Functional diversity in the trans-sialidase and mucin families in Trypanosoma cruzi

Trypanosomes are unable to synthesize the monosaccharide sialic acid, but some African trypanosomes and the American Trypanosoma cruzi can incorporate sialic acid derived from the host. To do so, T. cruzi expresses a trans-sialidase, an enzyme that catalyzes the transfer of sialic acid from host glycoconjugates to mucin-like molecules located on the parasite surface membrane. The importance of the process is indicated by the fact that T. cruzi has hundreds of genes encoding trans-sialidase, trans-sialidase-like proteins and mucin core proteins. Sequence divergence of members of these families has resulted in some molecules having functions unrelated to the acquisition of sialic acid. In this article, Alberto Frasch reviews the structure and possible function of the proteins making up these families.     

The tubulin genes of Trypanosoma cruzi

The organization of the alpha- and beta-tubulin genes in the genome of Trypanosoma cruzi have been analysed by Southern blotting using tubulin probes derived from Trypanosoma brucei. The tubulin array appears to be more complex in this organism than in other members of the same family. Some tubulin genes are tightly clustered in an alternating (alpha-beta)n array with a basic repeat unit length of 4.3 kb. However, other pairs of alternating alpha- and beta-tubulin sequences appear to be physically separated from the basic group. This finding indicates that the tubulin gene cluster present in T. cruzi is less perfectly conserved than in T. brucei. T. (Herpetosoma) rangeli is similar to T. (Schizotrypanum) cruzi in its tubulin gene organization whereas most of these genes are tandemly clustered in the genome of T. (Trypanozoon) evansi, with a basic repeat unit length of 3.6 kb as previously described for T. (Trypanozoon) brucei. Two overlapping recombinant clones containing T. cruzi tubulin sequences have been isolated from a genomic cosmid library of T. cruzi epimastigotes using the T. brucei tubulin probes. Partial sequencing of the T. cruzi beta-tubulin gene has confirmed its identity and shows more than 70% homology with the sea urchin, chicken and T. b. rhodesiense beta-tubulin reported gene sequences. Analysis of tubulin gene organization through the parasite life cycle does not show evidence of major rearrangements within the repeat unit. Several T. cruzi strains and cloned lines whilst sharing the 4.3-kb tubulin repeat unit, exhibited very variable tubulin gene organization with tubulin probes. These striking differences in the organization of this structural gene among T. cruzi strains and cloned lines suggest that the heterogeneity previously reported in parasite populations may be related to a very dynamic, diploid genome.     

Trypanosoma cruzi clonal diversity and the epidemiology of Chagas' disease

Chagas' disease is caused by the protozoan Trypanosoma cruzi and it has a variable clinical outcome. The basis for this variability relies in part on the complexity of the parasite population consisting of multiple clones displaying distinct biological properties. A major current challenge is to correlate parasite genetic variability with pathogenesis.     

Minicircle organization and diversity in Trypanosoma cruzi populations

Trypanosoma cruzi strains and isolates can be divided into at least two groups using biochemical and molecular markers such as isoenzymes, ribosomal DNA, mini-exon gene spacers and some maxicircle genes. Despite the accumulating evidence that these major groups are phylogenetically distinct, their kinetoplast minicircle overall organization (i.e. number of conserved regions per length of minicircle molecule) remains conserved in all T. cruzi isolates studied so far, including the two T. cruzi major lineages -T. cruzi I and T. cruzi II - and a third group of uncertain taxonomic status, T. cruzi ZIII. Thus far, despite the extensive intra- and inter-minicircle sequence polymorphism, no group clustering has been observed.     

Comparison of genes encoding Trypanosoma cruzi antigens

Trypanosoma cruzi, the causative agent of Chagas disease, simultaneously expresses several different surface antigens. This contrasts sharply with blood-stream forms o f the African trypanosomes, which display only one variant surface glycoprotein at a time. Over the past few years, the genes coding for a number of T. cruzi proteins recognized by sera from patients have been cloned and at least partially sequenced. However, some of those discovered in more than one laboratory have been given different names. Here, Carlos Frasch, Juan Cazzulo, Lena Aslund and UIf Pettersson try to systematize the literature available on these antigens, including what is known about their localization and function.     

Conservation of heat-shock proteins in Trypanosoma cruzi

Heat shock is an integral part of the life cycle of Trypanosoma cruzi. Here, Edson Rondinelli reviews the parasite's response to stress.     

Relevance of the diversity among members of the Trypanosoma cruzi trans-sialidase family analyzed with camelids single-domain antibodies

The sialic acid present in the protective surface mucin coat of Trypanosoma cruzi is added by a membrane anchored trans-sialidase (TcTS), a modified sialidase that is expressed from a large gene family. In this work, we analyzed single domain camelid antibodies produced against trans-sialidase. Llamas were immunized with a recombinant trans-sialidase and inhibitory single-domain antibody fragments were obtained by phage display selection, taking advantage of a screening strategy using an inhibition test instead of the classic binding assay. Four single domain antibodies displaying strong trans-sialidase inhibition activity against the recombinant enzyme were identified. They share the same complementarity-determining region 3 length (17 residues) and have very similar sequences. This result indicates that they likely derived from a unique clone. Probably there is only one structural solution for tight binding inhibitory antibodies against the TcTS used for immunization. To our surprise, this single domain antibody that inhibits the recombinant TcTS, failed to inhibit the enzymatic activity present in parasite extracts. Analysis of individual recombinant trans-sialidases showed that enzymes expressed from different genes were inhibited to different extents (from 8 to 98%) by the llama antibodies. Amino acid changes at key positions are likely to be responsible for the differences in inhibition found among the recombinant enzymes. These results suggest that the presence of a large and diverse trans-sialidase family might be required to prevent the inhibitory response against this essential enzyme and might thus constitute a novel strategy of T. cruzi to evade the host immune system.     

Trypanosoma cruzi TcSMUG L-surface Mucins Promote Development and Infectivity in the Triatomine Vector Rhodnius prolixus

Background

TcSMUG L products were recently identified as novel mucin-type glycoconjugates restricted to the surface of insect-dwelling epimastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. The remarkable conservation of their predicted mature N-terminal region, which is exposed to the extracellular milieu, suggests that TcSMUG L products may be involved in structural and/or functional aspects of the interaction with the insect vector.

Methodology and Principal Findings

Here, we investigated the putative roles of TcSMUG L mucins in both in vivo development and ex vivo attachment of epimastigotes to the luminal surface of the digestive tract of Rhodnius prolixus. Our results indicate that the exogenous addition of TcSMUG L N-terminal peptide, but not control T. cruzi mucin peptides, to the infected bloodmeal inhibited the development of parasites in R. prolixus in a dose-dependent manner. Pre-incubation of insect midguts with the TcSMUG L peptide impaired the ex vivo attachment of epimastigotes to the luminal surface epithelium, likely by competing out TcSMUG L binding sites on the luminal surface of the posterior midgut, as revealed by fluorescence microscopy.

Conclusion and Significance

Together, these observations indicate that TcSMUG L mucins are a determinant of both adhesion of T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine, and the infection of the insect vector, R. prolixus.     

HMG-like chromosomal proteins in Trypanosoma cruzi.

HMG-like chromosomal proteins from Trypanosoma cruzi were studied. Four HMG-like proteins, designated HMG A, HMG-B, HMG-C, and HMG-E, were isolated and found to have molecular weights of 35.5 kd, 27.5 kd, 21.8 kd and 10.4 kd, respectively. Immunological relatedness was demonstrated between the mammalian HMG 1,2 and the HMG-A and HMG-B from T. cruzi. The relative amounts of HMG-C and HMG-E proteins vary in T. cruzi depending to the proliferative stage of the cells. HMG-E protein is increased in proliferating cells when compared to its level in non-proliferating cells. HMG-C is increased in the non-proliferating cells. Probably, the shifts observed in the relative amounts of HMG-like proteins are related to the proliferating cells of this flagellate. The results are consistent with those described for other lower eukaryotes where the HMG-like proteins isolated are similar but not identical to HMG proteins from vertebrates.     

Trypanosoma cruzi surface mucins: host-dependent coat diversity

The surface of the protozoan parasite Trypanosoma cruzi is covered in mucins, which contribute to parasite protection and to the establishment of a persistent infection. Their importance is highlighted by the fact that the approximately 850 mucin-encoding genes comprise approximately 1% of the parasite genome and approximately 6% of all predicted T. cruzi genes. The coordinate expression of a large repertoire of mucins containing variable regions in the mammal-dwelling stages of the T. cruzi life cycle suggests a possible strategy to thwart the host immune response. Here, we discuss the expression profiling of T. cruzi mucins, the mechanisms leading to the acquisition of mucin diversity and the possible consequences of a mosaic surface coat in the interplay between parasite and host.     

Isolation and characterization of paraflagellar proteins from Trypanosoma cruzi

Two different Trypanosoma cruzi polypeptides, with masses of 70 and 68 kDa were purified and characterized in this work. These two polypeptides designated PAR 1 and PAR 2, respectively, co-purified during each step of the isolation procedure and were found to be located exclusively in T. cruzi flagella by indirect immunofluorescence. A pre-embedding immunoelectron microscopy procedure, with a gold-tagged secondary antibody, permitted direct identification of PAR 2 as a component of the T. cruzi paraflagellar rod. PAR 1 and PAR 2 were found to be immunologically distinct and showed no cross-reactivity with actin, tubulin, intermediate filament proteins, or other proteins present in mammalian cells. The results presented indicate that PAR 1 and PAR 2 are the major components of T. cruzi paraflagellar filaments, and that these filaments have no counterpart in mammalian cells.     

Identification of contractile vacuole proteins in Trypanosoma cruzi

Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism and provided information on the potential participation of adaptor protein complexes in their biogenesis.     

Trypanosoma cruzi: a gene family encoding chitin-binding-like proteins is posttranscriptionally regulated during metacyclogenesis.

The development of the representation of differential expression method has lead to the cloning of Trypanosoma cruzi stage-specific genes. We used this method to characterize a multicopy gene family differentially expressed during metacyclogenesis. The genomic and cDNA clones sequenced encoded three short cysteine-rich polypeptides, of two types, with predicted molecular masses of 7.1, 10.4, and 10.8 kDa. We searched GenBank for similar sequences and found that the sequences of these clones were similar to that encoding the wheat germ agglutinin protein. The region of similarity corresponds to the chitin-binding domain, with eight similarly positioned half-cysteines and conserved aromatic residues involved in chitin recognition. Multiple copies of the genes of this family are present on a high- molecular-mass chromosome. We studied the expression of genes of this family during metacyclogenesis by determining messenger RNA (mRNA) levels. The mRNAs for the members of this gene family were present in the total RNA fraction but were mobilized to the polysomal fraction of adhered (differentiating) epimastigotes during metacyclogenesis, with a peak of accumulation at 24 of differentiation. Polyclonal antisera were raised against a recombinant protein and a synthetic peptide. The specific sera obtained detected 7- and 11-kDa proteins in T. cruzi total protein extracts. The 11-kDa protein was present in similar amounts in the various cell populations, whereas the 7-kDa protein displayed differential synthesis during metacyclogenesis, with maximal levels in 24-h-adhered (differentiating) epimastigotes.     

Evolutionary history of Trypanosoma cruzi according to antigen genes

Trypanosoma cruzi, the agent of Chagas disease is associated with a very high clinical and epidemiological pleomorphism. This might be better understood through studies on the evolutionary history of the parasite. We explored here the value of antigen genes for the understanding of the evolution within T. cruzi. We selected 11 genes and 12 loci associated with different functions and considered to be involved in host-parasite interaction (cell adhesion, infection, molecular mimicry). The polymorphism of the respective genes in a sample representative of the diversity of T. cruzi was screened by PCR-RFLP and evolutionary relationships were inferred by phenetic analysis. Our results support the classification of T. cruzi in 2 major lineages and 6 discrete typing units (DTUs). The topology of the PCR-RFLP tree was the one that better fitted with the epidemiological features of the different DTUs: (i) lineage I, being encountered in sylvatic as well as domestic transmission cycles, (ii) IIa/c being associated with a sylvatic transmission cycle and (iii) IIb/d/e being associated with a domestic transmission cycle. Our study also supported the hypothesis that the evolutionary history of T. cruzi has been shaped by a series of hybridization events in the framework of a predominant clonal evolution pattern.     

Genetic diversity and kinetic properties of Trypanosoma cruzi dihydroorotate dehydrogenase isoforms

Dihydroorotate dehydrogenase (DHOD) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and is essential in Trypanosoma cruzi, the parasitic protist causing Chagas' disease. T. cruzi and human DHOD have different biochemical properties, including the electron acceptor capacities and cellular localization, suggesting that T. cruzi DHOD may be a potential chemotherapeutic target against Chagas' disease. Here, we report nucleotide sequence polymorphisms of T. cruzi DHOD genes and the kinetic properties of the recombinant enzymes. T. cruzi Tulahuen strain possesses three DHODgenes: DHOD1 and DHOD2, involved in the pyrimidine biosynthetic (pyr) gene cluster on an 800 and a 1000 kb chromosomal DNA, respectively, and DHOD3, located on an 800 kb DNA. The open reading frames of all three DHOD genes are comprised of 942 bp, and encode proteins of 314 amino acids. The three DHOD genes differ by 26 nucleotides, resulting in replacement of 8 amino acid residues. In contrast, all residues critical for constituting the active site are conserved among the three proteins. Recombinant T. cruzi DHOD1 and DHOD2 expressed in E. coli possess similar enzymatic properties, including optimal pH, optimal temperature, Vmax, and Km for dihydroorotate and fumarate. In contrast, DHOD3 had a higher Vmax and Km for both substrates. Orotate competitively inhibited all three DHOD enzymes to a comparable level. These results suggest that, despite their genetic variations, kinetic properties of the three T. cruziDHODs are conserved. Our findings facilitate further exploitation of T. cruzi DHOD inhibitors, as chemotherapeutic agents against Chagas' disease.     

Surface proteins in different isolates of Trypanosoma cruzi epimastigotes

Summary Epimastigotes from several Trypanosoma cruzi stocks were labeled by iodination with Chloramine T and their proteins detected by gel electrophoresis and autoradiography.The labeled proteins from the parasite surface were detected after immunoprecipitation with antisera against fixed trypanosomes or from infected rabbits. These antisera were able to recognize one or more proteins in all T. cruzi isolates analyzed, but the individual patterns differed from each other. Variations in the surface protein patterns were also observed in two Tulahuen stocks kept during several years under different conditions. Growth medium as well as the stage of growth at which the parasites were collected had also an effect upon the relative amount of the observed labeled proteins.