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A C Frasch

Publications and source records attributed to A C Frasch.

At least 37 records · Page 2Linked to original sources
PubMed1998

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.

Amino Acid Sequence↗
PubMed1997

Long-lasting antibodies detected by a trans-sialidase inhibition assay of sera from parasite-free, serologically cured chagasic patients.

A test based on the inhibition by antibodies of the trans-sialidase was used to analyze infection by Trypanosoma cruzi, the agent of Chagas' disease. Sera collected during the longitudinal follow-up of benznidazole-treated acutely and congenitally infected patients became negative for T. cruzi as determined by tests presently used to assess cure; however, the sera remained positive for T. cruzi by the trans-sialidase inhibition assay (TIA) up to 14 years after treatment. Therefore, TIA is a highly sensitive marker for previous T. cruzi infection.

Adult↗
PubMed1997

The trans-sialidase of Trypanosoma cruzi is anchored by two different lipids.

The trans-sialidase from the trypomastigote stage of Trypanosoma cruzi was metabolically labeled with [3H]-palmitic acid and purified by immunoprecipitation with a monoclonal antibody. The action of PI-PLC on the immunoprecipitate released a lipid that was analyzed by TLC. Lyso-1-O-hexadecylglycerol and N-palmitoyl-sphinganine were obtained in a 1:3 ratio. A comparison with the GPI anchors present in the different stages of T. cruzi was made.

Animals↗
PubMed1997

Trypanosoma rangeli sialidase: cloning, expression and similarity to T. cruzi trans-sialidase.

Sialidases are hydrolytic enzymes present from virus to higher eukaryotes, catalyzing the removal of sialic acid from glycoconjugates. Some protozoa Trypanosomatidae secrete high levels of sialidase into the medium. We have now purified the secreted sialidase from Trypanosoma rangeli. Its N-terminal sequence reveals 100% identity with the corresponding region of the trans-sialidase from T. cruzi. Trans-sialidase, although homologous to viral and bacterial sialidases, displays a novel sialyltransferase activity and is involved in host cell invasion. Several homologous transsialidase-like genes were cloned from genomic DNA of T. rangeli, and grouped in three subfamilies. Active sialidase-encoding genes were found in one of them. The recombinant sialidase shows similar properties to those of the native enzyme, including undetectable trans-sialidase activity. Nevertheless, it has an overall identity of 68.9% with the catalytic domain of T. cruzi trans-sialidase, increasing to 86.7% admitting conservative substitutions. Only three other eukaryotic sialidases have been previously cloned, none of them showing significant homology to trans-sialidase. The isolation of a highly similar sialidase is relevant to further identify the molecular determinants allowing trans-sialidase activity. As a first approach, chimeric constructs between sialidase and trans-sialidase were generated, one of them rendering a sialidase with three times lower Km than the natural enzyme.

Amino Acid Sequence↗
PubMed1996

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.

Amino Acids↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

Mapping the Trypanosoma cruzi genome: analyses of representative cosmid libraries.

In order to generate contiguous cosmid coverage of the genome of the protozoan parasite Trypanosoma cruzi for large-scale sequence analysis, a cosmid library of 36864 individual, primary clones was generated. Total genomic DNA of the reference strain CL Brener was fragmented both by partial digestion with MboI and by physical shearing. For cloning, a modified cosmid vector was used that simplifies analyses such as restriction mapping. The library's representation is about 25 genome equivalents, assuming a size of 55 Mb per haploid genome. No chimerism of inserts in the clones could be detected. The colinearity between cosmid inserts and genomic DNA was verified. Also, hybridizations to the gel-separated karyotype of the organism were carried out as a quality check. Gridded onto two nylon filters, the library was analyzed with a variety of probes. Apart from being used for combined physical and transcriptional mapping of the genome, library filters and clones are also available to interested parties.

Animals↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

Medium scale production and purification to homogeneity of a recombinant trans-sialidase from Trypanosoma cruzi.

Trypanosoma cruzi, the agent of Chagas' disease, presents an enzyme that catalyzes the transfer of sialic acid among glycoproteins and glycolipids known as trans-sialidase (TS), displaying some interesting features: 1) It differs from all other eucaryotic sialyltransferases, both kinetically and in substrate specificity and 2) it is involved in the parasite's mechanism of mammalian host cell invasion. We report here the production and purification to homogeneity of an enzymatically active recombinant TS (rTS) lacking the C-terminal amino acid repeats, using iminodiacetic metal affinity chromatography. Initial ratios of non-fusion recombinant versus total protein were very low in several expression systems tested, mainly due to high degradation rate. However, after purifying 1,330 times, we were able to obtain an essentially homogeneous preparation of rTS with a final yield of 29%. After minor changes, a modified protocol for a medium scale production was designed obtaining 0.5 mg of homogeneous rTS per liter of bacterial culture. The purified rTS behaved as a homogeneous protein in silver-stained denaturing gels, isoelectrofocusing and N-terminal sequencing having identical pH and temperature optima as the natural enzyme. Conditions to keep the rTS for long periods without a significant loss of activity were identified.

Animals↗
PubMed1995

The protozoan Trypanosoma cruzi has a family of genes resembling the mucin genes of mammalian cells.

Mucins are heavily O-glycosylated Thr/Ser/Pro-rich molecules. Given their relevant functions, mucins and their genes have been mainly studied in higher eukaryotes. In the protozoan parasite Trypanosoma cruzi, mucin-like glycoproteins were shown to play an important role in the interaction with the surface of the mammalian cell during the invasion process. We show now that this parasite has a family of putative mucin genes, whose organization resembles the one present in mammalian cells. Different parasite isolates have different sets of genes, as defined by their central domain. Central domains, rich in codons for Thr and/or Ser and Pro residues, are made up of either a variable number of repeat units in tandem or non-repetitive sequences. Conversely, 5'- and 3'-ends from different genes in different isolates have similar sequences, suggesting their common origin. Comparison of deduced amino acid sequences revealed that all members of the family have the same putative signal peptide on the N terminus and a putative sequence for glycophosphatidylinositol anchoring on the C terminus. The deduced molecular mass of the core proteins is small (from 17 to 21 kDa), in agreement with the 1-kilobase size of the mRNA detected. Putative mucin genes in T. cruzi are located on large chromosomal bands of about 1.6-2.2 megabase pairs.

Amino Acid Sequence↗
PubMed1995

A single tyrosine differentiates active and inactive Trypanosoma cruzi trans-sialidases.

Several genes encode members of the Trypanosoma cruzi (Tc) trans-sialidase (TS) family. These proteins contain an enzymatic domain on the N terminus, the only one required for TS activity, and an antigenic domain (SAPA (shed acute phase antigen) amino acid (aa) repeats) on the C terminus. Only some members of this glycoprotein family are enzymatically active. The complete sequence of two clones encoding the enzymatic domain of active and inactive protein from each of two Tc strains has now been obtained. Comparison of these sequences showed a limited divergence among them: 20 out of the 642 deduced aa in the enzymatic domain were found to differ. From these 20 aa, only one was found to be essential for enzymatic activity. A Tyr342 residue is deduced in both active proteins while a His342 is present in both inactive ones. This naturally occurring Tyr342-->His substitution completely abolished the TS activity. In addition to Tyr342, a second deduced aa, Pro231, was found to be necessary for full enzymatic TS activity; a Pro231-->Ala change rendered the TS protein partially active. Fourteen aa residues, including Tyr342, out of the 16 aa in the active site of a sialidase from Salmonella typhimurium are present at the same or very similar positions in the Tc TS.

Animals↗
PubMed1995

Chromosome specific markers reveal conserved linkage groups in spite of extensive chromosomal size variation in Trypanosoma cruzi.

The karyotypes of three cloned stocks, CL Brener (CL), CA I/72 (CA) and Sylvio X10/7 (X10), of Trypanosoma cruzi were studied by pulsed-field gel electrophoresis followed by ethidium bromide staining and hybridization with 35 different probes, 30 of which identified single chromosomes. The chromosome-specific probes identified between 26 and 31 chromosomal bands in the three cloned stocks, corresponding to 20 unique chromosomes in CL and 19 in CA and X10. Considering the DNA content of the parasite, it was predicted that the markers recognise at least half of all T. cruzi chromosomes. A majority of identified chromosomes showed large differences in size among different strains, in some cases by up to 50%. Interestingly, CL had in general larger chromosomes than the two other studied cloned stocks. Several of the markers showed linkage and nine different linkage groups were identified, each comprising 2-4 markers. The linkage between the markers was maintained in 8 of the 9 linkage groups when a panel comprising 26 different T. cruzi strains representing major T. cruzi populations was tested. One linkage group was found to be maintained in some strains but not in others. This result shows that chromosomal rearrangements occur in the T. cruzi genome, albeit with a low frequency. Repetitive DNA, both non-coding and in one case coding, was more abundant in the cloned stock CL Brener than in CA and X10. The information presented will make it possible to select chromosomes for the construction of physical chromosomal maps required for the T. cruzi genome project.

Animals↗
PubMed1995

Trypanosoma cruzi exoantigen is a member of a 160 kDa gene family.

During the chronic stage of Chagas disease a 160 kDa antigen appears in the blood of patients and remains detectable many years after the onset of the disease. This antigen is secreted by the trypomastigote form of the parasite while it is undetectable in the epimastigote form. We report here that the chronic 160 kDa exoantigen is encoded by a gene family (CEA 160 family). We describe the cloning and partial nucleotide sequence of a gene (CEA 160-1) belonging to the CEA160 family. Comparison of the gene sequence with other sequences present in the databases revealed homologies with several Trypanosoma cruzi surface antigens. Highest amino acid identity (59%) was with members of a family containing epitopes that mimic nervous tissues (Van Voorhis et al. 1993). Another related group (18-22% amino acid identity) comprises proteins of 85 or 160 kDa sharing an amino acid motif that is conserved among bacterial neuraminidases (Fouts et al. 1991; Pollevick et al. 1991; Kahn et al. 1991; Takle & Cross, 1991; Franco et al. 1993). The amino acid identities with the different antigens were not homogeneously distributed. Regions of higher identity (40-60%) were grouped in the central region of each protein.

Amino Acid Sequence↗
PubMed1994

Trans-sialidase, SAPA amino acid repeats and the relationship between Trypanosoma cruzi and the mammalian host.

During invasion of multicellular organisms, protozoan parasites expose functional molecules that become targets for the host immune response. Recent research on Trypanosoma cruzi, the agent of Chagas' disease, suggests a new model of how the parasite might deal with this problem. Several antigens of T. cruzi have tandemly repeated amino acid motifs in molecules with as yet unknown functions. In two cases, these repeats are in molecules with a defined structure or function. Both proteins are implicated in the invasion of host-cells by the parasite. One of these is the core protein of a putative mucin-like glycoprotein that has Thr/Pro-rich repeats which, by themselves, might define the structure of a highly O-glycosylated molecule. The other protein is SAPA/trans-sialidase/neuraminidase, a molecule able to transfer sialic acid, that has so far only been described in trypanosomes. The amino acid repeats present in SAPA/transsialidase/neuraminidase are unrelated to the enzymic activity and constitute an immunodominant C-terminal domain. The N-terminal domain of SAPA/trans-sialidase/neuraminidase controls the enzymic activity since a recombinant molecule lacking the repeats conserves trans-sialidase activity. That both domains are functionally independent is also indicated by experiments that show that antibodies directed against the amino acid repeats are unable to inhibit trans-sialidase activity. A large number of proteins having trans-sialidase related sequences but lacking enzymic activity are also present in the surface membrane of the parasite. The immunodominant SAPA/trans-sialidase/neuraminidase repeats, together with the complex network of cross-reacting epitopes present in related but enzymatically inactive proteins might contribute to the delay in mounting an effective antibody response.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Antibodies inhibiting Trypanosoma cruzi trans-sialidase activity in sera from human infections.

Trans-sialidase, an enzyme that transfers sialic acid among macromolecules, has been implicated in invasion of host cells by Trypanosoma cruzi, the agent of Chagas' disease. Most antibodies produced in natural and experimental infections are directed to the highly antigenic C-terminal domain (shed acute-phase antigen). These antibodies do not inhibit the trans-sialidase activity, which is present in the N-terminal domain of the molecule. Antibodies able to inhibit trans-sialidase in sera from human infections have been found. TIA (trans-sialidase inhibition assay) was positive in sera from patients with acute and chronic infections. Healthy and congenitally infected infants born to mothers with Chagas' disease were also TIA-positive, but the antibody titers diminished within months after birth or after treatment. Thus, antibodies neutralizing trans-sialidase are detectable in most forms of T. cruzi human infections, and TIA may be useful in the diagnosis of Chagas' disease.

Acute Disease↗