ROSA MARIA CHURA CHAMBI

ROSA MARIA CHURA CHAMBI

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Leptolysin, a Leptospira secreted metalloprotease of the pappalysin family with broad-spectrum activity
2022 - 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.
Inactivation of the antimicrobial peptide LL-37 by pathogenic Leptospira
2021 - OLIVEIRA, PRISCILA N.; COURROL, DANIELLA S.; CHURA-CHAMBI, ROSA M.; MORGANTI, LIGIA; SOUZA, GISELE O.; FRANZOLIN, MARCIA R.; WUNDER JUNIOR, ELSIO A.; HEINEMANN, MARCOS B.; BARBOSA, ANGELA S.
Leptospires are aerobic, Gram-negative spirochetes with a high invasive capacity. Pathogenic leptospires secrete proteases that inactivate a variety of host’s proteins including molecules of the extracellular matrix and of the human complement system. This strategy, used by several pathogens of medical importance, contributes to bacterial invasion and immune evasion. In the current work we present evidence that Leptospira proteases also target human cathelicidin (LL-37), an antimicrobial peptide that plays an important role in the innate immune response. By using six Leptospira strains, four pathogenic and two saprophytic, we demonstrated that proteases present in the supernatants of pathogenic strains were capable of degrading LL-37 in a time-dependent manner, whereas proteolytic degradation was not observed with the supernatants of the two saprophytic strains. Inactivation of LL-37 was prevented by using the 1,10-phenanthroline inhibitor, thus suggesting the involvement of metalloproteinases in this process. In addition, the antibacterial activity of LL-37 against two Leptospira strains was evaluated. Compared to the saprophytic strain, a greater resistance of the pathogenic strain to the action of the peptide was observed. Our data suggest that the capacity to inactivate the host defense peptide LL-37 may be part of the virulence arsenal of pathogenic Leptospira, and we hypothesize that its inactivation by the bacteria may influence the outcome of the disease.
Leptospira interrogans thermolysin refolded at high pressure and alkaline pH displays proteolytic activity against complement C3
2018 - CHURA-CHAMBI, ROSA M.; FRAGA, TATIANA R.; SILVA, LUDMILA B. da; YAMAMOTO, BRUNO B.; ISAAC, LOURDES; BARBOSA, ANGELA S.; MORGANTI, LIGIA
Enzymes from the thermolysin family are crucial factors in the pathogenesis of several diseases caused by bacteria and are potential targets for therapeutic interventions. Thermolysin encoded by the gene LIC13322 of the causative agent of leptospirosis, Leptospira interrogans, was shown to cleave proteins from the Complement System. However, the production of this recombinant protein using traditional refolding processes with high levels of denaturing reagents for thermolysin inclusion bodies (TL-IBs) solubilization results in poor recovery and low proteolytic activity probably due to improper refolding of the protein. Based on the assumption that leptospiral proteases play a crucial role during infection, the aim of this work was to obtain a functional recombinant thermolysin for future studies on the role of these metalloproteases on leptospiral infection. The association of high hydrostatic pressure (HHP) and alkaline pH was utilized for thermolysin refolding. Incubation of a suspension of TL-IBs at HHP and a pH of 11.0 is non-denaturing but effective for thermolysin solubilization. Soluble protein does not reaggregate by dialysis to pH 8.0. A volumetric yield of 46 mg thermolysin/L of bacterial culture and a yield of near 100% in relation to the total thermolysin present in TL-IBs were obtained. SEC-purified thermolysin suffers fragmentation, likely due to autoproteolysis and presents proteolytic activity against complement C3 α-chain, possibly by a generation of a C3b-like molecule. The proteolytic activity of thermolysin against C3 was time and dose-dependent. The experience gained in this study shall help to establish efficient HHP-based processes for refolding of bioactive proteins from IBs.
Investigation on solubilization protocols in the refolding of the thiredoxin TsnC from Xylella fastidiosa by high hydrostatic pressure approach
2015 - LEMKE, LAURA S.; CHURA-CHAMBI, ROSA M.; RODRIGUES, DANIELLA; CUSSIOL, JOSE R.R.; MALAVASI, NATALIA V.; ALEGRIA, THIAGO G.P.; SOARES NETTO, LUIS E.; MORGANTI, LIGIA
Application of high hydrostatic pressure for recovery of biologically active cruzain from recombinant inclusion bodies
2011 - CHAMBI, ROSA M.C.; LEMKE, LAURA S.; VALLEJO, NATALIA M.; JULIANO NETO, LUIZ; MORGANTI, LIGIA
The effect of temperature on protein refolding at high pressure enhanced green fluorescent protein as a model
2014 - MALAVASI, N.V.; CORDEIRO, Y.; RODRIGUES, D.; CHURA-CHAMBI, R.M.; LEMKE, L.S.; MORGANTI, L.
An analysis of the factors that affect the dissociation of inclusion bodies and the refolding of endostatin under high pressure
2013 - CHURA-CHAMBI, R.M.; CORDEIRO, Y.; MALAVASI, N.V.; LEMKE, L.S.; RODRIGUES, D.; MORGANTI, L.
Refolding of endostatin from inclusion bodies using high hydrostatic pressure
2008 - CHURA-CHAMBI, ROSA M.; GENOVA, LUIS A.; AFFONSO, REGINA; MORGANTI, LIGIA
Refolding on the recombinant protein OmpA70 from Leptospira interrogans from inclusion bodies using high hydrostatic pressure and partial characterization of its immunological properties
2010 - FRAGA, TATIANA R.; CHURA-CHAMBI, ROSA M.; GONCALEZ, AMANE P.; MORAIS, ZENAIDE M.; VASCONCELLOS, SILVIO A.; MORGANTI, LIGIA; MARTINS, ELIZABETH A.L.
Refolding by high pressure of a toxin containing seven disulfide bonds: bothropstoxin-1 from Bothrops jararacussu
2011 - BALDUINO, KELI N.; SPENCER, PATRICK J.; MALAVASI, NATALIA V.; CHURA-CHAMBI, ROSA M.; LEMKE, LAURA S.; MORGANTI, LIGIA