Mapping of B-Cell Epitopes in a Trypanosoma cruzi Immunodominant Antigen Expressed in Natural Infections

Tc40 is an immunodominant antigen present in natural Trypanosoma cruzi infections. This immunogen was thoroughly mapped by using overlapping amino acid sequences identified by gene cloning and chemical peptide synthesis. To map continuous epitopes of the Tc40 antigen, an epitope expression library was constructed and screened with sera from human chagasic patients. A major, linear B-cell epitope spanning residues 403 to 426 (PAKAAAPPAA) was identified in the central domain of Tc40. A synthetic peptide spanning this region reacted strongly with 89.8% of the serum samples from T. cruzi-infected individuals. This indicates that the main antigenic site is defined by the linear sequence of the peptide rather than a conformation-dependent structure. The major B-cell epitope of Tc40 shares a high degree of sequence identity with T. cruzi ribosomal and RNA binding proteins, suggesting the existence of cross-reactivity among these molecules.     

Peptide-based analysis of the amino acid sequence important to the immunoregulatory function of Trypanosoma cruzi Tc52 virulence factor

The intracellular protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas' disease. We have previously identified a T. cruzi-released protein called Tc52, which is crucial for parasite survival and virulence. In the present study, we attempted to define the Tc52 epitope(s) responsible for its immunoregulatory function. A naturally occurring major peptide fragment of molecular mass 28 kDa (Tc28k) was identified, which was localized in the C-terminal portion of Tc52 and was inhibitory for T-cell activation. Synthetic peptides corresponding to amino acid sequences in Tc52 were evaluated for their ability to modulate T-cell proliferation and cytokine production. Results obtained using five peptides spanning the N-terminal or C-terminal domain of the Tc52 protein indicated that the activity mapped to Tc52 residues 432-445. Moreover, it was found that the peptide, when coupled to a carrier protein (ovalbumin), exhibited increased inhibitory activity on T-lymphocyte activation. Incubation with 8 nm ovalbumin-coupled peptide 432-445 resulted in approximately the same levels (>75%) of inhibition of T-cell proliferation as 5 micro g/ml Tc28k. Furthermore, we showed that the coupled peptide significantly down-regulated the secretion of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). Likewise, in immunized mice, the coupled peptide 432-445 was a very poor B- and T-cell antigen compared with the other Tc52-derived peptides. These results suggest that the immunomodulatory portion of the T. cruzi Tc52 virulent factor may reside, at least in part, in a conserved sequence within its C-terminal domain, which could minimize its antigenicity.     

Development of an equine herpesvirus type 4-specific enzyme-linked immunosorbent assay using a B-cell epitope as an antigen

The equine herpesvirus type 4 (EHV-4)-specific region of glycoprotein G has served as an antigen for serodiagnosis and seroepizootic studies of EHV-4 infection (B. S. Crabb and M. J. Studdert, J. Virol. 67:6332-6338, 1993; S. Yasunaga, K. Maeda, T. Matsumura, K. Kai, H. Iwata, and T. Inoue, J. Vet. Med. Sci. 60:1133-137, 1998; S. Yasunaga, K. Maeda, T. Matsumura, T. Kondo, and K. Kai, J. Vet. Med. Sci. 62:687-691, 2000). Here we identified a major B-cell epitope in the type-specific region of EHV-4 and applied it as an antigen in enzyme-linked immunosorbent assays (ELISAs). A 24-amino-acid repeat sequence expressed as a glutathione S-transferase fusion protein specifically reacted as well as the type-specific region with sera from foals infected with EHV-4. Five synthetic peptides (12-mer peptides) in the repeat sequence were included as ELISA antigens. The results indicated that the 12-mer peptide MKNNPIYSEGSL contained a major B-cell epitope specific for EHV-4 infection. Inclusion of this 12-mer peptide in ELISAs for an epidemiological study specifically detected EHV-4 infection in the field. These results indicated that the 12-mer epitope was responsible for the type-specific antibody response and therefore is useful for seroepizootic studies and serodiagnosis of EHV-4 infection.     

The reactivity of naturally sensitized human CD4 cells and IgG antibodies to synthetic peptides derived from the amino terminal sequences of a 3800 MW Streptococcus mutans antigen.

Natural immunity to synthetic peptides (SP) derived from the sequences of a 3800 MW streptococcal antigen (SA) was found in human subjects. Significant serum IgG antibodies were detected both to the native SA and to peptides consisting of residues 3-13, 1-15 and 1-20. Inhibition studies confirmed cross-reactivity between the native SA and SP. A series of short peptides with deletions at the amino and carboxy termini were then tested to determine the sequence of B-cell epitopes. Residues 8-13 and 1-6 bound significant serum IgG antibodies, but residues 8-13 were more effective and consistent in inhibiting human antibodies than residues 1-6. These results suggest that residues 8-13 constitute a major B-cell epitope but that residues 1-6 may represent a minor B-cell epitope. The human CD4 subset of T cells was then examined by stimulating the cells with SA or SP and measuring the uptake of [3H]thymidine [( 3H]TdR). The cells were found to be sensitized in vivo to both the native SA and the SP and cross-reactivity between the SA and SP was shown by enrichment and depletion experiments on antigen-coated monocytes. As with the B-cell epitope, the series of short peptides was used to stimulate CD4 cells, in order to determine the T-cell epitope. Residues 6-15 were the shortest SP which stimulated significant [3H]TdR uptake and this peptide was designated as a T-cell epitope. The results suggest that natural oral immunization with Streptococcus mutans induces serum antibodies and T-cell sensitization to a peptide in which a T-cell epitope (residues 6-15) overlaps with a B-cell epitope (residues 8-13). Furthermore, a comparison between linear and cycled peptides suggests that unlike immunogenicity which is commonly enhanced by the more rigid cyclized peptides, antigenicity is favoured by linear peptides. This was evident not only for antibodies but also for T-cell proliferative responses.     

Stage-specific surface antigens of metacyclic trypomastigotes of Trypanosoma cruzi identified by monoclonal antibodies

Stage-specific Trypanosoma cruzi surface antigens were characterized by using monoclonal antibodies (MAb) which bind specifically to the metacyclic trypomastigotes derived from either the insect vector or acellular cultures. A protein with an apparent molecular weight of 90,000 was detected by the MAb 5E7 on the surface of cultured metacyclics of four strains of T. cruzi: G, CL, Y and Tulahuen. The MAb 1G7, which binds to an epitope of the 90 kDa antigen distinct from that recognized by the MAb 5E7, reacted with metacyclics of the G and Tulahuen strains but not of the Y or CL strain. A polypeptide of approximately 82 kDa, identified by the MAb 3F6, was found in the metacyclics of all four T. cruzi strains. The MAb 3F6 also detected a 75 kDa antigen in the G strain metacyclics. The stage-specific MAb and the polyclonal antibodies from mice protected against acute T. cruzi infection by immunization with killed G metacyclics identified the same set of major surface proteins of G metacyclic trypomastigotes.     

Identification of surface-exposed linear B-cell epitopes of the nonfimbrial adhesin CS31A of Escherichia coli by using overlapping peptides and antipeptide antibodies.

As a first step toward the design of an epitope vaccine, by using the nonfimbrial adhesin CS31A of Escherichia coli as a carrier, a low-resolution topological and epitope map of the CS31A subunit was developed by using solid-phase peptide synthesis and polyclonal rabbit antibodies raised against both native and denatured proteins. Peptides constituting antigenic epitopes on the major subunit (ClpG) of the multimeric CS31A antigen were identified by examining the binding of the antibodies to 249 overlapping nonapeptides covering the amino acid sequence of ClpG. With antibodies raised against denatured ClpG subunit, seven major epitope regions, corresponding to residues 10 to 18, 45 to 58, 88 to 107, 148 to 172, 187 to 196, 212 to 219, and 235 to 241, were located. Most of the epitopes were hydrophilic and were located in variable regions, residing largely in loop regions at the boundaries of secondary structural elements of ClpG. In contrast, antibodies raised against native CS31A antigen reacted only with the peptide AVNPNA (positions 179 to 184), demonstrating that this peptide was the only linear B-cell epitope of the native protein. The different immunogenic profiles of native CS31A antigen and denatured ClpG indicated that the denaturation process resulted in marked conformational changes in the protein, which could expose epitopes hidden or absent in native CS31A. To identify the surface-exposed epitopes, nine peptides covering the dominant antigenic regions of ClpG were synthesized and used to prepare site-specific antibodies. Antipeptide antibodies were tested, in a competitive enzyme-linked immunosorbent assay (ELISA), for cross-reactivity with native CS31A and denatured ClpG subunit. Four of these antipeptide antibodies bound to the native protein in an accessibility ELISA, indicating that residues 44 to 56, 174 to 190, 185 to 199, and 235 to 249 were surface exposed on CS31A. These data indicate that an immunodominant surface-exposed linear epitope was present in the region from positions 179 to 184 of ClpG in the native CS31A antigen on intact bacterial cells and suggest that the four surface-exposed epitopes constitute potential sites for insertions or substitutions with heterologous peptides.     

Towards a Taenia solium cysticercosis vaccine: an epitope shared by Taenia crassiceps and Taenia solium protects mice against experimental cysticercosis

The Taenia crassiceps recombinant antigen KETc7 has been shown to be effective as a vaccine against experimental murine cysticercosis, a laboratory model used to test potentially promising molecules against porcine Taenia solium cysticercosis. Based on the deduced amino acid sequence of this proline-rich polypeptide, three fragments, GK-1, GK-2, and GK-3, were chemically synthesized in linear form. Of the three peptides, only GK-1 induced sterile protection against T. crassiceps cysticercosis in 40 to 70% of BALB/cAnN male mice. GK-1 is an 18-amino-acid peptide which contains at least one B-cell epitope, as demonstrated by its ability to induce an antibody response to the peptide and T. crassiceps antigen without need of a carrier protein. Immunofluorescence studies revealed that anti-GK1 antibodies strongly react with the native protein in the tegument of T. crassiceps and also with anatomical structures of T. solium eggs, oncospheres, cysticercus, and tapeworm. GK-1 also contains at least one T-cell epitope, capable of stimulating the proliferation of CD8(+) and to a lower extent CD4(+) T cells primed either with the free peptide or T. crassiceps total antigen. The supernatant of the stimulated cells contained high levels of gamma interferon and low levels of interleukin-4. Similar results were obtained with T cells tested for intracellular cytokine production, an indication of the peptide's capacity to induce an inflammatory response. The remarkable protection induced by GK-1 immunization, its physicochemical properties, and its presence in all developmental stages of T. solium point to this synthetic peptide as a strong candidate in the construction of a synthetic vaccine against T. solium pig cysticercosis.     

Identification of another B-cell epitope in the type-specific region of equine herpesvirus 4 glycoprotein G

Recently, a novel 12-mer B-cell epitope, MKNNPIYSEGSL, in the type-specific region of equine herpesvirus 1 (EHV-1) glycoprotein G (gG) was identified and used as an antigen for enzyme-linked immunosorbent assay (Maeda et al., J. Clin. Microbiol. 42:1095-1098, 2004). Although our prototype strain, TH20p, possesses two repeat sequences containing the B-cell epitope, the EHV-4 NS80567 strain has two repeat sequences that are not identical. One repeat sequence stretch contained the B-cell epitope, while the other contained the 11-mer, MKNNPVYSESL (underlining indicates a different amino acid). In this study, heterogeneity of the type-specific region was compared among Japanese EHV-4 isolates. The 11-mer peptide, MKNNPVYSESL, specifically reacted with sera from horses naturally infected with EHV-4 but not with sera from horses experimentally infected with EHV-4 TH20p. The 11-mer peptide may be another B-cell epitope in the type-specific region.     

Immunoparasitological Studies of Trypanosoma Cruzi Low Virulence Clones from Panama: Humoral Immune Responses and Antigenic Cross-Reactions with Trypanosoma Rangeli in Experimentally Infected Mice

The kinetics of humoral immune responses were investigated in mice experimentally infected with five clones of Trypanosoma cruzi isolated from different sources in Panama. Sera were collected at different timepoints post-infection. ELISA and IHA tests were used to detect antibodies against T. cruzi epimastigote antigens. The levels of T. cruzi specific antibodies increased during the course of infection; at day 90 post-infection the range was between 1:5120 and 1: 10240. A high correlation was evident between ELISA and IHA results. Western blots revealed that these antibodies recognized polypeptides of 81, 76 and 71 KDa during the first weeks and 81, 76, 71, 50, 40, 28 and 12 KDa after 30–50 days. Only minor differences in antigen recognition patterns were demonstrated, suggesting that the major antigens may be represented in all clones. T. rangeli antigens were also recognized by T. cruzi seropositive sera. However, an ELISA test using antigens isolated from a genomic expression library of T. cruzi revealed that a hyperimmune rabbit serum against T. rangeli was unable to recognize the repeat sequence of SAPA (Shed Acute Phase Antigen) peptides but did recognize a number of other T. cruzi synthetic peptide antigens. The importance of these findings, in the context of Chagas' disease, is discussed.     

Monospecific Polyclonal Anti-Anti-Idiotypic Antibodies to the Carboxyterminal Undecapeptide of the SV40 Large Tumour Antigen

The murine monoclonal antibody PAb1605 defines an epitope, peptide Lys(698)-Thr(708) (KT), on the carboxyterminus of the tumour(T)antigen of SV40-transformed cells. In vivo and in vitro experiments had shown that this sequence represents an epitope for both humoral and cellular immune responses. When injected into rabbits PAb1605 induces anti-idiotypic antibodies (Ab-2). Ab-2β (internal image type) was purified by adsorption chromatography and characterized by the ability of KT to compete with the binding of ab-2 with ab-1. Murine anti-anti-idiotypic antibodies (ab-3) were obtained by immunization of mice with ab-2β. Both ab-1 and ab-3 JgG showed affinities to immunoprecipitated SV40 T antigen by immunoblot analysis and to nuclear SV40 T antigen by the immunofluorescence assay. The binding of ab-3 to SV40 T antigen was completely inhibited by competition with KT. We conclude that the polyclonal ab-3 is of the ab-3 subtype and specific for only one epitope which is represented by KT and defined by ab-1. The results demonstrate that the specificity for a defined peptide epitope of an antibody was conserved even after two consecutive steps of anti-idiotypic-antibody formation in two host species. Since this postulate of network theory could be verified for a sequence of a tumour-associated antigen which represents a B- and T cell epitope, this model is of great interest for further tumour immunological studies.     

The SA85-1.1 protein of the Trypanosoma cruzi trans-sialidase superfamily is a dominant T-cell antigen

Trypanosoma cruzi currently infects 18 million people, and 30% of those infected develop a chronic inflammatory process that causes significant morbidity or mortality. The major histocompatibility complex class II (MHC-II)-restricted T-cell response is critical to the control of the infection and to the ensuing inflammatory pathology. The specific epitopes or major antigens of this response have not been identified. The parasite simultaneously expresses variant members of the trans-sialidase superfamily. To begin to analyze the MHC-II response to these variant proteins, the response to a single surface protein, SA85-1.1, was initiated. These studies have demonstrated that a biased gamma interferon (IFN-gamma) response to the SA85-1.1 protein develops during T. cruzi infection. In addition, adoptive transfer of a CD4 clone that recognizes an SA85-1.1 epitope, named epitope 1, and immunization with a peptide encoding epitope 1 were protective and suggested that epitope 1 may be immunodominant. In this report IFN-gamma intracellular staining demonstrated that splenocytes from acutely and chronically infected mice, incubated with SA85-1.1 protein or peptides that encode epitope 1, result in IFN-gamma synthesis by 4 to 6% of the splenic CD4 cells. These data indicate that during T. cruzi infection epitope 1 is a major epitope and that 4 to 6% of the CD4 cells are stimulated by a single trans-sialidase superfamily epitope and suggest that a combination of trans-sialidase superfamily proteins combines to stimulate a majority of CD4 cells. These data suggest that during T. cruzi infection the CD4 response to the trans-sialidase superfamily is critical to the protective response and to the ensuing chronic inflammatory pathology.     

An antigenic peptide inducing cross-reacting antibodies inhibiting the interaction of Streptococcus mutans PAc with human salivary components.

A 190-kDa surface protein antigen (PAc) of Streptococcus mutans, in particular the A region of this molecule, may be implicated in the induction of dental caries via an interaction with salivary components. For this reason, it was probably used successfully as an antigenic component for experimental vaccination to prevent dental caries in animals. While developing a synthetic peptide vaccine for dental caries, as reported herein, we have identified a unique peptide, TYEAALKQYEADL, as a candidate vaccinal immunogen. The amino acid sequence of this peptide completely corresponds to the sequence of a B-cell epitope in the A region of PAc and additionally contains its own T-cell epitope for B10.D2 mice within the molecule. This peptide strongly induces the production of only cross-reacting antibodies against PAc. In addition, as demonstrated by surface plasmon resonance analysis using the BIAcore system, these cross-reacting antibodies inhibit approximately 50% of the binding of fluid-phase salivary components to immobilized recombinant PAc.     

Peptide libraries for T cell epitope screening and characterization

Screening with synthetic peptide libraries is the fastest and most efficient method for locating helper T cell (Th) and cytotoxic T cell (Tc) epitopes in proteins of known sequence. Epitope detection and analysis can be performed on clones, lines or freshly isolated ex-vivo T cells. Novel approaches to peptide library design ensure that the peptides are presented in a format which is optimized for Th or Tc epitope detection and analysis. The major histocompatibility complex (MHC) binding motif can be determined by binding or functional assays using analogs of the minimal active sequence.     

Specific epitope-induced conversion of CD8+ memory cells into effector cytotoxic T lymphocytes in vitro: presentation of peptide antigen by CD8+ T cells.

The requirements for the conversion of CD8+ memory T cells into effector class I major histocompatibility complex (MHC) Kd-restricted cytotoxic T (Tc) cells in vitro have been studied. Purified CD8+ splenocytes from influenza A/WSN-primed BALB/c (H-2d) mice stimulated with a synthetic nucleoprotein peptide 147-158 R156- (NPP) alone generated Tc cells specific for influenza virus-infected target cells. No additional requirements for accessory cells or their lymphokine products were necessary indicating that peptide antigen (Ag) in association with Kd was presented on CD8+ T cells. The evidence for presentation of NPP by CD8+ T cells was supported by the use of CD8+ memory T cells from semiallogeneic bone marrow radiation chimeras of P1----F1 type (H-2b----[H-2d x H-2b]F1). Memory CD8+ splenocytes from A/WSN-immune chimeras did not develop into secondary effector Tc cells as a result of a 4-day culture with NPP alone, however, were able to do so if NPP was presented by Kd-bearing Ag-presenting cells. In addition, these results exclude the possibility of direct recognition of free NPP molecules by the specific T cell receptor of CD8+ memory T cells. CD8+ memory splenocytes (H-2b) from chimeras were also able to develop into functionally active Tc cells as a result of presentation of Db-restricted synthetic peptide (NP 366-374) with a sequence derived from influenza virus nucleoprotein with high affinity for Db MHC class I molecules. Blockade of endogenously produced interleukin 2 (IL-2) activity by anti-IL-2 or anti-IL-2 receptor monoclonal antibody in the culture of CD8+ memory T cells during a 4-day NPP stimulation completely abolished Tc cell generation, indicating that the utilization of this lymphokine is absolutely required for the secondary Tc cell development. These findings demonstrate that CD8+ memory T cells per se are able to recognize the restimulating epitope as a result of its presentation by CD8+ T cells and develop into cytolytically active and highly specific Tc cells with no requirements for other cellular helper components or their lymphokine products.     

Antibody recognition of a neutralization epitope on the major outer membrane protein of Chlamydia trachomatis.

Two BALB/c mice were immunized with serovar C Chlamydia trachomatis elementary bodies, and 63 hybridomas producing monoclonal antibodies to C. trachomatis were recovered. Eight hybridomas which were specific for an identical peptide epitope (AGLQND) in serovar C major outer membrane protein variable domain I were identified. Detailed immunochemical study of the antigen-antibody interaction and genetic characterization of the antibody variable-region gene sequences showed that distinct B-cell clonal lineages were elicited by the epitope sequence. Since each antibody had a distinct pattern of fine specificity for recognition of the epitope and displayed different degrees of cross-reactivity with a related serovar (serovar A), we conclude that B-cell recognition of an immunodominant neutralization epitope can be pleiotropic. Differences in B-cell recognition of a neutralization epitope may delay the emergence by mutation of antigenic-drift variants of the C. trachomatis major outer membrane protein.     

Signal peptides and trans-membrane regions are broadly immunogenic and have high CD8+ T cell epitope densities: Implications for vaccine development

Cell mediated immune response has a major role in controlling the elimination of infectious agents. The rational design of sub-unit peptide vaccines against intracellular pathogens or cancer requires the use of antigenic sequence/s that can induce highly potent, long lasting and antigen-specific responses in the majority of the population. A promising peptide selection strategy is the detection of multi-epitope peptide sequences with an ability to bind multiple MHC alleles. While past research sought the best epitopes based on their specific antigenicity, we ask whether specific defined domains have high epitope densities. Signal peptides and trans-membrane domains were found to have exceptionally high epitope densities. The improved MHC binding of these domains relies on their hydrophobic nature and, in signal peptides, also on their specific sequence. The high epitope density of SP was computed using in-silico methods and corroborated by the high percentage of identified SP epitope in the IEDB (immune epitope database). The enhanced immunogenicity of SP was then experimentally confirmed using a panel of nine peptides derived from Mycobacterium tuberculosis (MTb) proteins used in human PBMC proliferation assays and T cell lines functional assays. Our results show the exceptionally high antigen specific response rates and population coverage to SP sequences compared with non-SP peptide antigens derived from the same proteins. The results suggest a novel scheme for the rational design of T cell vaccines using a domain based rather than an epitope based approach.     

Cytotoxic T lymphocyte escape variants, induced mutations, and synthetic peptides define a dominant H-2Kb-restricted determinant in simian virus 40 tumor antigen

Immunization of C57BL/6 mice with syngeneic cells transformed by simian virus 40 large T antigen (SV40 T ag) induces the generation of T antigen-specific cytotoxic T lymphocytes (CTL) which are restricted by the major histocompatibility class I antigens H-2Db and H-2Kb. Previous studies have shown that the H-2Db-restricted CTL response is directed to at least three distinct epitopes (I, II/III, and V) in the SV40 T antigen which have been precisely mapped using deletion mutagenesis and overlapping synthetic peptides. Although in vivo the CTL response to SV40 T antigen is dominated by the H-2Kb class I antigen, the precise location of the H-2Kb-restricted epitope(s) was not known, and whether there was multiplicity of H-2Kb-restricted epitopes remained unclear. In this study, we have defined the minimal recognition epitope for the SV40-specific H-2Kb-restricted CTL clone Y-4 as T antigen residues 404-411 by using T antigen deletion and point mutants and synthetic peptides. DNA sequence analysis of the region encoding residues 404-411 from the T antigens expressed in three independently isolated CTL clone Y-4 escape variants identified inactivating mutations capable of abrogating CTL recognition. Estimation of CTL precursor (CTLp) frequencies by limiting dilution analysis revealed that CTLp specific for epitope IV represent a large percentage of the total CTL response elicited by the intact T antigen in H-2b mice. Immunization of B6 mice with cells expressing a T antigen derivative deleted of residues 404-411 revealed that site IV represents the only immunodominant H-2Kb-restricted epitope within T antigen.     

T-cell, adhesion, and B-cell epitopes of the cell surface Streptococcus mutans protein antigen I/II.

The T-cell and antibody responses to a cell surface streptococcal antigen (SA I/II) were investigated in naturally sensitized humans. Serum antibody responses were directed predominantly to the N-terminal (residues 39 to 481) and central (residues 816 to 1213) regions of SA I/II which may be involved in bacterial adhesion to salivary receptors. T-cell responses were also directed predominantly towards the central region. The linear peptide relationship of the immunodominant and minor T- and B-cell as well as adhesion epitopes was mapped within residues 816 to 1213. Immunodominant T-cell and B-cell epitopes were identified within residues 803 to 853, which were separated in linear sequence from the adhesion epitopes (residues 1005 to 1044). Adhesion epitopes overlapped with minor B- and T-cell epitopes (residues 1005 to 1054 and 1085 to 1134). An immunodominant promiscuous T-cell epitope (residues 985 to 1004) was adjacent to an adhesion epitope (residues 1005 to 1024). The limited B-cell response to adhesion epitopes is consistent with the success of Streptococcus mutans in colonizing the oral cavity. The strategy of T-cell, adhesion, and B-cell epitope mapping has revealed a general approach for identifying components of subunit vaccines which may focus responses to critical functional determinants. Such epitopes of SA I/II may constitute the components of a subunit vaccine against dental caries.     

Trypanosoma cruzi antigen that interacts with the beta1-adrenergic receptor and modifies myocardial contractile activity

Previously, we have demonstrated that plasma membranes from the parasite Trypanosoma cruzi (T. cruzi) recognize and adhere to host cells through parasite surface attachment molecules that have affinity for beta(1)-adrenergic receptors (beta(1)-ARs) on target organs. In this report we identify a parasite protein that not only interacts with beta(1)-ARs, but also displays beta-agonist-like activity. We demonstrate that a recombinant maltose binding protein fusion of Tc13 Tul (MBP-Tc13 Tul), a member of the T. cruzi antigen 13 family of surface antigen proteins, competes for binding sites with the beta-adrenergic receptor antagonist [125I]-CYP on membranes purified both from CHO cells expressing human beta(1)-ARs and from rat atria. The competition is prevented by pre-treating MBP-Tc13 Tul with antibodies directed against the EPKSA repeat domain of Tc13 Tul, implicating this portion of the molecule in binding to the beta(1)-AR. Furthermore, MBP-Tc13 Tul activates rat myocardial beta(1)-ARs, resulting in synthesis of cyclic adenosine monophosphate (cAMP) and an increase in cardiac contractility. These biological effects are selectively suppressed by the beta(1)-AR antagonist atenolol, by a synthetic peptide corresponding to the second extracellular loop of the human beta(1)-AR, and by the anti-EPKSA repeat antibodies. These results imply that the Tc13 Tul cell-surface antigen of T. cruzi plays a central role in misregulating the beta(1)-AR following parasite infection, and may be a causative factor of dysautonomic syndrome described in Chagas' disease.