ROSA MARIA CHURA CHAMBI

ROSA MARIA CHURA CHAMBI

Página do item completo

Resultados de Busca

Agora exibindo 1 - 10 de 41

Proteolytic activity of secreted proteases from pathogenic leptospires and effects on phagocytosis by murine macrophages
2023 - AMAMURA, THAIS; COURROL, DANIELLA; BARBOSA, ANGELA; SILVA JÚNIOR, ILDEFONSO; SILVA, TIAGO da; MIDON, LEONARDO; HEINEMANN, MARCOS B.; CHAMBI, ROSA C.; MORGANTI, LIGIA; ISAAC, LOURDES
Leptospirosis is a zoonosis caused by spirochete bacteria that belong to the genus Leptospira. This disease represents a serious public health problem, especially in developing countries with tropical and subtropical temperatures. Pathogenic leptospires escape from the Complement System, a property that permits them to survive in vitro when in contact with normal human serum (NHS). In a previous study carried out by our group, it was observed that culture supernatants from different pathogenic species of leptospires (SPL) contain proteases that cleave many Complement proteins, including the central molecule C3 and its fragments C3b and iC3b. Our hypothesis is that these proteases, could decrease the phagocytic clearance of leptospires. Using flow cytometry, we observed decreased amounts of CR3 and CR4 in murine peritoneal macrophages treated with SPL for 24 h. By confocal microscopy, we observed reduction in TLR2, CD11b and CD206 levels when these cells were treated with SPL and recombinant thermolysin for 24 h. Furthermore, opsonins such as C3b/iC3b deposited on the surface of pathogenic leptospires were observed to be completely degraded in the presence of SPL or recombinant thermolysin. Finally, we decided to investigate the phagocytosis of pathogenic leptospires by macrophages in the presence of these proteases. We observed an increase of phagocytosis of leptospires opsonized with normal mouse serum even when macrophages were treated with the proteases. However, when opsonized bacteria were also incubated with SPL, recombinant thermolysin and recombinant leptolysin., there was a decline in leptospires phagocytosis. This suggests that the proteolytic activity can affect phagocytosis by peritoneal macrophages mainly through the degradation of opsonins deposited in the membrane of leptospires. These observations lead us to suggest that proteases secreted by pathogenic leptospires could degrade opsonins present in normal serum or deposited in the bacterial membrane as well as cleave or inhibit macrophage surface molecules. Therefore, these proteases could interfere with the recognition and internalization by murine macrophages, favoring the spread of leptospires in the host.
Human growth hormone inclusion bodies present native‑like secondary and tertiary structures which can be preserved by mild solubilization for refolding
2022 - CHURA-CHAMBI, ROSA M.; FARAH, CHUCK S.; MORGANTI, LIGIA
Background: Native-like secondary structures and biological activity have been described for proteins in inclusion bodies (IBs). Tertiary structure analysis, however, is hampered due to the necessity of mild solubilization conditions. Denaturing reagents used for IBs solubilization generally lead to the loss of these structures and to consequent reaggregation due to intermolecular interactions among exposed hydrophobic domains after removal of the solubilization reagent. The use of mild, non-denaturing solubilization processes that maintain existing structures could allow tertiary structure analysis and increase the efficiency of refolding. Results: In this study we use a variety of biophysical methods to analyze protein structure in human growth hormone IBs (hGH-IBs). hGH-IBs present native-like secondary and tertiary structures, as shown by far and near-UV CD analysis. hGH-IBs present similar λmax intrinsic Trp fluorescence to the native protein (334 nm), indicative of a native-like tertiary structure. Similar fluorescence behavior was also obtained for hGH solubilized from IBs and native hGH at pH 10.0 and 2.5 kbar and after decompression. hGH-IBs expressed in E. coli were extracted to high yield and purity (95%) and solubilized using non-denaturing conditions [2.4 kbar, 0.25 M arginine (pH 10), 10 mM DTT]. After decompression, the protein was incubated at pH 7.4 in the presence of the glutathione-oxidized glutathione (GSH-GSSG) pair which led to intramolecular disulfide bond formation and refolded hGH (81% yield). Conclusions: We have shown that hGH-IBs present native-like secondary and tertiary structures and that non-denaturing methods that aim to preserve them can lead to high yields of refolded protein. It is likely that the refolding process described can be extended to different proteins and may be particularly useful to reduce the pH required for alkaline solubilization.
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.
Recombinant PilS
2022 - 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.
High level SARS-CoV-2 nucleocapsid refolding using mild condition for inclusion bodies solubilization
2022 - CHURA-CHAMBI, ROSA M.; PRIETO-DA-SILVA, ALVARO R. de B.; DI LELA, MATHEUS M.; OLIVEIRA, JOAO E.; ABREU, PATRICIA E.A.; MEIRELES, LUCIANA R.; ANDRADE JUNIOR, HEITOR F. de; MORGANTI, LIGIA
SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.
Prime-boost with Chikungunya virus E2 envelope protein combined with Poly (I:C) induces specific humoral and cellular immune responses
2021 - AMARAL, MARCELO P.; COIRADA, FERNANDA C.; APOSTOLICO, JULIANA de S.; TOMITA, NADIA; FERNANDES, EDGAR R.; SOUZA, HIGO F.S.; CHURA-CHAMBI, ROSA M.; MORGANTI, LIGIA; BOSCARDIN, SILVIA B.; ROSA, DANIELA S.
Chikungunya virus (CHIKV) is an arbovirus transmitted to humans mainly by the bite of infected Aedes aegypti and Aedes albopictus mosquitoes. CHIKV illness is characterized by fever and long-lasting arthritic symptoms, and in some cases it is a deadly disease. The CHIKV envelope E2 (E2CHIKV) glycoprotein is crucial for virus attachment to the cell. Furthermore, E2CHIKV is the immunodominant protein and the main target of neutralizing antibodies. To date, there is no available prophylactic vaccine or specific treatment against CHIKV infection. Here, we designed and produced a DNA vaccine and a recombinant protein containing a consensus sequence of E2CHIKV. C57BL/6 mice immunized twice with the E2CHIKV recombinant protein in the presence of the adjuvant Poly (I:C) induced the highest E2CHIKV-specific humoral and cellular immune responses, while the immunization with the homologous DNA vaccine pVAX-E2CHIKV was able to induce specific IFN-γ producing cells. The heterologous prime-boost strategy was also able to induce specific cellular and humoral immune responses that were, in general, lower than the responses induced by the homologous E2CHIKV recombinant protein immunization. Furthermore, recombinant E2CHIKV induced the highest titers of neutralizing antibodies. Collectively, we believe this is the first report to analyze E2CHIKV-specific humoral and cellular immune responses after immunization with E2CHIKV recombinant protein and DNA pVAX-E2CHIKV vaccine platforms.
Rational selection of hidden epitopes for a molecularly imprinted electrochemical sensor in the recognition of heat-denatured dengue NS1 protein
2021 - SILVA, MATHEUS S.; TAVARES, ANA P.M.; COELHO, LUIZ F.L.; DIAS, LIGIA E.M.F.; CHURA-CHAMBI, ROSA M.; FONSECA, FLAVIO G. da; SALES, MARIA G.F.; FIGUEIREDO, EDUARDO C.
Rational selection of predicted peptides to be employed as templates in molecular imprinting was carried out for the heat-denatured non-structural protein 1 (NS1) of dengue virus (DENV). Conservation analysis among 301 sequences of Brazilian isolates of DENV and zika virus (ZIKV) NS1 was carried out by UniProtKB, and peptide selection was based on in silico data of the conservational, structural and immunogenic properties of the sequences. The selected peptide (from dengue 1 NS1) was synthesized and employed as a template in the electropolymerization of polyaminophenol-imprinted films on the surface of carbon screen-printed electrodes. Heat denaturation of the protein was carried out prior to analysis, in order to expose its internal hidden epitopes. After removal of the template, the molecularly imprinted cavities were able to rebind to the whole denatured protein as determined by electrochemical impedance spectroscopy. This label-free sensor was efficient to distinguish the NS1 of DENV from the NS1 of ZIKV. Additionally, the sensor was also selective for dengue NS1, in comparison with human serum immunoglobulin G and human serum albumin. Additionally, the device was able to detect the DENV NS1 at concentrations from 50 to 200 μg L−1 (RSD below 5.04%, r = 0.9678) in diluted human serum samples. The calculated LOD and LOQ were, respectively, 29.3 and 88.7 μg L−1 and each sensor could be used for six sequential cycles with the same performance.
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.
Enhanced immune responses and protective immunity to Zika virus induced by a DNA vaccine encoding a chimeric NS1 fused with type 1 Herpes virus gD protein
2020 - PEREIRA, LENNON R.; ALVES, RUBENS P. dos S.; SALES, NATIELY S.; ANDREATA-SANTOS, ROBERT; VENCESLAU-CARVALHO, ALEXIA A.; PEREIRA, SAMUEL S.; CASTRO-AMARANTE, MARIA F.; RODRIGUES-JESUS, MONICA J.; FAVARO, MARIANNA T. de P.; CHURA-CHAMBI, ROSA M.; MORGANTI, LIGIA; FERREIRA, LUIS C. de S.
Zika virus (ZIKV) is a globally-distributed flavivirus transmitted to humans by Aedes mosquitoes, usually causing mild symptoms that may evolve to severe conditions, including neurological alterations, such as neonatal microcephaly and Guillain-Barré syndrome. Due to the absence of specific and effective preventive methods, we designed a new subunit vaccine based on a DNA vector (pgDNS1-ZIKV) encoding the non-structural protein 1 (NS1) genetically fused to the Herpes Simplex Virus (HSV) glycoprotein D (gD) protein. Recombinant plasmids were replicated in Escherichia coli and the expression of the target protein was confirmed in transfected HEK293 cells. C57BL/6 and AB6 (IFNAR1–/–) mice were i.m. immunized by electroporation in order to evaluate pgDNS1-ZIKV immunogenicity. After two doses, high NS1-specific IgG antibody titers were measured in serum samples collected from pgDNS1-ZIKV-immunized mice. The NS1-specific antibodies were capable to bind the native protein expressed in infected mammalian cells. Immunization with pgDNS1-ZIKV increased both humoral and cellular immune responses regarding mice immunized with a ZIKV NS1 encoding vaccine. Immunization with pgDNS1-ZIKV reduced viremia and morbidity scores leading to enhanced survival of immunodeficient AB6 mice challenged with a lethal virus load. These results give support to the use of ZIKV NS1 as a target antigen and further demonstrate the relevant adjuvant effects of HSV-1 gD.
Natural versus recombinant viral antigens in SARS-CoV-2 serology
2020 - MEIRELES, LUCIANA R.; SILVA, ANGELICA M.F. da; CARVALHO, CAMILA A.; KESPER, NORIVAL; GALISTEO JUNIOR, ANDRES J.; SOARES, CAMILA P.; ARAUJO, DANIELLE B.; DURIGON, EDISON L.; OLIVEIRA, DANIELLE B.L.; MORGANTI, LIGIA; CHURA-CHAMBI, ROSA M.; ANDRADE JUNIOR, HEITOR F. de
OBJECTIVES: COVID-19 is a public health emergency of international concern whose detection in recovered asymptomatic patients is dependent on accurate diagnosis as it enables the estimation of the susceptibility of the population to the infection. This demand has resulted in the development of several commercial assays employing recombinant proteins, but the results of these assays are not reliable as they do not involve comparison with natural viral antigens. We independently used the SARS-CoV-2 whole viral antigen (WVA) and recombinant nucleocapsid protein (rNP) to develop in-house ELISAs for IgG detection; the results of these ELISAs were then compared to obtain reliable results. METHODS: WVA and rNP ELISAs were performed on COVID-19 negative sera from patients before the pandemic in Brazil, and on RT-qPCR-positive or SARS-CoV-2-IgG against rNP and IgG against WVA–positive samples from recently infected patients in Sao Paulo, Brazil. RESULTS: Both ELISAs detected a large fraction of infected patients but exhibited certain drawbacks. Higher signals and lower numbers of false-negatives were observed in rNP ELISA; however, a higher fraction of falsepositives was observed in control groups. A high number of false-negatives was observed with WVA ELISA. Correlating the results of rNP and WVA ELISAs resulted in improved performance for COVID-19 diagnosis. CONCLUSION: The choice of antigen is an important aspect in optimizing the laboratory diagnosis of COVID-19. The use of rNP ELISA for the detection of anti-SARS-CoV-2 IgG antibodies seems promising, but comparison of the results with those of WVA ELISA is crucial for accurate test development prior to commercialization. IgG serology using several assays, and with the spectral patterns of SARS-CoV-2, resulted in confusing information that must be clarified before the establishment of diagnostic serology criteria.