Trypanosoma cruzi surface mucins with exposed variant epitopes.

The protozoan parasite Trypanosoma cruzi, the agent of Chagas disease, has a large number of mucin molecules on its surface, whose expression is regulated during the life cycle. These mucins are the main acceptors of sialic acid, a monosaccharide that is required by the parasite to infect and survive in the mammalian host. A large mucin-like gene family named TcMUC containing about 500 members has been identified previously in T. cruzi. TcMUC can be divided into two subfamilies according to the presence or absence of tandem repeats in the central region of the genes. In this work, T. cruzi parasites were transfected with one tagged member of each subfamily. Only the product from the gene with repeats was highly O-glycosylated in vivo. The O-linked oligosaccharides consisted mainly of beta-d-Galp(1-->4)GlcNAc and beta-d-Galp(1-->4)[beta-d-Galp(1-->6)]-d-GlcNAc. The same glycosyl moieties were found in endogenous mucins. The mature product was anchored by glycosylphosphatidylinositol to the plasma membrane and exposed to the medium. Sera from infected mice recognized the recombinant product of one repeats-containing gene thus showing that they are expressed during the infection. TcMUC genes encode a hypervariable region at the N terminus. We now show that the hypervariable region is indeed present in the exposed mature N termini of the mucins because sera from infected hosts recognized peptides having sequences from this region. The results are discussed in comparison with the mucins from the insect stages of the parasite (Di Noia, J. M., D'Orso, I., Sánchez, D. O., and Frasch, A. C. C. (2000) J. Biol. Chem. 275, 10218-10227) which do not have variable regions.

Vector development for the expression of foreign proteins in the vaccine strain Brucella abortus S19.

A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae.

High diversity in mucin genes and mucin molecules in Trypanosoma cruzi.

Mucins are highly O-glycosylated molecules which in mammalian cells accomplish essential functions, like cytoprotection and cell-cell interactions. In the protozoan parasite Trypanosoma cruzi, mucin-related glycoproteins have been shown to play a relevant role in the interaction with and invasion of host cells. We have previously reported a family of mucin-like genes in T. cruzi whose overall structure resembled that of mammalian mucin genes. We have now analyzed the relationship between these genes and mucin proteins. A monoclonal antibody specific for a mucin sugar epitope and a polyclonal serum directed to peptide epitopes in a MUC gene-encoded recombinant protein, detected identical bands in three out of seven strains of T. cruzi. Immunoprecipitation experiments confirmed these results. When expressed in eukaryotic cells, the MUC gene product is post-translationally modified, most likely, through extensive O-glycosylation. Gene sequencing showed that the central domains encoding the repeated sequences with the consensus T8KP2, varies in number from 1 to 10, and the number of Thr residues in each repeat could be 7, 8, or 10. A run of 16 to 18 Thr residues was present in some, but not all, MUC gene-derived sequences. Direct compositional analysis of mucin core proteins showed that Thr residues are much more frequent than Ser residues. The same fact occurs in MUC gene-derived protein sequences. Molecular mass determinations of the 35-kDa glycoproteins further extend the heterogeneity of the family to the natural mucin molecules. Difficulties in assigning each of the several MUC genes identified to a mucin product arise from the high diversity and partial sequence conservation of the members of this family.

A putative pyruvate dehydrogenase alpha subunit gene from Trypanosoma cruzi.

A full-length DNA clone encoding a putative pyruvate dehydrogenase alpha subunit (E1 alpha) gene was isolated from a Trypanosoma cruzi (RA strain) DNA library. Sequencing of this clone revealed it to encode a 378 amino acid protein (M(r) 42774) with high sequence similarity to E1 alpha obtained from different sources. The highest score is obtained with human E1 alpha: 43,3% similarity. Southern blot analysis is consistent with the existence of a single copy of this putative T. cruzi E1 alpha gene per haploid genome in different parasite strains. Expression of this gene was demonstrated by Northern blot analysis and its trans-splicing acceptor site was identified by Polymerase Chain Reaction-mediated amplification of its cDNA.

Effect of primary structure modifications in Trypanosoma cruzi neuraminidase/trans-sialidase activities.

Neuraminidases have been implicated in various processes involving the interaction of pathogens and their receptor cells. Trypanosoma cruzi, the agent of Chagas disease, has an unusual neuraminidase, able to transfer bound alpha(2-3)sialic acid to a suitable acceptor rather than to water: the trans-sialidase (TcTS). This enzyme is encoded by a family of several homologous genes, some of them rendering inactive the products. We have shown that enzymatically active proteins have Tyr in position 342, whereas inactive TcTS contain a His342. We have now mutated this Tyr residue to Phe or Thr. Both mutant proteins resulted in enzymatically inactive products. Chimeras expressing parts of Salmonella typhimurium neuraminidase (NANH) and TcTS were constructed. Only the construct containing the complete NANH molecule fused to the last 272 residues of TcTS had neuraminidase activity. However this construct did not present TcTS activity. This finding suggests that other residues present in the homology region are required for TcTS activity.

Direct detection of Vibrio cholerae in stool samples.

A direct method to detect Vibrio cholerae in stool samples was developed by using a PCR procedure that did not require a DNA purification step. Dilution (1/100) of stool samples prevented inhibition of the reaction by contaminants, and two consecutive PCRs, the second one with a nested primer, achieved the desired sensitivity. Comparison of the results obtained from stool swab samples processed by the two-step PCR and by an enzyme-linked immunosorbent assay using GM1 as the capture molecule showed that the former is more sensitive and gave positive results even when V. cholerae was not culturable or dead.

An unusually small gene encoding a putative mucin-like glycoprotein in Trypanosoma cruzi.

The gene encoding a putative core protein of a mucin-like glycoprotein was identified in Trypanosoma cruzi. It contains five repeats of eleven amino acids each, eight of which are Thr and two of which are Pro residues. These Thr-Pro-rich repeats resemble the ones in the human MUC2 gene encoding mucin.

Identification of the gene(s) coding for the trans-sialidase of Trypanosoma cruzi.

The gene(s) encoding the Trypanosoma cruzi shed-acute-phase-antigen (SAPA) has a 5' end encoding a region containing two totally and two partially conserved Ser-X-Asp-X-Gly-X-Thr-Trp motifs which are present in bacterial neuraminidases, and a 3' end encoding tandemly repeated units of 12 amino acids. It is now reported that 54-87% of the total neuraminidase activity present in the parasite could be immunoprecipitated with polyclonal or monoclonal antibodies against the repeated amino acid units of SAPA. These immunoprecipitates also had greater than 80% of the trans-sialidase activity of the parasite. SAPA used sialyllactose, fetuin and 4-methylumbelliferyl-sialic acid as substrate donors. In the presence of a suitable acceptor molecule (lactose) the sialic acid residues were transferred to the disaccharide, whereas in the absence of acceptors the residues were transferred to water. If relatively inefficient acceptors (maltose or cellobiose) were added to the incubation mixtures, the sialic acid units were transferred both to the disaccharides and to water. It is concluded that a major T. cruzi antigen has both the trans-sialidase and the neuraminidase activities of the parasite. Both activities are probably located on the N-terminus of SAPA since antibodies directed against the C-terminus, which contains the repeated amino acid units, do not affect the enzymatic activities.

The expression of the major shed Trypanosoma cruzi antigen results from the developmentally-regulated transcription of a small gene family.

To better understand the mechanisms involved in the developmental expression of Trypanosoma cruzi antigens we examined the gene structure and transcription properties of the major shed trypomastigote antigen (SAPA). We report in this paper that SAPA is encoded by a small family of at lest 6 genes which differ mainly in the length of a repeat region made up of tandemly arranged 36-bp repeat units. SAPA genes are located distant from chromosomal telomeres as inferred from their insensitivity to Bal31 nuclease treatment. Furthermore, Northern blot and S1 protection analyses strongly support the fact that most (or all) SAPA genes are transcribed in the infective form of the parasite.

Variable number of repeat units in genes encoding Trypanosoma cruzi antigens.

The genes encoding a protein antigenic during the acute phase (SAPA) and another one antigenic during the chronic phase (antigen 30) of human Chagas' disease were analyzed in 15 Trypanosoma cruzi isolates and clones collected in distant geographical regions. These two genes had tandem repeats which were present in all parasites tested. However, large differences in the length of restriction fragments were observed among isolates. This was readily explained by variations in the number of repeat units present in homologous genes. This result was confirmed after analysis of 3 members of the SAPA gene family. In the case of antigen 30, we propose that differences in the number of repeat units result in size differences in the corresponding RNAs and proteins, which explains the large size heterogeneity in otherwise homologous T. cruzi antigens.