Trypanosoma evansi: Paraflagellar rod protein 1 and 2 are similar but lack common B cell epitopes

In an attempt to identify invariant proteins with vaccine potential against African trypanosomes, we investigated the existence of PFR1 protein in Trypanosoma evansi and compared its B cell epitope with that of PFR2 protein of T. evansi using Western blotting and immuno-precipitation assays. The PFR1 gene of T. evansi was amplified by RT-PCR using primers designed based on the open reading frame of PFR1 gene of Trypanosoma brucei. The cloned PFR1 gene of T.evansi was similar to PFR1 genes of T. brucei and Trypanosoma cruzi. The expressed protein from the PFR1 gene was 68.4% homologous to the PFR2 protein of T. evansi, and showed 99.8%, 87%, 77.9% and 77.5% homologous to the PFR1 protein of T. brucei, T. cruzi, Leishmania mexicana and Leishmania major, respectively. Western blot and immuno-precipitation assays showed that antibodies raised against PFR1 and 2 proteins in BALB/c mice recognized the PFR1 and 2 proteins, respectively, with no cross-reactivity. Immuno-agglutination assay showed trypanolytic properties of the anti-PFR1, anti-PFR2 and anti-native PFR sera. These results suggest that PFR1 and PFR2 proteins are components of native PFR antigen and do not share common B cell epitopes.     

Unveiling the Intracellular Survival Gene Kit of Trypanosomatid Parasites

Trypanosomatids are unicellular protozoans of medical and economical relevance since they are the etiologic agents of infectious diseases in humans as well as livestock. Whereas Trypanosoma cruzi and different species of Leishmania are obligate intracellular parasites, Trypanosoma brucei and other trypanosomatids develop extracellularly throughout their entire life cycle. After their genomes have been sequenced, various comparative genomic studies aimed at identifying sequences involved with host cell invasion and intracellular survival have been described. However, for only a handful of genes, most of them present exclusively in the T. cruzi or Leishmania genomes, has there been any experimental evidence associating them with intracellular parasitism. With the increasing number of published complete genome sequences of members of the trypanosomatid family, including not only different Trypanosoma and Leishmania strains and subspecies but also trypanosomatids that do not infect humans or other mammals, we may now be able to contemplate a slightly better picture regarding the specific set of parasite factors that defines each organism''s mode of living and the associated disease phenotypes. Here, we review the studies concerning T. cruzi and Leishmania genes that have been implicated with cell invasion and intracellular parasitism and also summarize the wealth of new information regarding the mode of living of intracellular parasites that is resulting from comparative genome studies that are based on increasingly larger trypanosomatid genome datasets.     

A high-throughput cloning system for reverse genetics in <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Background  

The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.     

A Cell-surface Phylome for African Trypanosomes

The cell surface of Trypanosoma brucei, like many protistan blood parasites, is crucial for mediating host-parasite interactions and is instrumental to the initiation, maintenance and severity of infection. Previous comparisons with the related trypanosomatid parasites T. cruzi and Leishmania major suggest that the cell-surface proteome of T. brucei is largely taxon-specific. Here we compare genes predicted to encode cell surface proteins of T. brucei with those from two related African trypanosomes, T. congolense and T. vivax. We created a cell surface phylome (CSP) by estimating phylogenies for 79 gene families with putative surface functions to understand the more recent evolution of African trypanosome surface architecture. Our findings demonstrate that the transferrin receptor genes essential for bloodstream survival in T. brucei are conserved in T. congolense but absent from T. vivax and include an expanded gene family of insect stage-specific surface glycoproteins that includes many currently uncharacterized genes. We also identify species-specific features and innovations and confirm that these include most expression site-associated genes (ESAGs) in T. brucei, which are absent from T. congolense and T. vivax. The CSP presents the first global picture of the origins and dynamics of cell surface architecture in African trypanosomes, representing the principal differences in genomic repertoire between African trypanosome species and provides a basis from which to explore the developmental and pathological differences in surface architectures. All data can be accessed at: http://www.genedb.org/Page/trypanosoma_surface_phylome.     

Quinoxaline derivatives as potential antitrypanosomal and antileishmanial agents

Continuous efforts have been made to discover new drugs for the treatment of Chagas’ disease, human African trypanosomiasis, and leishmaniasis. We have previously reported the synthesis and antileishmanial and antitrypanosomal (Y strain) properties of 2,3-disubstituted quinoxalines. Considering their promising antiparasitic potential, the present study was conducted to expand our search and take advantage of high-throughput assays to investigate the effects of quinoxaline derivatives against Leishmania donovani, Trypanosoma brucei, and Trypanosoma cruzi (Tulahuen strain). These compounds were active against the kinetoplastid parasites that were evaluated. The 2-chloro-3-methylsulfoxylsulfonyl and 2-chloro-3-methylsulfinyl quinoxalines were the most potent, and some of these derivatives were even more active than the reference drugs. Although the 2,3-diaryl-substituted quinoxalines were not active against all of the parasites, they were active against T. brucei and intracellular amastigotes of T. cruzi, without interfering with mammalian cell viability. These compounds presented encouraging results that will guide our future studies on in vivo bioassays towards the mode of action.     

Ebsulfur Is a Benzisothiazolone Cytocidal Inhibitor Targeting the Trypanothione Reductase of Trypanosoma brucei

Trypanosoma brucei is the causing agent of African trypanosomiasis. These parasites possess a unique thiol redox system required for DNA synthesis and defense against oxidative stress. It includes trypanothione and trypanothione reductase (TryR) instead of the thioredoxin and glutaredoxin systems of mammalian hosts. Here, we show that the benzisothiazolone compound ebsulfur (EbS), a sulfur analogue of ebselen, is a potent inhibitor of T. brucei growth with a favorable selectivity index over mammalian cells. EbS inhibited the TryR activity and decreased non-protein thiol levels in cultured parasites. The inhibition of TryR by EbS was irreversible and NADPH-dependent. EbS formed a complex with TryR and caused oxidation and inactivation of the enzyme. EbS was more toxic for T. brucei than for Trypanosoma cruzi, probably due to lower levels of TryR and trypanothione in T. brucei. Furthermore, inhibition of TryR produced high intracellular reactive oxygen species. Hydrogen peroxide, known to be constitutively high in T. brucei, enhanced the EbS inhibition of TryR. The elevation of reactive oxygen species production in parasites caused by EbS induced a programmed cell death. Soluble EbS analogues were synthesized and cured T. brucei brucei infection in mice when used together with nifurtimox. Altogether, EbS and EbS analogues disrupt the trypanothione system, hampering the defense against oxidative stress. Thus, EbS is a promising lead for development of drugs against African trypanosomiasis.     

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.     

2,4-Diaminopyrimidines as potent inhibitors of Trypanosoma brucei and identification of molecular targets by a chemical proteomics approach

Background

There is an urgent need to develop new, safe and effective treatments for human African trypanosomiasis (HAT) because current drugs have extremely poor safety profiles and are difficult to administer. Here we report the discovery of 2,4-diaminopyrimidines, exemplified by 4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid phenylamide (SCYX-5070), as potent inhibitors of Trypanosoma brucei and the related trypanosomatid protozoans Leishmania spp.

Methodology/Principal Findings

In this work we show that loss of T. brucei viability following SCYX-5070 exposure was dependent on compound concentration and incubation time. Pulse incubation of T. brucei with SCYX-5070 demonstrates that a short period of exposure (10–12 hrs) is required to produce irreversible effects on survival or commit the parasites to death. SCYX-5070 cured an acute trypanosomiasis infection in mice without exhibiting signs of compound related acute or chronic toxicity. To identify the molecular target(s) responsible for the mechanism of action of 2,4-diaminopyrimidines against trypanosomatid protozoa, a representative analogue was immobilized on a solid matrix (sepharose) and used to isolate target proteins from parasite extracts. Mitogen-activated protein kinases (MAPKs) and cdc2-related kinases (CRKs) were identified as the major proteins specifically bound to the immobilized compound, suggesting their participation in the pharmacological effects of 2,4-diaminopyrimidines against trypanosomatid protozoan parasites.

Conclusions/Significance

Results show that 2,4-diaminopyrimidines have a good in vitro and in vivo pharmacological profile against trypanosomatid protozoans and that MAPKs and CRKs are potential molecular targets of these compounds. The 2,4-diminipyrimidines may serve as suitable leads for the development of novel treatments for HAT.     

Comparative analysis of editosome proteins in trypanosomatids

Detailed comparisons of 16 editosome proteins from Trypanosoma brucei, Trypanosoma cruzi and Leishmania major identified protein motifs associated with catalysis and protein or nucleic acid interactions that suggest their functions in RNA editing. Five related proteins with RNase III-like motifs also contain a U1-like zinc finger and either dsRBM or Pumilio motifs. These proteins may provide the endoribonuclease function in editing. Two other related proteins, at least one of which is associated with U-specific 3′ exonuclease activity, contain two putative nuclease motifs. Thus, editosomes contain a plethora of nucleases or proteins presumably derived from nucleases. Five additional related proteins, three of which have zinc fingers, each contain a motif associated with an OB fold; the TUTases have C-terminal folds reminiscent of RNA binding motifs, thus indicating the presence of numerous nucleic acid and/or protein binding domains, as do the two RNA ligases and a RNA helicase, which provide for additional catalytic steps in editing. These data indicate that trypanosomatid RNA editing is orchestrated by a variety of domains for catalysis, molecular interaction and structure. These domains are generally conserved within other protein families, but some are found in novel combinations in the editosome proteins.     

Conserved repeats in the kinetoplast maxicircle divergent region of Leishmania sp. and Leptomonas seymouri

The maxicircle control region [also termed divergent region (DR)] composed of various repeat elements remains the most poorly studied part of the kinetoplast genome. Only three extensive DR sequences demonstrating no significant similarity were available for trypanosomatids (Leishmania tarentolae, Crithidia oncopelti, Trypanosoma brucei). Recently, extensive DR sequences have been obtained for Leishmania major and Trypanosoma cruzi. In this work we have sequenced DR fragments of Leishmania turanica, Leishmania mexicana, Leishmania chagasi and two monogenetic trypanosomatids Leptomonas seymouri and Leptomonas collosoma. With the emergence of the additional extensive sequences some conserved features of DR structure become evident. A conserved palindromic sequence has been revealed in the DRs of the studied Leishmania species, L. seymouri, and T. cruzi. The overall DR structure appears to be similar in all the Leishmania species, their relative L. seymouri, and T. brucei: long relatively GC-rich repeats are interspersed with clusters of short AT-rich repeats. C. oncopelti, L. collosoma, and T. cruzi have a completely different DR structure. Identification of conserved sequences and invariable structural features of the DR may further our understanding of the functioning of this important genome fragment.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at Nucleotide sequence data reported in this paper are available in the GenBank™, EMBL and DDBJ databases under the accession numbers DQ107351, DQ107352, DQ107354-DQ107358, DQ239759-DQ239765, DQ492251-DQ492256.     

The Hsp70 chaperones of the Tritryps are characterized by unusual features and novel members

Proteins belonging to the Hsp70 class of molecular chaperones are highly conserved and ubiquitous, performing an essential role in the maintenance of cellular homeostasis in almost all known organisms. Trypanosoma brucei, Trypanosoma cruzi and Leishmania major are human parasites collectively known as the Tritryps. The Tritryps undergo extensive morphological changes during their life cycles, largely triggered by the marked differences between conditions in their insect vector and human host. Hsp70s are synthesised in response to these marked changes in environment and are proposed to be required for these parasites to successfully transition between differentiation stages while remaining viable and infective. While the Tritryps Hsp70 complement consists of homologues of all the major eukaryotic Hsp70s, there are a number of novel members, and some unique structural features. This review critically evaluates the current knowledge on the Tritryps Hsp70 proteins with an emphasis on T. brucei, and highlights some novel and previously unstudied aspects of these multifaceted molecular chaperones.     

A comparative analysis of trypanosomatid SNARE proteins

The Kinetoplastida are flagellated protozoa evolutionary distant and divergent from yeast and humans. Kinetoplastida include trypanosomatids, and a number of important pathogens. Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. inflict significant morbidity and mortality on humans and livestock as the etiological agents of human African trypanosomiasis, Chagas' disease and leishmaniasis respectively. For all of these organisms, intracellular trafficking is vital for maintenance of the host–pathogen interface, modulation/evasion of host immune system responses and nutrient uptake. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are critical components of the intracellular trafficking machinery in eukaryotes, mediating membrane fusion and contributing to organelle specificity. We asked how the SNARE complement evolved across the trypanosomatids. An in silico search of the predicted proteomes of T. b. brucei and T. cruzi was used to identify candidate SNARE sequences. Phylogenetic analysis, including comparisons with yeast and human SNAREs, allowed assignment of trypanosomatid SNAREs to the Q or R subclass, as well as identification of several SNAREs orthologous with those of opisthokonts. Only limited variation in number and identity of SNAREs was found, with Leishmania major having 27 and T. brucei 26, suggesting a stable SNARE complement post-speciation. Expression analysis of T. brucei SNAREs revealed significant differential expression between mammalian and insect infective forms, especially within R and Qb-SNARE subclasses, suggesting possible roles in adaptation to different environments. For trypanosome SNAREs with clear orthologs in opisthokonts, the subcellular localization of TbVAMP7C is endosomal while both TbSyn5 and TbSyn16B are at the Golgi complex, which suggests conservation of localization and possibly also function. Despite highly distinct life styles, the complement of trypanosomatid SNAREs is quite stable between the three pathogenic lineages, suggesting establishment in the last common ancestor of trypanosomes and Leishmania. Developmental changes to SNARE mRNA levels between blood steam and procyclic life stages suggest that trypanosomes modulate SNARE functions via expression. Finally, the locations of some conserved SNAREs have been retained across the eukaryotic lineage.     

Transient Superdiffusion and Long-Range Correlations in the Motility Patterns of Trypanosomatid Flagellate Protozoa

We report on a diffusive analysis of the motion of flagellate protozoa species. These parasites are the etiological agents of neglected tropical diseases: leishmaniasis caused by Leishmania amazonensis and Leishmania braziliensis, African sleeping sickness caused by Trypanosoma brucei, and Chagas disease caused by Trypanosoma cruzi. By tracking the positions of these parasites and evaluating the variance related to the radial positions, we find that their motions are characterized by a short-time transient superdiffusive behavior. Also, the probability distributions of the radial positions are self-similar and can be approximated by a stretched Gaussian distribution. We further investigate the probability distributions of the radial velocities of individual trajectories. Among several candidates, we find that the generalized gamma distribution shows a good agreement with these distributions. The velocity time series have long-range correlations, displaying a strong persistent behavior (Hurst exponents close to one). The prevalence of “universal” patterns across all analyzed species indicates that similar mechanisms may be ruling the motion of these parasites, despite their differences in morphological traits. In addition, further analysis of these patterns could become a useful tool for investigating the activity of new candidate drugs against these and others neglected tropical diseases.     

The Acidocalcisome Vacuolar Transporter Chaperone 4 Catalyzes the Synthesis of Polyphosphate in Insect‐stages of Trypanosoma brucei and T. cruzi

Polyphosphate is a polymer of inorganic phosphate found in both prokaryotes and eukaryotes. Polyphosphate typically accumulates in acidic, calcium‐rich organelles known as acidocalcisomes, and recent research demonstrated that vacuolar transporter chaperone 4 catalyzes its synthesis in yeast. The human pathogens Trypanosoma brucei and T. cruzi possess vacuolar transporter chaperone 4 homologs. We demonstrate that T. cruzi vacuolar transporter chaperone 4 localizes to acidocalcisomes of epimastigotes by immunofluorescence and immuno‐electron microscopy and that the recombinant catalytic region of the T. cruzi enzyme is a polyphosphate kinase. RNA interference of the T. brucei enzyme in procyclic form parasites reduced short chain polyphosphate levels and resulted in accumulation of pyrophosphate. These results suggest that this trypanosome enzyme is an important component of a polyphosphate synthase complex that utilizes ATP to synthesize and translocate polyphosphate to acidocalcisomes in insect stages of these parasites.     

New Pyrano-4H-benzo[g]chromene-5,10-diones with Antiparasitic and Antioxidant Activities

New pyranonaphthoquinone derivatives were synthesized and investigated for their activity against Trypanosoma brucei, Leishmania major, and Toxoplasma gondii parasites. The pentafluorophenyl derivative was efficacious against T. brucei with single digit micromolar EC₅₀ values and against T. gondii with even sub-micromolar values. The 3-chloro-4,5-dimethoxyphenyl derivative showed an activity against amastigotes of Leishmania major parasites comparable to that of amphotericin B. In addition, antioxidant activities were observed for the bromophenyl derivatives, and their redox behavior was studied by cyclovoltammetry. Anti-parasitic and antioxidative activities of the new naphthoquinone derivatives appear uncorrelated.     

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.     

Trypanosoma cruzi: defined medium for continuous cultivation of virulent parasites.

A liquid medium was developed for the continuous cultivation of Trypanosoma cruzi. Among the several highly purified macromolecules tested only bovine liver catalase, horseradish peroxidase, lactoperoxidase, and bovine hemoglobin supported the continuous growth, at high yield, of mice-virulent Trypanosoma cruzi; other hemoproteins were inactive. Bovine liver catalase showed optimal Trypanosoma cruzi growth-promoting activity, parasites reaching 20 × 10⁶ parasites/ml (95% epimastigotes) at about 10 days in most of the 45 subpassages to date. Furthermore, this protein in the incubation medium provided all the amino acid requirements of actively growing parasites, thus eliminating the need for exogeneous free amino acids. Additional experiments revealed that the hemoprotein's growth-promoting activity was independent of any enzymatic activity and that reconstituting the exact protein composition by means of exogeneous amino acids did not support parasite multiplication, suggesting the importance of the primary structure of the active proteins for growth-promoting activity. These active macromolecules supported the multiplication of five different strains of Trypanosoma cruzi, but did not support Leishmania brasiliensis or Leishmania mexicana proliferation, suggesting species specificity.     

Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)

Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major), insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis) and plants (Phytomonas serpens). A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids.