Relevance of the Diversity among Members of the Trypanosoma Cruzi Trans-Sialidase Family Analyzed with Camelids Single-Domain Antibodies

The sialic acid present in the protective surface mucin coat of Trypanosoma cruzi is added by a membrane anchored trans-sialidase (TcTS), a modified sialidase that is expressed from a large gene family. In this work, we analyzed single domain camelid antibodies produced against trans-sialidase. Llamas were immunized with a recombinant trans-sialidase and inhibitory single-domain antibody fragments were obtained by phage display selection, taking advantage of a screening strategy using an inhibition test instead of the classic binding assay. Four single domain antibodies displaying strong trans-sialidase inhibition activity against the recombinant enzyme were identified. They share the same complementarity-determining region 3 length (17 residues) and have very similar sequences. This result indicates that they likely derived from a unique clone. Probably there is only one structural solution for tight binding inhibitory antibodies against the TcTS used for immunization. To our surprise, this single domain antibody that inhibits the recombinant TcTS, failed to inhibit the enzymatic activity present in parasite extracts. Analysis of individual recombinant trans-sialidases showed that enzymes expressed from different genes were inhibited to different extents (from 8 to 98%) by the llama antibodies. Amino acid changes at key positions are likely to be responsible for the differences in inhibition found among the recombinant enzymes. These results suggest that the presence of a large and diverse trans-sialidase family might be required to prevent the inhibitory response against this essential enzyme and might thus constitute a novel strategy of T. cruzi to evade the host immune system

Genetic predisposition and cellular basis for ischemia-induced ST-segment changes and arrhythmias.

Ventricular tachycardia and fibrillation (VT/VF) complicating Brugada syndrome, a genetic disorder linked to SCN5A mutations, and VF complicating acute myocardial infarction (AMI) have both been linked to phase 2 reentry. Because of these mechanistic similarities in arrhythmogenesis, we examined the contribution of SCN5A mutations to VT/VF complicating AMI. Nineteen consecutive patients developing VF during AMI were enrolled. Wild-type (WT) and mutant SCN5A genes were co-expressed with SCN1B in TSA201 cells and studied using whole-cell patch-clamp techniques. One missense mutation (G400A) in SCN5A was detected in a conserved region among the cohort of 19 patients. A H558R polymorphism was detected on the same allele. Unlike the other 18 patients who each developed 1-2 VF episodes during acute MI, the mutation carrier developed six episodes of VT/VF within the first 12 hours. All VT/VF episodes were associated with ST segment changes and were initiated by short-coupled extrasystoles. We describe the first sodium channel mutation to be associated with the development of an arrhythmic storm during acute ischemia. These findings suggest that a loss of function in SCN5A may predispose to ischemia induced arrhythmic storm. These results could be very useful for forensic implications regarding genetic screening in relatives

Trypanosoma cruzi TcSMUG L-surface Mucins Promote Development and Infectivity in the Triatomine Vector Rhodnius prolixus

Made available in DSpace on 2015-08-19T13:49:31Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) eloi_garcia_etal_IOC_2013.pdf: 6467561 bytes, checksum: 3d08464f54865b8fee87e5456fed719c (MD5) Previous issue date: 2013Universidade Federal Fluminense. Instituto de Biologia. Departamento de Biologia Geral. Laboratório de Biologia de Insetos. Niterói, RJ, Brasil / Instituto Nacional de Entomologia Molecular (INCT-EM, CNPq). Brasil.Universidade Federal Fluminense. Instituto de Biologia. Departamento de Biologia Geral. Laboratório de Biologia de Insetos. Niterói, RJ, Brasil.Instituto Nacional de Entomologia Molecular (INCT-EM, CNPq). Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Universidade Estadual do Norte Fluminense - Horto. Centro de Biocieˆncias e Biotecnologia. Laborato´ rio de Biologia Celular e Tecidual. Campos dos Goytacases, RJ, Brasil.Universidade Federal Fluminense. Instituto de Biologia. Departamento de Biologia Geral. Laboratório de Biologia de Insetos. Niterói, RJ, Brasil / Instituto Nacional de Entomologia Molecular (INCT-EM, CNPq). Brasil.Universidad Nacional de San Martín (UNSAM) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Instituto de Investigaciones Biotecnológicas ‘‘Dr Rodolfo Ugalde’’. Campus UNSAM. Buenos Aires, Argentina.Universidad Nacional de San Martín (UNSAM) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Instituto de Investigaciones Biotecnológicas ‘‘Dr Rodolfo Ugalde’’. Campus UNSAM. Buenos Aires, Argentina.nstituto Nacional de Entomologia Molecular (INCT-EM, CNPq). Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Universidad Nacional de San Martín (UNSAM) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Instituto de Investigaciones Biotecnológicas ‘‘Dr Rodolfo Ugalde’’. Campus UNSAM. Buenos Aires, Argentina.Background: TcSMUG L products were recently identified as novel mucin-type glycoconjugates restricted to the surface of insect-dwelling epimastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. The remarkable conservation of their predicted mature N-terminal region, which is exposed to the extracellular milieu, suggests that TcSMUG L products may be involved in structural and/or functional aspects of the interaction with the insect vector. Methodology and Principal Findings: Here, we investigated the putative roles of TcSMUG L mucins in both in vivo development and ex vivo attachment of epimastigotes to the luminal surface of the digestive tract of Rhodnius prolixus. Our results indicate that the exogenous addition of TcSMUG L N-terminal peptide, but not control T. cruzi mucin peptides, to the infected bloodmeal inhibited the development of parasites in R. prolixus in a dose-dependent manner. Pre-incubation of insect midguts with the TcSMUG L peptide impaired the ex vivo attachment of epimastigotes to the luminal surface epithelium, likely by competing out TcSMUG L binding sites on the luminal surface of the posterior midgut, as revealed by fluorescence microscopy. Conclusion and Significance: Together, these observations indicate that TcSMUG L mucins are a determinant of both adhesion of T. cruzto the posterior midgut epithelial cells of the triatomine, and the infection of the insect vector, R. prolixus