Trypanosomatid protozoa: survey of acetylornithinase and ornithine acetyltransferase

Trypanosomatid protozoa (Crithidia deanei, C. deanei aposymbiotic, C. oncopelti, C. fasciculata, C. acanthocephali, Leptomonas seymouri, L. collosoma, L. samueli, Herpetomonas samuelpessoai, H. sp., H. megaseliae, H. muscarum muscarum, Leishmania donovani, L. braziliensis, Trypanosoma cruzi, T. conorhini and T. mega) were examined for the presence of acetylornithinase (EC 3.5.1.16) and ornithine acetyltransferase (EC 2.3.1.35). As a rule, species of the genus Crithidia presented one of the two enzymes for the conversion of acetylornithine into ornithine. Crithidia fasciculata and C. acanthocephali presented acetylornithinase, while C. deanei and C. oncopelti, species harboring symbionts, presented ornithine acetyltransferase. The enzyme was absent in the aposymbiotic strain of C. deanei, which suggests that the enzyme belongs to the symbiont. Among the other trypanosomatids examined only Herpetomonas samuelpessoai presented acetylomithinase. The participation of acetylornithinase and ornithine acetyltransferase in the metabolism of trypanosomatids is discussed in the light of their nutritional requirements and possession of enzymes of the arginineornithine metabolism.     

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.     

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.     

Influence of leishmanolysin-like molecules of Herpetomonas samuelpessoai on the interaction with macrophages

Monoxenous trypanosomatids usually have an invertebrate as the only host in their life cycles, however, they have been found repeatedly in plants and/or mammals. To succeed in colonizing a vertebrate host, the parasite must quickly adapt to drastic changes in the environment (e.g. temperature), which reflect the conditions found in the insect and mammalian hosts. Leishmanolysin is a metalloprotease ubiquitously distributed in trypanosomatids, playing a myriad of functions. In Herpetomonas samuelpessoai, an insect trypanosomatid, the leishmanolysin-like molecule was implicated in the nutrition and insect adhesion. Herein, we showed that leishmanolysin expression is equally expressed in H. samuelpessoai parasites submitted to insect (26 °C) and mammalian (37 °C) temperatures. Also, the parasites grown in both temperatures interacted at similar rates with macrophages. Finally, we showed that leishmanolysin is involved in crucial steps in the interaction of H. samuelpessoai cells with macrophages, since the treatment with either anti-leishmanolysin antibodies or metalloprotease inhibitor 1,10-phenanthroline significantly reduced the association index. Similarly, the treatment of the macrophages with purified leishmanolysin promoted a powerful reduction in the association index, suggesting the direct involvement of macrophage receptors. These results suggest that H. samuelpessoai leishmanolysin molecules are not modulated by temperature and are involved in the interaction with mammalian cells.     

Infection rates and pathogenicity of trypanosomatid gut parasites in the water strider Gerris odontogaster (Zett.) (Heteroptera: Gerridae)

Summary Trypanosomatid flagellates are common protozoan gut parasites of a wide range of insect species. Water striders (Gerridae) harbour the trypanosomatid Blastocrithidia gerridis. Three different populations of the water strider Gerris odontogaster in northern Sweden were sampled to assess the infection rate dynamics of trypanosomatids. The initially very low infection rates (0%–15%) early in the season were followed by a rapid increase during the reproductive period of the water striders, reaching very high levels (80%–90%). The pathogenic effects of trypanosomatids on G. odontogaster adults were studied in laboratory experiments. The parasites caused a general reduction of host vigour. Male skating endurance was negatively correlated with the intensity of the trypanosomatid infection. However, infection of trypanosomatids increased the mortality among adults only when the water striders were subjected to food stress. The trypanosomatids did not reduce the fecundity of females provided with food. We suggest that trypanosomatid gut parasites may be an important mortality factor in water strider populations. Since the pathogenicity of the parasites is enhanced by stress, parasitism by trypanosomatids may contribute to the regulation of host populations.     

Molecular Epidemiology of Trypanosomatids and <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> in Primates from Peru

We determined the prevalence rate and risk of infection of Trypanosoma cruzi and other trypanosomatids in Peruvian non-human primates (NHPs) in the wild (n = 126) and in different captive conditions (n = 183). Blood samples were collected on filter paper, FTA cards, or EDTA tubes and tested using a nested PCR protocol targeting the 24Sα rRNA gene. Main risk factors associated with trypanosomatid and T. cruzi infection were genus and the human–animal context (wild vs captive animals). Wild NHPs had higher prevalence of both trypanosomatids (64.3 vs 27.9%, P < 0.001) and T. cruzi (8.7 vs 3.3%, P = 0.057), compared to captive NHPs, suggesting that parasite transmission in NHPs occurs more actively in the sylvatic cycle. In terms of primate family, Pitheciidae had the highest trypanosomatid prevalence (20/22, 90.9%) and Cebidae had the highest T. cruzi prevalence (15/117, 12.8%). T. cruzi and trypanosomatids are common in Peruvian NHPs and could pose a health risk to human and animals that has not been properly studied.     

Developmentally Regulated Protein Expression Mediated by Dimethylsulfoxide in Herpetomonas samuelpessoai

We have analyzed the total cell extract, cell surface, and secretory protein profiles related to cellular differentiation triggered by dimethylsulfoxide in the insect trypanosomatid Herpetomonas samuelpessoai. The flagellates were cultivated in chemically defined conditions in the absence or in the presence of 4% DMSO, and the resolved protein bands were detected by SDS-PAGE gels and avidin-Western blotting. The cell-associated proteins showed a complex pattern of around 40 silver-staining bands ranging from 15 to 150 kDa. There were generally minor quantitative differences in the protein profile between the non-treated and the DMSO-treated cells. The cell-surface protein profile revealed by the incubation of live parasites with biotin showed a decrease in the expression of the 120 kDa biotinylated polypeptide observed in the DMSO-treated cells when compared with untreated ones. However, control samples of both systems showed an endogenous biotinylated polypeptide of 63 kDa which also presented gelatinolytic activity. The trypanosomatids released at least 10 polypeptides to the culture medium. A low molecular mass exopolypeptide (35 kDa) was found exclusively in untreated cells, whereas a high-molecular-mass exopolypetide (250 kDa) was preferentially found in DMSO-treated cells. Received: 12 July 2000 / Accepted: 14 August 2000     

Genome of the Avirulent Human-Infective Trypanosome—Trypanosoma rangeli

Background

Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.

Methodology/Principal Findings

The T. rangeli haploid genome is ∼24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heat-shock proteins.

Conclusions/Significance

Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets.     

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.     

Isolation and Properties of Flagella of Trypanosomatids*

SYNOPSIS. A procedure is described for the isolation of flagella of Crithidia fasciculata. Herpetomonas samuelpessoai and Leishmania tarentolae in a highly purified state and giving reasonably good yield. The 3 types of flagella give a similar electrophoretic pattern of proteins. It is shown that H. samuelpessoai and, to a lesser extent, C. fasciculata flagella confer protection against Trypanosoma cruzi infection.     

<Emphasis Type="Italic">Herpetomonas samuelpessoai:</Emphasis> Dimethylsulfoxide-Induced Differentiation Is Influenced by Proteinase Expression

In this study, we analyzed the influence of proteinase expression on the cellular differentiation of Herpetomonas samuelpessoai. Along cellular differentiation, which was induced by dimethylsulfoxide (DMSO), the trypanosomatids secreted several molecules with variable proteolytic activity. All of them were inhibited by 10 mM 1,10-phenanthroline, suggesting that they are zinc-metalloproteinases. Analysis of parasite extracts revealed the occurrence of a 63-kDa metalloproteinase and a 45-kDa cysteine proteinase. After extraction with Triton X-114 followed by water-detergent partition, the 63-kDa component was present in both aqueous and detergent phases, which indicated that this enzyme may be distributed over different cellular compartments including membrane domains. The 45-kDa component, however, presented hydrophilic properties and was predominantly expressed by DMSO non-treated parasites, suggesting that proteinases may be involved in the process of cellular differentiation in H. samuelpessoai. This was confirmed by the fact that a cysteine proteinase inhibitor abrogated parasite differentiation. The role of proteinases and their relevance in the differentiation of H. samuelpessoai are discussed. Received: 15 February 2002 / Accepted: 27 March 2002     

A fluorescence-based reporter substrate for monitoring RNA editing in trypanosomatid pathogens

RNA editing regulates mitochondrial gene expression in trypanosomatid pathogens by creating functional mRNAs. It is catalyzed by a multi-protein complex (the editosome), and is found to be essential in both insect stage and mammalian blood stream form of Trypanosoma brucei. This particular form of RNA editing is unique to trypanosomatids, and thus provides a suitable drug target in trypanosomatid pathogens. Here, we demonstrate the feasibility of a rapid and sensitive fluorescence-based reporter assay to monitor RNA editing based on ribozyme activity. We could validate our new assay using previously identified inhibitors against the essential RNA editing ligase. The principle advantages of this assay are: (i) the use of non-radioactively labeled materials, (ii) sensitivity afforded by fluorescence instrumentation applicable to high-throughput screening of chemical inhibitors against the essential editosome and (iii) a rapid and convenient ‘mix and measure’ type of assay in low volume with a high signal to noise ratio. This assay should enhance rapid identification and characterization of the editosome inhibitors primarily based on the overall composition of the editosomes from T. brucei. These inhibitors could also be tested against the editosomes from the closely related pathogens including T. cruzi and Leishmania species.     

Genetically different isolates of Trypanosoma cruzi elicit different infection dynamics in raccoons (Procyon lotor) and Virginia opossums (Didelphis virginiana)

Trypanosoma cruzi is a genetically and biologically diverse species. In the current study we determined T. cruzi infection dynamics in two common North American reservoirs, Virginia opossums (Didelphis virginiana) and raccoons (Procyon lotor). Based on previous molecular and culture data from naturally-exposed animals, we hypothesised that raccoons would have a longer patent period than opossums, and raccoons would be competent reservoirs for both genotypes T. cruzi I (TcI) and TcIIa, while opossums would only serve as hosts for TcI. Individuals (n = 2 or 3) of each species were inoculated with 1 × 10⁶ culture-derived T. cruzi trypomastigotes of TcIIa (North American (NA) – raccoon), TcI (NA – opossum), TcIIb (South American – human), or both TcI and TcIIa. Parasitemias in opossums gradually increased and declined rapidly, whereas parasitemias peaked sooner in raccoons and they maintained relatively high parasitemia for 5 weeks. Raccoons became infected with all three T. cruzi strains, while opossums only became infected with TcI and TcIIb. Although opossums were susceptible to TcIIb, infection dynamics were dramatically different compared with TcI. Opossums inoculated with TcIIb seroconverted, but parasitemia duration was short and only detectable by PCR. In addition, raccoons seroconverted sooner (3–7 days post inoculation) than opossums (10 days post inoculation). These data suggest that infection dynamics of various T. cruzi strains can differ considerably in different wildlife hosts.     

Detection of matrix metallopeptidase-9-like proteins in Trypanosoma cruzi

In this study, the cell-associated and extracellular peptidases of Trypanosoma cruzi grown in modified Roitman’s complex (MRC) medium were analyzed by measuring peptidase activity in gelatin-containing zymograms. Our results showed that the cell-associated peptidases as well as peptidases extracellularly released by T. cruzi displayed two distinct proteolytic classes: cysteine and metallopeptidase activities. The major cysteine peptidase, cruzipain, synthesized by T. cruzi cells was detected in cellular parasite content, as a 50 kDa reactive polypeptide, after probing with anti-cruzipain antibody. In addition, metallo-type peptidases belonging to the matrix metallopeptidase-9 (MMP-9) family were revealed, after Western blotting, as a 97 kDa protein band in cellular extract and an 85 kDa polypeptide in both cellular and secreted parasite extracts. The MMP-9-like activity present in cells and spent culture medium was immunoprecipitated by an anti-MMP-9 polyclonal antibody. The surface location of MMP-9-like proteins in T. cruzi was also evidenced by means of flow cytometry analysis. Furthermore, doxycycline that has direct MMP-9 inhibiting properties in vitro, inhibited MMP-9-like activities in gel zymography, immunoprecipitation and flow cytometry analyses. This is the first report of the presence of MMP-9-like molecules in T. cruzi. The presence of a matrix extracellular-degrading enzyme may play a role in the T. cruzi-host cell interaction, making this enzyme a potential target for future drug development against this pathogenic trypanosomatid.     

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.     

Evidence for an association of the early light-inducible protein (ELIP) of pea with photosystem II

The precursor to the nuclear-coded 17 kDa early light-inducible protein (ELIP) of pea has been transported into isolated intact chloroplasts. The location of the mature protein in the thylakoid membranes was investigated after using cleavable crosslinkers such as DSP and SAND in conjunction with immuno-fractionation methods and by application of mild detergent fractionation. We show that ELIP is integrated into the membranes via the unstacked stroma thylakoids. After isolation of protein complexes by solubilization of membranes with Triton X-100 and sucrose density-gradient centrifugation the crosslinked ELIP comigrates with the PS II core complex. Using SAND we identified ELIP as a 41–51 kDa crosslinked product while with DSP four products of 80 kDa, 70 kDa, 50–42 kDa and 23–21 kDa were found. The immunoprecipitation data suggested that the D₁-protein of the PS II complex is one of the ELIP partners in crosslinked products.Abbreviations chl chlorophyll - D₁ herbicide-binding protein - DSP dithiobis-(succinimidylpropionate) - ELIP early light-inducible protein - LHC I and LHC II light-harvesting chlorophyll a/b complex associated with photosystem I or II - PAGE polyacrylamide gel electrophoresis - poly(A)-rich RNA polyadenyd mRNA - PS I and PS II photosystems I and II - SAND sulfosuccinimidyl 2-(m-azido-o-nitro-benzamido)-ethyl-1,3-dithiopropionate - Triton X-100 octylphenoxypolyethoxyethanol     

Detection of Weakly Expressed Trypanosoma cruzi Membrane Proteins Using High‐Performance Probes

Membrane proteins in trypanosomatids are, in general, weakly expressed and confirmation of their subcellular localization frequently requires their overexpression with epitope tags. However, overexpression can lead to mislocalization of the probes. Viswanathan et al. (Nat. Methods, 2015, 12:568) described high performance tags for localization of weakly expressed proteins. We report here the use of these protein tags, named “spaghetti monster,” for CRISPR/Cas9‐mediated C‐terminal endogenous tagging of Trypanosoma cruzi to localize two weakly expressed transient receptor potential channels to acidic compartments. The results indicate that this method will improve the detection of membrane proteins in T. cruzi.