Immunoprophylactic properties of the Corynebacterium pseudotuberculosis-derived MBP:PLD:CP40 fusion protein.

Caseous lymphadenitis (CLA) is a disease that affects small ruminants, and the best way to prevent its spread on a herd is through immunoprophylaxis. Thus, we aimed to evaluate the MBP:PLD:CP40 fusion protein as a new CLA immunogen. The fusion protein was constructed by combining Corynebacterium pseudotuberculosis PLD and CP40 proteins with maltose-binding protein (MBP) as an intrinsic adjuvant. The antigenicity, allergenic potential, prediction of B epitopes, binding to MHC receptors, and docking on the Toll-Like 2 receptor were evaluated in silico. MBP:PLD:CP40 was expressed and purified. 40 BALB/c were divided into four groups (G1 - control, G2 - Saponin, G3 - MBP:PLD:CP40, and G4 - rPLD + rCP40). Total IgG, IgG1, and IgG2a were quantified, and the expressions of cytokines after splenocyte in vitro stimulation were assessed. Mice were challenged 42 days after the first immunization. The in silico analysis showed that MBP:PLD:CP40 has immunogenic potential, does not have allergic properties, and can dock on the TRL2 receptor. MBP:PLD:CP40 stimulated the production of IgG1 antibodies in a fivefold proportion to IgG2a, and TNF and IL-17 were significantly expressed in response to the antigenic stimuli. When rPLD and rCP40 were used together for immunization, they could induce IFN-γ and IL-12, but with no detectable antibody production. The G3 and G4 groups presented a survival of 57.14% and 42.86%, respectively, while the G1 and G2 mice were all dead 15 days after the challenge. MBP:PLD:CP40 partially protected the mice against C. pseudotuberculosis infection and can be considered a potential new CLA immunogen. KEY POINTS: • The fusion protein induced more IgG1 than IgG2a antibodies; • The fusion protein also induced the expression of the TNF and IL-17 cytokines; • Mice inoculated with MBP:PLD:CP40 presented a 57.14% survival.

Argentinian small ruminant lentivirus (SRLV) p55gag antigen fused to maltose binding protein to use in SRLV serological confirmatory diagnosis.

The complete gag gene from small ruminant lentiviruses (SRLV) encodes for a polyprotein of 55 kDa, known as p55gag. p55gag presents multiple antigenic epitopes, which can be recognized by antibodies, increasing the opportunity to detect SRLV-positive animals. Therefore, this polyprotein is considered an excellent candidate to use in diagnostic tests to detect antibodies against SRLV. Different studies have suggested that the selection of the recombinant antigen, which must be representative of the virus strains circulating in the test population, is crucial to avoid false negative results. Thus, the use of proteins from different viral strains isolated from goats or sheep of a given region or country may be a useful strategy to increase the ability to detect SRLV-infected animals. In the present study, the pMAL-p5X vector was used to express and purify p55gag (now called rp55gag for recombinant polyprotein 55 gag). The cloned gene was inserted downstream from the malE gene of Escherichia coli, which encodes a maltose-binding protein (MBP), resulting in the expression of an MBP fusion protein. The complete gag gene was amplified by RT-PCR. Finally, after digestion, the product was cloned into the pMAL-p5X vector and used to transform E. coli ER2325 cells. After the purification of MBP-rp55gag by affinity chromatography, the eluted fraction was observed by SDS-PAGE and Western Blot (WB). The WB was carried out with 85 serum samples from small ruminants previously analysed and compared by two commercial ELISAs. The results show that 76 of the serum samples were concordant with those by both ELISAs. Regarding the other nine serum samples, which showed discordant results between both ELISAs, were positive by WB. The results thus show that the rp55gag could be considered as an antigen in a confirmatory diagnostic assay to detect SRLV by WB. For this purpose, a future study with a high number of sera to determine the test specificity and sensitivity, using the p55gag of the circulating strain in Argentina will be necessary.

Characterisation of the interaction of guanine nucleotides with ribosomal GTPase Lsg1.

Ribosome biogenesis in eukaryotes requires the participation of several transactivation factors that are involved in the modification, assembly, transport and quality control of the ribosomal subunits. One of these factors is the Large subunit GTPase 1 (Lsg1), a protein that acts as the release factor for the export adaptor named Nonsense-mediated mRNA decay 3 protein (Nmd3) and facilitates the incorporation of the last structural protein uL16 into the 60S subunit. Here, we characterised the recombinant yeast Lsg1 and studied its catalysis and binding properties for guanine nucleotides. We described the interaction of Lsg1 with guanine nucleotides alone and in the presence of the complex Nmd3•60S using fluorescence spectroscopy. Lsg1 has a greater affinity for GTP than for GDP suggesting that in the cell cytoplasm it exists mainly bound to the former. In the presence of 60S subunits loaded with Nmd3, the affinity of Lsg1 for both nucleotides increases but to a larger extent towards GTP. From this observation together with the excess of GTP present in the cytoplasm of exponentially growing cells over that of GDP, we can infer that the pre-ribosomal particle composed by Nmd3•60S acts as a GTP Stabilising Factor for Lsg1. Additionally, Lsg1 undergoes different conformational changes depending on its binding partner or the guanine nucleotides it interacts with. Steady-state kinetic analysis of free Lsg1 indicated slow GTP hydrolysis with values of kcat 1 min-1 and Km of 34 µM.

Enzyme-linked immunosorbent assay and agar gel immunodiffusion assay for diagnosis of equine infectious anemia employing p26 protein fused to the maltose-binding protein.

A codon-optimized equine infectious anemia virus p26 gene was fused to a maltose-binding protein (MBP) and expressed in Escherichia coli for use as an antigen in agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) for diagnosis of equine infectious anemia. An analysis of analytical sensitivity and specificity showed that the antigen MBP-p26rec reacted positively with a reference World Organization for Animal Health serum and demonstrated no cross-reaction against sera from vaccinated animals in either test. The diagnostic characteristics were evaluated and presented excellent values. The AGIDrec showed 100% sensitivity and specificity, and the ELISArec showed 100% sensitivity and 99.64% specificity. In addition, MBP-p26rec was stabile after three years of storage at 4 °C, maintaining its immunoreactivity.

Recombinant 3-Hydroxy 3-Methyl Glutaryl-CoA Reductase from Candida glabrata (Rec-CgHMGR) Obtained by Heterologous Expression, as a Novel Therapeutic Target Model for Testing Synthetic Drugs.

The enzyme 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGR) is a glycoprotein of the endoplasmic reticulum that participates in the mevalonate pathway, the precursor of cholesterol in human and ergosterol in fungi. This enzyme has three domains: transmembrane, binding, and soluble. In this study, we expressed and purified the soluble fraction of the HMGR enzyme from Candida glabrata (CgHMGR) in an Escherichia coli heterologous system and used it as a model for studying its inhibitory activity. The soluble fraction of CgHMGR was fused to the maltose binding protein (MBP), purified, and characterized. Optimal pH was 8.0, and its optimal temperature activity was 37 °C. The k m and V max for the HMG-CoA were 6.5 µM and 2.26 × 10-3 µM min-1, respectively. Recombinant CgHMGR was inhibited by simvastatin presenting an IC50 at 14.5 µM. In conclusion, our findings suggest that the recombinant HMGR version from C. glabrata may be used as a study model system for HMGR inhibitors such as statins and newly synthesized inhibitor compounds that might be used in the treatment of hypercholesterolemia or mycosis.

Plant heat shock protein 90 as carrier-adjuvant for immunization against a reporter antigen.

Here, we evaluated the modulation of the immune response induced by Hsp90 of Nicotiana benthamiana (NbHsp90.3) against the Maltose Binding Protein (MBP) as a reporter antigen. Equimolar quantities of recombinant proteins were administered in mice as follows: MBP alone (MBP group), a mixture of MBP and rNbHsp90.3 (MBP+rNbHsp90.3 group) and the fusion of MBP to rNbHsp90.3 (MBP-rNbHsp90.3 group). The covalent linkage between NbHsp90.3 and MBP to bring a fusion protein was essential to induce the strong specific antibody response with predominance of IgG2a. Eighty-four days after the first immunization, splenocyte proliferation from MBP-rNbHsp90.3-immunized mice was consistently higher than that from MBP and MBP+rNbHsp90.3 groups. In addition, splenocytes from MBP-rNbHsp90.3 immunized mice produced higher levels of IFN-γ than controls. Finally, both formulations with rNbHsp90.3 significantly enhanced the MHC class I expression levels, but only rNbHsp90.3 covalent bound to MBP induced a specific cellular immune response against MBP measured as increased percentage of CD8(+) T cells. Taken together, these results suggest that plant HSP90s could be incorporated as adjuvants in vaccines that require the generation of a Th1 response along with a CD8 cytotoxic cell response to confer immunity.

Comparative fusion expression of maize SINAT5 in two different strains of Escherichia coli.

SINAT5 is a plant E3 ligase that regulates auxin signaling and root morphogenesis by ubiquitination of the NAC1 protein. Consequently, it may be a putative regulator of aspects of plant development cycles that are controlled by auxin. Efficient production, purification and correctly folded form of this protein are important requirements for functional studies. We produced and quantitatively compared fusion expression of the "maltose binding protein (mbp)-maize sinat5" construct in two different strains of Escherichia coli. One-step purification of fused products gave about 33 mg protein/L bacterial cell culture for E. coli TB1 cells and approximately 18 mg protein/L bacterial cell culture for E. coli DH5α cells. Continuous expression of the fused product and similarity of growth patterns were observed in both cultures.

Kinetics and docking studies of two potential new inhibitors of the nucleoside hydrolase from Leishmania donovani.

In this study the recombinant enzyme nucleoside hydrolase of Leishmania donovani (rLdNH) was expressed in Escherichia coli in connection with maltose binding protein (MBP). The rLdNH-MBP showed efficient a significant in vitro activity with inosine as substrate. From the coupled reaction with xanthine oxidase (XO) it was possible to determine the kinetic constants of rLdNH-MBP as K(M) (434 ± 109 µM) and V(max) (0.20 ± 0.02 µM). In addition, two nucleoside analogs (compounds 1 and 2) were tested as prototypes of rLdNH inhibitors. These compounds presented high affinity for the enzyme with K(i) values of 1.6 ± 0.2 and 17.0 ± 2.1 µM, respectively, as well as 271 and 26 folds higher than the affinity constant found for inosine. We also determined the type of enzyme inhibition, using double-reciprocal plot for these two compounds and the results confirmed a competitive inhibition. Additional docking studies showed the binding manner of compounds 1 and 2 inside the active site of LdNH revealing the essential residues for an effective inhibition. These results confirm that compounds 1 and 2 are strong rLdNH-MBP inhibitors.

Maltose-binding protein isolated from Escherichia coli induces Toll-like receptor 2-mediated viability in U937 cells.

BACKGROUND: Stimulation of Toll-like receptors (TLRs) by microbial products has been utilised to potentiate immune responses against haematologic malignancies. The maltose-binding protein (MBP) of Escherichia coli could induce the activation of immune cells via TLR4. The aim of the present study was to investigate whether TLRs mediated the biological effects of MBP on U937 and Jurkat cells in vitro. METHODS We observed the effect of MBP on U937 and Jurkat cells by using the WST, cell cycle analysis and morphological observation. Further, cells were stimulated with MBP for indicated times and doses, and detected by RT-PCR, western blotting, immunohistochemistry and immunofluorescence staining to investigate the mechanisms involved in cell viability. RESULTS: MBP enhanced the viability of U937 and Jurkat cells, and the effects were blocked by anti-TLR2, but not anti-TLR4 in U937 cells. Further studies confirmed that MBP was able to directly bind to U937 and Jurkat cells and modulate TLR expression. The effects of MBP depended on the activation of NF-κB and MAP kinase in U937 and Jurkat cells. CONCLUSIONS: Our results demonstrated that MBP could directly promote U937 cell viability via TLR2. It suggested that MBP may be used as an adjuvant for participating in the immunotherapy of haematologic malignancies.

Purification of MBP-EnvZ fusion proteins using an automated system.

Bacteria use two-component signal transduction systems to detect and respond to environmental changes. These systems have been studied systematically in Escherichia coli as a model organism. Most of the signal transduction systems present in E. coli are conserved in related pathogenic bacteria; however, differences in regulation by these systems have been reported from one bacterial species to another [Oropeza, R., and Calva, E. (2009). The cysteine 354 and 277 residues of Salmonella enterica serovar Typhi EnvZ are determinants of autophosphorylation and OmpR phosphorylation. FEMS Microbiol. Lett.292, 282-290]. Our laboratory has been interested in studying the OmpR/EnvZ two-component system in S. enterica. In S. enterica serovar Typhi (Typhi), it regulates the expression of the porin genes, namely ompC, ompF, ompS1, and ompS2. OmpR proteins are identical between E. coli and Typhi, but several differences exist between the EnvZ proteins. To define whether some differences in porin regulation are due to changes on EnvZ, we decided to overexpress and purify E. coli, Typhi, and S. enterica serovar Typhimurium (Typhimurium) EnvZ proteins fused to the maltose-binding protein (MBP) as a purification tag. Differences in the autophosphorylation level of these proteins were evidenced. Hence, considering the differences at the amino acid level between E. coli and Typhi EnvZ proteins, several mutations were introduced in the Typhi EnvZ protein in order to try to find the amino acids affecting the enzymatic activity of the protein. We found that Cys354 plays an important role in defining the enzymatic activity of this histidine kinase. Here, we report the automated purification of a collection of MBP-EnvZ fusions using a mini-chromatography commercial system, but adapting an amylose affinity column packed by ourselves.

Crystallization, data collection and data processing of maltose-binding protein (MalE) from the phytopathogen Xanthomonas axonopodis pv. citri.

Maltose-binding protein is the periplasmic component of the ABC transporter responsible for the uptake of maltose/maltodextrins. The Xanthomonas axonopodis pv. citri maltose-binding protein MalE has been crystallized at 293 K using the hanging-drop vapour-diffusion method. The crystal belonged to the primitive hexagonal space group P6(1)22, with unit-cell parameters a = 123.59, b = 123.59, c = 304.20 A, and contained two molecules in the asymetric unit. It diffracted to 2.24 A resolution.

Production of polyclonal antibodies against the human respiratory syncytial virus nucleoprotein and phosphoprotein expressed in Escherichia coli.

The nucleoprotein (N) and the phosphoprotein (P) of the human respiratory syncytial virus (HRSV), A2 strain, were cloned into pMAL-c2e vector. The proteins were expressed fused with the maltose-binding protein (MBP) and were preferentially found in the soluble fraction of the bacterial lysate. After their purification using amylose resin, almost no other protein was detected in SDS-PAGE. The fused proteins were cleaved by digestion with enterokinase and then used as antigens for BALB/c mice immunization. The obtained polyclonal antibodies were tested against HRSV infected cells in immunofluorescence assays. The results indicate that the antibodies generated against the recombinant proteins were able to recognize the virus proteins. We now intend to purify the cleaved N and P proteins and use them in structural studies. The recombinant proteins will also be tested as potential inducers of a protective immunity against the HRSV.

Purification and in vitro characterization of the maltose-binding protein of the plant pathogen Xanthomonas citri.

The uptake of maltose and maltodextrins in gram-negative bacteria is mediated by an ATP-dependent transport complex composed of a periplasmic maltose-binding protein (MBP) and membrane-associated proteins responsible for the formation of a membrane pore and generation of energy to drive the translocation process. In this work, we report the purification and in vitro functional analysis of MBP, encoded by the malE gene, of the plant pathogen Xanthomonas citri, responsible for the canker disease affecting citrus plants throughout the world. The X. citri MBP is composed of 456 amino acids, displaying a low amino acid identity (16% throughout the sequence) compared to the Escherichia coli K12 ortholog. The X. citri malE gene was cloned into a pET28a vector, and the encoded protein was expressed and purified by affinity chromatography as a His-tag N-terminal fusion peptide produced by the E. coli BL21 strain. Enhanced levels of soluble protein were achieved with static cultures kept overnight at 23 degrees C. Ability to bind immobilized amylose, the emission of intrinsic fluorescence and circular dichroism spectra indicated that the purified recombinant protein preserved both conformation and biological activity of the native protein. The availability of the recombinant MBP will contribute to the functional and structural analysis of the maltose and maltodextrin uptake system of the plant pathogen X. citri.

Overexpression, purification, and biochemical characterization of GumC, an enzyme involved in the biosynthesis of exopolysaccharide by Xylella fastidiosa.

GumC is one of nine enzymes involved in the biosynthesis of fastidian gum, an exopolysaccharide produced by Xylella fastidiosa that may be linked directly to the pathogenicity of the microorganism. GumC may be responsible for gum polymerization or secretion through the membrane of X. fastidiosa. To perform structure and functions studies, we developed an expression system for the production of GumC as a fusion protein with maltose binding protein (MBP) using pMAL-c2x vector. The GumC-MBP fusion protein was expressed as a 94 kDa protein, which strongly reacts with anti-MBP antibodies. GumC-MBP was isolated by affinity chromatography through an amylose column and used to produce antibodies against the fusion protein. After the enzymatic cleavage of MBP, GumC was purified on a Q Sepharose Fast Flow column. GumC showed a molecular weight corresponding to the expected one (52 kDa) and its N-terminal sequence was identical to that deduced from the DNA. The shape of the circular dichroism spectrum was compatible with a folded protein that contains alpha-helical regions in its structure. Therefore, in this study we describe, for the first time, the production of GumC recombinant protein.

The diagnostic performance of recombinant Trypanosoma cruzi ribosomal P2beta protein is influenced by its expression system.

In the present work, we have determined the effect of expression vectors and their corresponding host bacteria on the antigenic performance of Trypanosoma cruzi P2beta (TcP2beta) full-length recombinant protein. The gene encoding the TcP2beta ribosomal protein was cloned in pMAL-c2 and pET-32a vectors that allow the expression of high levels of soluble fusion proteins. A panel of 32 positive and 32 negative sera was assayed with the purified proteins expressed using pMal-c2 (TcP2beta-MBP) and pET-32a (TcP2beta-TRX) vectors and with MBP and TRX purified from pMAL-c2 and pET-32a vectors, respectively. The antigenic behavior of each TcP2beta recombinant protein differed in the diagnostic performance in terms of DI(+) (93.7 for TcP2beta-MBP vs 100% for TcP2beta-TRX), in DI(-) (90.5 for TcP2beta-MBP vs 100% for TcP2beta-TRX) and in cross-reaction with negative sera. To determine if the higher reactivity of expressed pMAL-c2 protein was due to folding during protein expression or to a steric effect related to the protein adsorption at the titration plate, the reactivity of sera against soluble proteins was assessed by ELISA inhibition assays. As each soluble protein preserved its level of reactivity, we concluded that differences in reactivity were due to intrinsic characteristics of the proteins and not to differences in patterns of adsorption to the plates.

Properties of GDP-mannose pyrophosphorylase, a critical enzyme and drug target in Leishmania mexicana.

Leishmania parasites synthesize a range of mannose-containing glycoconjugates thought to be essential for virulence in the mammalian host and sandfly vector. A prerequisite for the synthesis of these molecules is the availability of the activated mannose donor, GDP-Man, the product of the catalysis of mannose-1-phosphate and GTP by GDP-mannose pyrophosphorylase (GDP-MP). In contrast to the lethal phenotype in fungi, the deletion of the gene in Leishmania mexicana did not affect parasite viability but led to a total loss of virulence, making GDP-MP an ideal target for anti-Leishmania drug development. We show by immunofluorescence and subcellular fractionation that GDP-MP is a cytoplasmic protein, and we describe a colorimetric activity assay suitable for the high throughput screening of small molecule inhibitors. We expressed recombinant GDP-MP as a fusion with maltose-binding protein and separated the enzyme from maltose-binding protein by thrombin cleavage, ion-exchange, and size exclusion chromatography. Size exclusion chromatography and analytical ultracentrifugation studies demonstrate that GDP-MP self-associates to form an enzymatically active and stable hexamer. However, sedimentation studies show that the GDP-MP hexamer dissociates to trimers and monomers in a time-dependent manner, at low protein concentrations, at low ionic strength, and at alkaline pH. Circular dichroism spectroscopy reveals that GDP-MP is comprised of mixed alpha/beta structure, similar to its closest related homologue, N-acetyl-glucoseamine-1-phosphate uridyltransferase (Glmu) from Streptococcus pneumoniae. Our studies provide insight into the structure of a novel target for the development of anti-Leishmania drugs.

Effect of Urtica dioica agglutinin and Arabidopsis thaliana Chia4 chitinase on the protozoan Phytomonas françai.

The genus Phytomonas is responsible for many diseases in different crop plant species. The finding that chitin is an exposed cell surface polysaccharide in Phytomonas françai and the observation that chitinases can inhibit fungal growth raises expectations about the potential effect of plant chitinases on the P. françai cell membrane surface. The plant chitinases Urtica dioica agglutinin (UDA) and Arabidopsis thaliana Chia4 (ATCHIT4) proteins were over-expressed in bacteria and the interaction between these proteins and P. françai surface was analyzed by immunocytochemistry. We showed that UDA and ATCHIT4 proteins can interact with surface-exposed chitin from P. françai.

The effect of IFN-gamma and TNF-alpha on the eosinophilic differentiation and NADPH oxidase activation of human HL-60 clone 15 cells.

The aim of this study was to investigate the effect of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on NADPH oxidase activity and gp91-phox gene expression in HL-60 clone 15 cells as they differentiate along the eosinophilic lineage. The results were compared to the eosoniphilic inducers interleukin-5 (IL-5) and butyric acid. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) or IL-5 (200 pM) caused a significant increase in the expression of the eosinophil peroxidase (EPO) and the major basic protein (MBP) genes. Similar results were observed when the cells were cultured with 0.5 mM butyric acid for 5 days. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) also caused a significant increase in superoxide release by HL-60 clone 15 cells after 2 days compared with control or with butyric acid-induced cells. After 5 days, these cytokines and butyric acid induced an even stronger release of superoxide. HL-60 clone 15 cells cultured with IFN-gamma and TNF-alpha for 2 days showed a significant increase in gp91-phox gene expression. We conclude that IFN-gamma and TNF-alpha are sufficient to induce the differentiation of HL-60 clone 15 cells to the eosinophilic lineage and to upregulate gp91-phox gene expression and activity of the NADPH oxidase system.

Cloning and molecular characterization of the Hevea brasiliensis allergen Hev b 11, a class I chitinase.

BACKGROUND: In the last 10 years type-I allergy against proteins from Hevea brasiliensis latex has become an acknowledged medical issue. Fruit-allergic patients represent one risk group for developing latex allergy. Class I chitinases have been identified from chestnut, avocado and banana as relevant allergens. The chitin binding (hevein) domain from these class I chitinases has been postulated to bear the important IgE binding epitopes. OBJECTIVE: To clone the cDNA of an allergenic latex class I chitinase, to express the recombinant protein and to determine its IgE cross-reactivity with hevein (Hev b 6.02). METHODS: A full-length cDNA coding for a class I chitinase has been isolated from Hevea latex RNA by reverse transcription followed by PCR. The chitinase encoding sequence has been subcloned into the pMAL expression vector and expressed in E. coli as a fusion protein to maltose binding protein. The highly enriched recombinant protein fraction has been tested for its IgE binding capacity in immunoblots and ELISA. Furthermore, the pathogenesis-related function of the recombinant protein was tested in a fungal growth inhibition assay. RESULTS: The Hevea brasiliensis latex chitinase, designated Hev b 11, displays 70% identity to the endochitinase from avocado and its hevein-domain 58% to hevein (Hev b 6.02). The recombinant Hev b 11-maltose binding protein is recognized by latex- and fruit-allergic patients with IgE binding in both, ELISA and immunoblots. Pre-incubation of sera with rHev b 11-maltose binding protein showed an overall 16% inhibition of subsequent binding to rHev b 6.02-maltose binding protein on solid phase. The growth of F. oxysporum was inhibited in a dose dependent manner by addition of rHev b 11-maltose binding protein to the culture. CONCLUSIONS: Hev b 11, a class I chitinase, is another allergen from Hevea latex with a chitin binding domain and displays a different IgE binding capacity compared with hevein.

Antibodies against the carboxyl-terminal end of the Trypanosoma cruzi ribosomal P proteins are pathogenic.

Sera from patients with chronic Chagas heart disease recognize the carboxyl-terminal regions of the Trypanosoma cruzi ribosomal P proteins defined by B cell epitopes P013 (EDDDDDFGMGALF) and R13 (EEEDDDMGFGLFD) corresponding to the T. cruzi ribosomal P0 (TcP0) and P2beta (TcP2beta) proteins, respectively. It has been hypothesized that both epitopes may induce antibodies that cross-react and stimulate the beta1-adrenoreceptor. However, no proof as to their pathogenicity has been obtained. We investigated the consequences of immunizing mice with either TcP0 or TcP2beta proteins. Of 24 immunized animals, 16 generated antibodies against the carboxyl-terminal end of the corresponding protein, 13 of which showed an altered ECG (P<0.001, 81%). Immunization with TcP0 induced anti-P013 antibodies that bind to and stimulate cardiac G-protein-coupled receptors and are linked to the induction of supraventricular arrhythmia, repolarization, and conduction abnormalities as monitored by serial electrocardiographic analysis. In contrast, immunization with TcP2beta generated anti-R13 antibodies with an exclusive beta1-adrenergic-stimulating activity whose appearance strictly correlated with the recording of supraventricular tachycardia and death. These findings demonstrate that anti-P antibodies are arrhythmogenic in the setting of a normal heart, since no inflammatory lesions or fibrosis were evident to light microscopic examination.