M. Carmen Thomas

DNA immunization with Trypanosoma cruzi HSP70 fused to the KMP11 protein elicits a cytotoxic and humoral immune response against the antigen and leads to protection.

ABSTRACT Murine immunization with Trypanosoma cruzi KMP11-HSP70fused genes but not the KMP11 gene alone elicited both an immunoglobulin G2a long-lasting humoral immune response against KMP11 protein and activation of CD8+ cytotoxic T lymphocytes specific for two KMP11 peptides containing A2 motifs. Moreover, protection against the parasite challenge was observed after immunization with the chimeric gene.

Risk Factors and Primary Prevention of Congenital Chagas Disease in a Nonendemic Country

BACKGROUND In this longitudinal cohort study we evaluated the congenital transmission of Chagas disease (CD) in a nonendemic area. The aim of this work was to analyze the predictive value of a Trypanosoma cruzi-positive polymerase chain reaction (PCR) result in pregnant women for the diagnosis of vertical transmission and to evaluate the use of PCR as a tool for early detection of infection. METHODS The offspring of 59 seropositive pregnant mothers were followed up. The parasitological status of mothers was studied by PCR in a total of 64 pregnancies; 10 of these women had received treatment before pregnancy. Sixty-five infants (including a pair of twins) were monitored at 0, 6, 9, and 12 months of age by PCR and serology. In cases of congenital transmission, hemoculture and parasite lineage typing were performed. RESULTS Nine infants had acquired CD congenitally. This represents a transmission rate of 13.8% among seropositive mothers (9 infected newborns of 65 total live births). All infants were infected with T. cruzi discrete typing unit V strain. A statistically significant correlation was found between T. cruzi vertical transmission and a positive PCR result during pregnancy (31%; 9 infected newborns in 29 live births). No infected infants were detected among 10 mothers who were treated before they became pregnant, compared with 16.4% (9 of 55 live births) among untreated mothers. CONCLUSIONS PCR is a useful tool for the detection of congenital CD, and the treatment of infected women of childbearing age seems to be useful for preventing vertical transmission.

Biological markers for evaluating therapeutic efficacy in Chagas disease, a systematic review

The most neglected aspects of Chagas disease (CD) have been patient care and treatment. Despite recent progress in the development of potentially improved drugs, there is no consensus among different research groups on the lack of therapeutic response markers to evaluate efficacy of newly proposed drugs early after treatment. A systematic review of current evidence regarding molecules which are potential biomarkers for therapeutic response has been conducted using quality assessment and target responses as primary criteria. The review provides a panorama of the cumulative evidence and specific needs for development of a battery of complementary biomarkers which together fulfill ideal or acceptable criteria to evaluate early responses to treatment for chronic CD. There are several marker candidates which together may fulfill acceptable criteria to indicate the efficacy of a trypanocidal treatment. Data from ongoing studies are considered essential to improve assessment of existing markers and to identify those for early follow-up of treated patients.

The immunization of A2/Kb transgenic mice with the KMP11-HSP70 fusion protein induces CTL response against human cells expressing the T. cruzi KMP11 antigen: identification of A2-restricted epitopes

Cytotoxic T lymphocyte response against Jurkat-A2/K(b) cells expressing the T. cruzi KMP11 protein has been evaluated after immunization of C57BL/6-A2/K(b) transgenic mice with the KMP11 and KMP11-HSP70 recombinant proteins. The results show that mice immunized with KMP11 covalently fused to the T. cruzi HSP70 protein, but not mice immunized with KMP11 alone, elicit a CTL response against the Jurkat-A2/K(b) cells expressing the KMP11 protein. The data also show that spleen cells from mice immunized with the fusion protein and stimulated with the K1 peptide induce lysis of both the Jurkat-A2/K(b) cells transfected with the KMP11 coding gene and the Jurkat-A2-K(b) cells pulsed with the K1 peptide. Splenocytes stimulated with the K3 peptide induce lysis of the Jurkat-A2/K(b) cells loaded with the K3 peptide but they do not recognize the target cells expressing the KMP11 protein. Similar results were obtained using lymph node from mice immunized with the peptides. Thus, we believe there are two cytotoxic T cell epitopes restricted to the A2 molecule (K1(KMP11) (4-12) and K3(KMP11) (41-50)) in the KMP11 protein, and suggest that the K1 peptide could be considered an immunodominant antigen whilst the K3 peptide may be regarded as a cryptic epitope. The fact that the CTL lines induced in B6-A2/K(b) mice recognize human cells expressing KMP11 protein, indicates that the KMP11 antigen fused to HSP70 could be a good candidate for the induction of immunoprotective cytotoxic responses against T. cruzi natural infection.

The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts

L1Tc is the best represented autonomous LINE of the Trypanosoma cruzi genome, throughout which several functional copies may exist. In this study, we show that the first 77 bp of L1Tc (Pr77) (also present in the T. cruzi non-autonomous retrotransposon NARTc, in the Trypanosoma brucei RIME/ingi elements, and in the T. cruzi, T. brucei and Leishmania major degenerate L1Tc/ingi-related elements [DIREs]) behave as a promoter element that activates gene transcription. The transcription rate promoted by Pr77 is 10–14-fold higher than that mediated by sequences located upstream from the T. cruzi tandemly repeated genes KMP11 and the GAPDH. The Pr77 promoter-derived mRNAs initiate at nucleotide +1 of L1Tc, are unspliced and translated. L1Tc transcripts show a moderate half life and are RNA pol II dependent. The presence of an internal promoter at the 5′ end of L1Tc favors the production of full-length L1Tc RNAs and reinforces the hypothesis that this mobile element may be naturally autonomous in its transposition.

The non-LTR (long terminal repeat) retrotransposon L1Tc from Trypanosoma cruzi codes for a protein with RNase H activity.

The deduced amino acid sequence of the region downstream of the reverse transcriptase (RT) motif of theTrypanosoma cruzi L1Tc non-LTR retrotransposon shows a significant homology with the sequence coding for proteins with RNase H activity from different organisms and retroelements. The 25-kDa His6-tagged recombinant protein bearing only the L1Tc RNase H domain, named RHL1Tc, exhibits RNase H activity as measured on the [3H]poly(rA)/poly(dT) hybrid used as substrate as well as on specific homologous and heterologous [32P]RNA/DNA hybrids. The mutation of the conserved aspartic acid at position 39 of the enzyme catalytic site, but not of the serine at position 56 (non-conservative amino acid), abolishes protein RNase H activity. The RNase H activity of the RHL1Tc protein is Mg2+-dependent, and it is also active in the presence of the Mn2+ ion. The optimal condition of RNase H activity is found at pH 8 and 37 °C, although it also has significant enzymatic activity at 19 °C and pH 6. However, it cannot be excluded that the RNase H activity level and its optimal conditions may be different from that of a protein containing both RT and RNase H domains.

Identification of an hepatitis delta virus-like ribozyme at the mRNA 5′-end of the L1Tc retrotransposon from Trypanosoma cruzi

L1Tc is a non-LTR LINE element from Trypanosoma cruzi that encodes its transposition machinery and bears an internal promoter. Herewith, we report the identification of an in vitro active hepatitis delta virus-like ribozyme located in the first 77 nt at the 5′-end of the L1Tc mRNA (L1TcRz). The data presented show that L1TcRz has a co-transcriptional function. Using gel-purified uncleaved RNA transcripts, the data presented indicate that the kinetics of the self-cleaving, in a magnesium-dependent reaction, fits to a two-phase decay curve. The cleavage point identified by primer extension takes place at +1 position of the element. The hydroxyl nature of the 5′-end of the 3′-fragment generated by the cleavage activity of L1TcRz was confirmed. Since we have previously described that the 77-nt long fragment located at the 5′-end of L1Tc has promoter activity, the existence of a ribozyme in L1Tc makes this element to be the first described non-LTR retroelement that has an internal promoter–ribozyme dual function. The L1Tc nucleotides located downstream of the ribozyme catalytic motif appear to inhibit its activity. This inhibition may be influenced by the existence of a specific L1Tc RNA conformation that is recognized by RNase P.

The biology and evolution of transposable elements in parasites.

Transposable elements (TEs) are dynamic elements that can reshape host genomes by generating rearrangements with the potential to create or disrupt genes, to shuffle existing genes, and to modulate their patterns of expression. In the genomes of parasites that infect mammals several TEs have been identified that probably have been maintained throughout evolution due to their contribution to gene function and regulation of gene expression. This review addresses how TEs are organized, how they colonize the genomes of mammalian parasites, the functional role these elements play in parasite biology, and the interactions between these elements and the parasite genome.

Trypanosoma cruzi paraflagellar rod proteins 2 and 3 contain immunodominant CD8+ T-cell epitopes that are recognized by cytotoxic T cells from Chagas disease patients

The protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas disease. To date, no vaccine is available for protection against T. cruzi infection. The CD8(+) T cells immune response against specific antigens has shown to efficiently control the spread of the parasite in murine experimental infection. However, data concerning CD8(+) response in Chagas patients are still restricted to a few epitopes. We have studied the existence of immunodominant CD8(+) T cell epitopes in the paraflagellar rod proteins 2 and 3 (PFR2 and PFR3) from T. cruzi in a mouse model, and analyzed their recognition by cytotoxic T lymphocytes from Chagas disease patients. Immunization of C57BL/6-A2/K(b) transgenic mice with plasmids coding for the fusion proteins PFR2-HSP70 and PFR3-HSP70 induced a specific CTL response against two PFRs epitopes (PFR2(449-457) and PFR3(481-489)), and showed specific lysis percentages of 24 and 12, respectively. Moreover, the PFR2(19-28), PFR2(156-163), PFR2(449-457), PFR3(428-436), PFR3(475-482) and PFR3(481-489) peptides were observed to have a high binding affinity to the HLA-A*02:01 molecule. Remarkably, these HLA-A*02:01-binding peptides are successfully processed and presented during natural infection by T. cruzi in the context of MHC class I as evidenced by using peptide-pulsed K562-A2 cells as antigen presenting cells. The T cells from Chagas disease chronic patients specific for PFR2/PFR3 selected CD8(+) epitopes showed a pro-inflammatory cytokine secretion profile (IFN-γ, TNF-α and IL-6). A positive Granzime B secretion was observed in three out of 16 patients in response to PFR2(156-163) and PFR2(449-457) peptides, two out of 11 patients in response to PFR2(19-28) peptide and one out of 14 and 11 patients in response to PFR3(428-436) and PFR3(481-489) peptides, respectively. The PFRs-specific cytotoxic activity in purified PBMC was only detected in patients in the indeterminate phase of the disease.

The L1Tc C-terminal domain from Trypanosoma cruzi non-long terminal repeat retrotransposon codes for a protein that bears two C2H2 zinc finger motifs and is endowed with nucleic acid chaperone activity.

ABSTRACT L1Tc, a non-long terminal repeat retrotransposon from Trypanosoma cruzi, is a 4.9-kb actively transcribed element which contains a single open reading frame coding for the machinery necessary for its autonomous retrotransposition. In this paper, we analyze the protein encoded by the L1Tc 3′ region, termed C2-L1Tc, which contains two zinc finger motifs similar to those present in the TFIIIA transcription factor family. C2-L1Tc binds nucleic acids with different affinities, such that RNA > tRNA > single-stranded DNA > double-stranded DNA, without any evidence for sequence specificity. C2-L1Tc also exhibits nucleic acid chaperone activity on different DNA templates that may participate in the mechanism of retrotransposition of the element. C2-L1Tc promotes annealing of complementary oligonucleotides, prevents melting of perfect DNA duplexes, and facilitates the strand exchange between DNAs to form the most stable duplex DNA in competitive displacement assays. Mapping of regions of C2-L1Tc using specific peptides showed that nucleic acid chaperone activity required a short basic sequence accompanied by a zinc finger motif or by another basic region such as RRR. Thus, a short basic polypeptide containing the two C2H2 motifs promotes formation of the most stable duplex DNA at a concentration only three times higher than that required for C2-L1Tc.