Reconhecimento molecular na doença de chagas do ponto de vista do parasita e do hospedeiro / Molecular recognition in Chagas disease from the point of view of the parasite and the host

A doença de Chagas, causada pelo parasita protozoário Trypanosoma cruzi, afeta milhões de pessoas, a maioria delas vivendo na América latina. Apesar dos avanços da medicina e da biotecnologia, ainda existem poucas opções de tratamento para indivíduos com a doença. Assim, é importante compreendermos os detalhes moleculares da infecção parasitária, para que novas alternativas terapêuticas e de diagnóstico possam ser desenvolvidas para esses pacientes. Neste trabalho estudamos esta doença em duas frentes, uma do ponto de vista do parasita, e a outra, da resposta do hospedeiro. Utilizando bioinformática, identifcamos um peptídeo conservado (denominado TS9) presente nas proteínas de superfície gp85/transsialidases do parasita. Este peptídeo é capaz de promover adesão celular e, na sua forma sintética, inibe a entrada do T. cruzi na célula hospedeira. Análise da estrutura proteica revelou que o peptídeo TS9 encontra-se num domínio do tipo laminina-G, lado-a-lado com o peptídeo FLY, outro peptídeo conservado desta grande família, previamente descrito pelo nosso grupo. Juntos, eles formam um sítio de adesão a citoqueratinas e proteínas de flamento intermediário. Na segunda parte, investigamos os antígenos e epítopos reconhecidos pelas imunoglobulinas de pacientes portadores da doença nas suas diferentes formas clínicas: assintomática e cardiomiopatias, leve ou grave. Criamos uma biblioteca de phage display contendo, virtualmente, todos os fragmentos proteicos existentes no T. cruzi, que foi varrida contra imunoglobulinas para a construção de um mapa da resposta humoral dos pacientes com a doença de Chagas. Nossos resultados mostram que a resposta dos pacientes é complexa, e mais de dois mil epítopos foram mapeados. Muitos deles, como os antígenos B13, SAPA e FRA já foram previamente descritos, validando nosso método. Porém, um grande número de novos epítopos, inclusive contra proteína descritas como hipotéticas ou sem função conhecida, também foram encontrados. Seus papéis na infecção e resposta imune da doença merecem, portanto, atenção. Em resumo, as abordagens e técnicas utilizadas nesta tese são inovadoras, e permitiram a identificação de peptídeos e moléculas que poderão ser úteis para o desenvolvimento de novos métodos diagnósticos e terapêuticos para a doença de Chagas

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, afects millions of people, most of them living in Latin America. Despite advances in medicine and biotechnology, there are still few treatment options for individuals with the disease. Thus, it is important to understand the molecular details of the parasitic infection, so that new therapeutic and diagnostic alternatives can be developed for these patients. In this work, we study this disease in two fronts, one from the point of view of the parasite, and the other, of the response of the host. Using bioinformatics, we identifed a conserved peptide (called TS9) present in the surface proteins gp85 / trans-sialidases of the parasite. This peptide is capable of promoting cell adhesion and, in its synthetic form, inhibits the entry of T. cruzi into the host cell. Analysis of the protein structure revealed that the TS9 peptide is in a laminin-G-like domain, side-by-side with the peptide FLY, another conserved peptide of this large family, previously described by our group. Together, they form an adhesion site to cytokeratins and intermediate flament proteins. In the second part, we investigated the antigens and epitopes recognized by the immunoglobulins of patients with the disease in their diferent clinical forms: asymptomatic and cardiomyopathies, mild or severe. We created a phage display library containing virtually all existing protein fragments in T. cruzi. This library was screened against immunoglobulins for the construction of a humoral response map of patients with Chagas disease. Our results show that the response of the patients is complex, and more than 2,000 epitopes have been mapped. Many of them, such as the B13, SAPA and FRA antigens have been previously described, validating our method. However, a large number of new epitopes, including many against proteins described as hypothetical or with no known function, were also found. Their roles in infection and immune response of the disease deserve, therefore, attention. In summary, the approaches and techniques used in this thesis are innovative and have allowed the identifcation of new peptides and molecules that may be useful for the development of new diagnostic and therapeutic methods for Chagas disease

Epitope mapping from real time kinetic studies - role of crosslinked disulphides and incidental interacting regions in affinity measurements: study with human chorionic gonadotropin and monoclonal antibodies.

Real time kinetic studies were used to map conformational epitopes in human chorionic gonadotropin (hCG) for two monoclonal antibodies (MAbs). The epitopes were identified in the regions (alpha 5--14 and alpha 55--62). The association rate constant (k+1) was found to be altered by chemical modification of hCG, and the ionic strength of the reaction medium. Based on these changes, we propose the presence of additional interactions away from the epitope- paratope region in the hCG-MAb reaction. We have identified such incidental interacting regions (IIRs) in hCG to be the loop region alpha 35--47 and alpha 60--84. The IIRs contribute significantly towards the KA of the interaction. Therefore, in a macromolecular interaction of hCG and its MAb, KA is determined not only by epitopeparatope interaction but also by the interaction of the nonepitopic-nonparatopic IIRs. However, the specificity of the interaction resides exclusively with the epitope-paratope pair.