast. Sequence analyses indicated that this protein, as well as other photolyases from Leishmania spp. and Trypanosoma brucei are related to single-stranded photolyases or cryptochromes DASH.     

Phosphoglucomutase is absent in Trypanosoma brucei and redundantly substituted by phosphomannomutase and phospho-N-acetylglucosamine mutase

The enzymes phosphomannomutase (PMM), phospho‐N‐acetylglucosamine mutase (PAGM) and phosphoglucomutase (PGM) reversibly catalyse the transfer of phosphate between the C6 and C1 hydroxyl groups of mannose, N‐acetylglucosamine and glucose respectively. Although genes for a candidate PMM and a PAGM enzymes have been found in the Trypanosoma brucei genome, there is, surprisingly, no candidate gene for PGM. The TbPMM and TbPAGM genes were cloned and expressed in Escherichia coli and the TbPMM enzyme was crystallized and its structure solved at 1.85 Å resolution. Antibodies to the recombinant proteins localized endogenous TbPMM to glycosomes in the bloodstream form of the parasite, while TbPAGM localized to both the cytosol and glycosomes. Both recombinant enzymes were able to interconvert glucose‐phosphates, as well as acting on their own definitive substrates. Analysis of sugar nucleotide levels in parasites with TbPMM or TbPAGM knocked down by RNA interference (RNAi) suggests that, in vivo, PGM activity is catalysed by both enzymes. This is the first example in any organism of PGM activity being completely replaced in this way and it explains why, uniquely, T. brucei has been able to lose its PGM gene. The RNAi data for TbPMM also showed that this is an essential gene for parasite growth.     

Genome research and evolution in trypanosomes

Trypanosoma brucei and Trypanosoma cruzi cause different human diseases. As strategies for immune evasion. T. brucei undergoes antigenic variation whereas T. cruzi becomes an intracellular organism. This fundamental difference is reflected by major differences in their genome organizations. Recent comparisons of their gene sequences indicate that these two trypanosome species are highly divergent evolutionarily.     

Interaction between cFLIPL and Itch,a ubiquitin ligase,is obstructed in Trypanosoma cruzi‐infected human cells

Death receptor‐mediated host cell apoptosis, a defense strategy for elimination by the immune system of parasite‐infected cells, is inhibited by Trypanosoma cruzi, the causative agent of Chagas' disease. It has previously been reported by us that, in infected cells, T. cruzi upregulates and exploits cFLIP_L, a mammalian inhibitor of death receptor signaling. Here it is shown that ubiquitination of cFLIP_L, leading to proteasomal degradation, is inhibited in parasite‐infected cells. The extent of expression of Itch, a protein thought to be an ubiquitin ligase for cFLIP_L, was found to be equivalent in T. cruzi‐infected and in uninfected cells. However, co‐immunoprecipitation analysis showed that the interaction between cFLIP_L and Itch is strongly inhibited in T. cruzi‐infected cells. This unique parasite strategy, which has not been reported in any other pathogen‐infected cells, may allow the host cell to accumulate cFLIP_L, eventually resulting in the inhibition of apoptosis of T. cruzi‐infected cells.     

Trypanosoma cruzi: single cell live imaging inside infected tissues

Although imaging the live Trypanosoma cruzi parasite is a routine technique in most laboratories, identification of the parasite in infected tissues and organs has been hindered by their intrinsic opaque nature. We describe a simple method for in vivo observation of live single‐cell Trypanosoma cruzi parasites inside mammalian host tissues. BALB/c or C57BL/6 mice infected with DsRed‐CL or GFP‐G trypomastigotes had their organs removed and sectioned with surgical blades. Ex vivo organ sections were observed under confocal microscopy. For the first time, this procedure enabled imaging of individual amastigotes, intermediate forms and motile trypomastigotes within infected tissues of mammalian hosts.     

Presence of an unusually high concentration of an ubiquitinated histone-like protein in Trypanosoma cruzi

The conjugation of ubiquitin to histones H2A and H2B has been established in higher eukaryotes and has been related to changes in chromatin organization. In Trypanosoma cruzi, no condensation of chromatin occurs during mitosis. In order to determine the presence of histone ubiquitination in T. cruzi epimastigotes, histones were extracted from chromatin and analyzed by three electrophoretic systems: acid-urea, triton-acid-urea and sodium-dodecyl-sulphate polyacrylamide gel. The immunochemical detection of ubiquitin-histone conjugates by Western blotting showed a strong reaction with a slow migrating band of M_r 19 kDa. The high percentage of ubiquitin-histone conjugates present in T. cruzi chromatin may be related to the inability of this parasite to condense chromatin into a 30 nm fiber. J. Cell. Biochem. 66:433–440, 1997. © 1997 Wiley-Liss, Inc.     

Cell cycle and cleavage events during in vitro cultivation of bloodstream forms of Trypanosoma lewisi,a zoonotic pathogen

Trypanosoma (Herpetosoma) lewisi is a globally distributed rat trypanosome, currently considered as a zoonotic pathogen; however, a detailed understanding of the morphological events occurring during the cell cycle is lacking. This study aimed to investigate the cell cycle morphology and cleavage events of Trypanosoma lewisi (T. lewisi) during in vitro cultivation. By establishing in vitro cultivation of T. lewisi at 37°C, various cell morphologies and stages could be observed. We have provided a quantitative analysis of the morphological events during T. lewisi proliferation. We confirmed a generation time of 12.14 ± 0.79 hours, which is similar to that in vivo (12.21 ± 0.14 hours). We also found that there are two distinct cell cycles, with a two-way transformation connection in the developmental status of this parasite, which was contrasted with the previous model of multiple division patterns seen in T. lewisi. We quantified the timing of cell cycle phases (G1_n, 0.56 U; S_n, 0.14 U; G2_n, 0.16 U; M, 0.06 U; C, 0.08 U; G1_k, 0.65 U; S_k, 0.10 U; G2_k, 0.17 U; D, 0.03 U; A, 0.05 U) and their morphological characteristics, particularly with respect to the position of kinetoplast(s) and nucleus/nuclei. Interestingly, we found that both nuclear synthesis initiation and segregation in T. lewisi occurred prior to kinetoplast, different to the order of replication found in Trypanosoma brucei and Trypanosoma cruzi, implicating a distinct cell cycle control mechanism in T. lewisi. We characterized the morphological events during the T. lewisi cell cycle and presented evidence to support the existence of two distinct cell cycles with two-way transformation between them. These results provide insights into the differentiation and evolution of this parasite and its related species.     

Megazol and its bioisostere 4H-1,2,4-triazole: comparing the trypanocidal,cytotoxic and genotoxic activities and their in vitro and in silico interactions with the Trypanosoma brucei nitroreductase enzyme

Megazol (7) is a 5-nitroimidazole that is highly active against Trypanosoma cruzi and Trypanosoma brucei, as well as drug-resistant forms of trypanosomiasis. Compound 7 is not used clinically due to its mutagenic and genotoxic properties, but has been largely used as a lead compound. Here, we compared the activity of 7 with its 4H-1,2,4-triazole bioisostere (8) in bloodstream forms of T. brucei and T. cruzi and evaluated their activation by T. brucei type I nitroreductase (TbNTR) enzyme. We also analysed the cytotoxic and genotoxic effects of these compounds in whole human blood using Comet and fluorescein diacetate/ethidium bromide assays. Although the only difference between 7 and 8 is the substitution of sulphur (in the thiadiazole in 7) for nitrogen (in the triazole in 8), the results indicated that 8 had poorer antiparasitic activity than 7 and was not genotoxic, whereas 7 presented this effect. The determination of Vmax indicated that although 8 was metabolised more rapidly than 7, it bounds to the TbNTR with better affinity, resulting in equivalent kcat/KM values. Docking assays of 7 and 8 performed within the active site of a homology model of the TbNTR indicating that 8 had greater affinity than 7.     

Trans-sialidase and mucins of Trypanosoma cruzi: an important interplay for the parasite

A dense glycocalix covers the surface of Trypanosoma cruzi, the agent of Chagas disease. Sialic acid in the surface of the parasite plays an important role in the infectious process, however, T. cruzi is unable to synthesize sialic acid or the usual donor CMP-sialic acid. Instead, T. cruzi expresses a unique enzyme, the trans-sialidase (TcTS) involved in the transfer of sialic acid from host glycoconjugates to mucins of the parasite. The mucins are the major glycoproteins in the insect stage epimastigotes and in the infective trypomastigotes. Both, the mucins and the TcTS are anchored to the plasma membrane by a glycosylphosphatidylinositol anchor. Thus, TcTS may be shed into the bloodstream of the mammal host by the action of a parasite phosphatidylinositol-phospholipase C, affecting the immune system. The composition and structure of the sugars in the parasite mucins is characteristic of each differentiation stage, also, interstrain variations were described for epimastigote mucins. This review focus on the characteristics of the interplay between the trans-sialidase and the mucins of T. cruzi and summarizes the known carbohydrate structures of the mucins.     

New Culture Medium for Maintenance of Tsetse Tissues and Growth of Trypanosomatids*

SYNOPSIS. A new culture medium (SM), based on the amino-acid composition of tsetse hemolymph and containing fetal bovine serum, was designed for the maintenance of tsetse organs and the cultivation of various trypanosomatids. For optimum growth 20% (v/v) serum was required. The medium supported prolonged peristalsis of the alimentary tract and salivary glands of pre-emerged Glossina morsitans morsitans. In established cultures, derived from bloodstream forms of pleomorphic Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense strains, inocula of ～ 10⁶ procyclics/ml yielded 4–5 × 10⁷ organisms/ml after 4 or 5 days of incubation at 28 C. Bloodstream forms of a cloned monomorphic T. b. brucei strain were also able to transform into procyclics, which, however, multiplied at a lower rate, with maximum yields of ～ 2 × 10⁷ after 5 days. Cultures of Trypanosoma congolense and of a nearly monomorphic Trypanosoma brucei gambiense strains could be established in SM medium only in the presence of tsetse alimentary tract. The procyclic trypomastigotes of these species, adapted to SM medium and able to grow in it without Glossina organs, gave maximum populations of ～ 4.5 × 10⁷ cells/ml. Promastigotes of Leishmania donovani, cultivated routinely in a diphasic Table's medium, multiplied actively upon being transferred into SM medium, producing yields of ～ 4 × 10⁷ cells/ml.     

Trypanosoma cruzi trans-sialidase in complex with a neutralizing antibody: structure/function studies towards the rational design of inhibitors

Trans-sialidase (TS), a virulence factor from Trypanosoma cruzi, is an enzyme playing key roles in the biology of this protozoan parasite. Absent from the mammalian host, it constitutes a potential target for the development of novel chemotherapeutic drugs, an urgent need to combat Chagas'' disease. TS is involved in host cell invasion and parasite survival in the bloodstream. However, TS is also actively shed by the parasite to the bloodstream, inducing systemic effects readily detected during the acute phase of the disease, in particular, hematological alterations and triggering of immune cells apoptosis, until specific neutralizing antibodies are elicited. These antibodies constitute the only known submicromolar inhibitor of TS''s catalytic activity. We now report the identification and detailed characterization of a neutralizing mouse monoclonal antibody (mAb 13G9), recognizing T. cruzi TS with high specificity and subnanomolar affinity. This mAb displays undetectable association with the T. cruzi superfamily of TS-like proteins or yet with the TS-related enzymes from Trypanosoma brucei or Trypanosoma rangeli. In immunofluorescence assays, mAb 13G9 labeled 100% of the parasites from the infective trypomastigote stage. This mAb also reduces parasite invasion of cultured cells and strongly inhibits parasite surface sialylation. The crystal structure of the mAb 13G9 antigen-binding fragment in complex with the globular region of T. cruzi TS was determined, revealing detailed molecular insights of the inhibition mechanism. Not occluding the enzyme''s catalytic site, the antibody performs a subtle action by inhibiting the movement of an assisting tyrosine (Y₁₁₉), whose mobility is known to play a key role in the trans-glycosidase mechanism. As an example of enzymatic inhibition involving non-catalytic residues that occupy sites distal from the substrate-binding pocket, this first near atomic characterization of a high affinity inhibitory molecule for TS provides a rational framework for novel strategies in the design of chemotherapeutic compounds.