ROSA MARIA CHURA CHAMBI
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Artigo IPEN-doc 29049 Leptolysin, a Leptospira secreted metalloprotease of the pappalysin family with broad-spectrum activity2022 - COURROL, DANIELLA dos S.; SILVA, CRISTIANE C.F. da; PRADO, LUAN G.; CHURA-CHAMBI, ROSA M.; MORGANTI, LIGIA; SOUZA, GISELE O. de; HEINEMANN, MARCOS B.; ISAAC, LOURDES; CONTE, FERNANDO P.; PORTARO, FERNANDA C.V.; RODRIGUES-DA-SILVA, RODRIGO N.; BARBOSA, ANGELA S.Extracellular proteolytic enzymes are produced by a variety of pathogenic microorganisms, and contribute to host colonization by modulating virulence. Here, we present a first characterization of leptolysin, a Leptospira metalloprotease of the pappalysin family identified in a previous exoproteomic study. Comparative molecular analysis of leptolysin with two other pappalysins from prokaryotes, ulilysin and mirolysin, reveals similarities regarding calcium, zinc, and arginine -binding sites conservation within the catalytic domain, but also discloses peculiarities. Variations observed in the primary and tertiary structures may reflect differences in primary specificities. Purified recombinant leptolysin of L. interrogans was obtained as a ~50 kDa protein. The protease exhibited maximal activity at pH 8.0 and 37°C, and hydrolytic activity was observed in the presence of different salts with maximum efficiency in NaCl. Substrate specificity was assessed using a small number of FRET peptides, and showed a marked preference for arginine residues at the P1 position. L. interrogans leptolysin proteolytic activity on proteinaceous substrates such as proteoglycans and plasma fibronectin was also evaluated. All proteins tested were efficiently degraded over time, confirming the protease´s broad-spectrum activity in vitro. In addition, leptolysin induced morphological alterations on HK-2 cells, which may be partially attributed to extracellular matrix (ECM) degradation. Hemorrhagic foci were observed in the dorsal skin of mice intradermally injected with leptolysin, as a plausible consequence of ECM disarray and vascular endothelium glycocalyx damage. Assuming that leptospiral proteases play an important role in all stages of the infectious process, characterizing their functional properties, substrates and mechanisms of action is of great importance for therapeutic purposes.Artigo IPEN-doc 28852 Recombinant PilS2022 - MUNHOZ, DANIELLE D.; SILVA, JESSIKA C.A.; FREITAS, NATALIA C.; IWAI, LEO K.; AIRES, KARINA A.; OZAKI, CHRISTIANE Y.; SOUZA, CRISTIANE S.; ROCHA, LETICIA B.; SILVA, MIRIAM A.; HENRIQUE, IZABELLA M.; ELIAS, WALDIR P.; CARVALHO, ENEAS; MORGANTI, LIGIA; CHURA-CHAMBI, ROSA M.; PIAZZA, ROXANE M.F.Pil-fimbriae is a type IV pili member, which is a remarkably versatile component with a wide variety of functions, including motility, attachment to different surfaces, electrical conductance, DNA acquisition, and secretion of a broad range of structurally distinct protein substrates. Despite the previous functional characterization of Pil, more studies are required to understand the regulation of Pil expression and production, since the exact mechanisms involved in these steps are still unknown. Therefore it is extremely important to have a protein with the correct secondary and tertiary structure that will enable an accurate characterization and a specific antisera generation. For this reason, the aim of this work was to generate potential tools for further investigations to comprehend the mechanisms involved in Pil regulation and its role in pathogenic E. coli infections with the obtaining of a precise native-like recombinant PilS and the corresponding antisera. The pilS gene was successfully cloned into an expression vector, and recombinant PilS (rPilS) was efficiently solubilized and purified by metal affinity chromatography. Protein characterization analyses indicated that rPilS presented native-like secondary and tertiary structures after the refolding process. The generated anti-rPilS sera efficiently recognized recombinant and native proteins from atypical enteropathogenic E. coli strains.