ROSA MARIA CHURA CHAMBI

ROSA MARIA CHURA CHAMBI

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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.
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.
Protein refolding based on high hydrostatic pressure and alkaline pH
2019 - CHURA-CHAMBI, ROSA M.; SILVA, CLEIDE M.R. da; PEREIRA, LENNON R.; BARTOLINI, PAOLO; FERREIRA, LUIS C. de S.; MORGANTI, LIGIA
In this study we evaluated the association of high hydrostatic pressure (HHP) and alkaline pH as a minimally denaturing condition for the solubilization of inclusion bodies (IBs) generated by recombinant proteins expressed by Escherichia coli strains. The method was successfully applied to a recombinant form of the dengue virus (DENV) non-structural protein 1 (NS1). The minimal pH for IBs solubilization at 1 bar was 12 while a pH of 10 was sufficient for solubilization at HHP: 2.4 kbar for 90 min and 0.4 kbar for 14 h 30 min. An optimal refolding condition was achieved by compression of IBs at HHP and pH 10.5 in the presence of arginine, oxidized and reduced glutathiones, providing much higher yields (up to 8-fold) than association of HHP and GdnHCl via an established protocol. The refolded NS1, 109 ± 9.5 mg/L bacterial culture was recovered mainly as monomer and dimer, corresponding up to 90% of the total protein and remaining immunologically active. The proposed conditions represent an alternative for the refolding of immunologically active recombinant proteins expressed as IBs.
Association of high pressure and alkaline condition for solubilization of inclusion bodies and refolding of the NS1 protein from zika virus
2018 - SILVA, CLEIDE M.R. da; CHURA-CHAMBI, ROSA M.; PEREIRA, LENNON R.; CORDEIRO, YRAIMA; FERREIRA, LUIS C. de S.; MORGANTI, LIGIA
Background: Proteins in inclusion bodies (IBs) present native-like secondary structures. However, chaotropic agents at denaturing concentrations, which are widely used for IB solubilization and subsequent refolding, unfold these secondary structures. Removal of the chaotropes frequently causes reaggregation and poor recovery of bioactive proteins. High hydrostatic pressure (HHP) and alkaline pH are two conditions that, in the presence of low level of chaotropes, have been described as non-denaturing solubilization agents. In the present study we evaluated the strategy of combination of HHP and alkaline pH on the solubilization of IB using as a model an antigenic form of the zika virus (ZIKV) non-structural 1 (NS1) protein. Results: Pressure-treatment (2.4 kbar) of NS1-IBs at a pH of 11.0 induced a low degree of NS1 unfolding and led to solubilization of the IBs, mainly into monomers. After dialysis at pH 8.5, NS1 was refolded and formed soluble oligomers. High (up to 68 mg/liter) NS1 concentrations were obtained by solubilization of NS1-IBs at pH 11 in the presence of arginine (Arg) with a final yield of approximately 80% of total protein content. The process proved to be efficient, quick and did not require further purification steps. Refolded NS1 preserved biological features regarding reactivity with antigen-specific antibodies, including sera of ZIKV-infected patients. The method resulted in an increase of approximately 30-fold over conventional IB solubilization-refolding methods. Conclusions: The present results represent an innovative non-denaturing protein refolding process by means of the concomitant use of HHP and alkaline pH. Application of the reported method allowed the recovery of ZIKV NS1 at a condition that maintained the antigenic properties of the protein.