@ips.pt
Escola Superior de Tecnologia do Barreiro / Departamento de Engenharia Química e Biológica
Instituto Politécnico de Setúbal
Biochemistry, Genetics and Molecular Biology, Biotechnology, Molecular Biology, Bioengineering
Scopus Publications
Gonçalo C. Justino, Catarina P. Nascimento, and Marta C. Justino
Wiley
A computational biochemistry laboratory, fitted for bioinformatics students, is presented. The molecular dynamics package GROMACS is used to prepare and simulate a solvated protein. Students analyze the trajectory with different available tools (GROMACS and VMD) to probe the structural stability of the protein during the simulation. Students are also required to make use of Python libraries and write their own code to probe non‐covalent interactions between the amino acid side chains. Based on these results, students characterize the system in a qualitatively approach but also assess the importance of each specific interaction through time. This work mobilizes biochemical concepts and programming skills, fostering critical thinking and group work and developing presenting skills.
Pedro C. Rosado, Ricardo Meyrelles, Ana M. Macatrão, Marta C. Justino, A. Gabriela Gomes, Maria F. Montemor, Marta M. Alves, Gonçalo C. Justino, Ana P.C. Ribeiro, and Karina Shimizu
Elsevier BV
Gonçalo C. Justino, Pedro F. Pinheiro, Alexandra P. S. Roseiro, Ana S. O. Knittel, João Gonçalves, Marta C. Justino, and M. Fernanda N. N. Carvalho
Royal Society of Chemistry (RSC)
This study identifies novel camphor-derived compounds that bind the CCR5 receptor and can be used as lead compounds for drug discovery.
Marta C. Justino, Margarida R. Parente, Ivo G. Boneca, and Lígia M. Saraiva
Wiley
Helicobacter pylori is a pathogen that infects the gastric mucosa of a large percentage of the human population worldwide, and predisposes to peptic ulceration and gastric cancer. Persistent colonization of humans by H. pylori triggers an inflammatory response that leads to the production of reactive nitrogen species. However, the mechanisms of H. pylori defence against nitrosative stress remain largely unknown. In this study, we show that the NADH‐flavin oxidoreductase FrxA of H. pylori, besides metabolizing nitrofurans and metronidazole, has S‐nitrosoglutathione reductase activity. In agreement with this, inactivation of the FrxA‐encoding gene resulted in a strain that was more sensitive to S‐nitrosoglutathione. FrxA was also shown to contribute to the proliferation of H. pylori in macrophages, which are key phagocytic cells of the mammalian innate immune system. Moreover, FrxA was shown to support the virulence of the pathogen upon mouse infection. Altogether, we provide evidence for a new function of FrxA that contributes to the successful chronic colonization ability that characterizes H. pylori.
Ana F. Tavares, Margarida R. Parente, Marta C. Justino, Mónica Oleastro, Lígia S. Nobre, and Lígia M. Saraiva
Public Library of Science (PLoS)
Helicobacter pylori is a pathogen that establishes long life infections responsible for chronic gastric ulcer diseases and a proved risk factor for gastric carcinoma. The therapeutic properties of carbon-monoxide releasing molecules (CORMs) led us to investigate their effect on H. pylori. We show that H. pylori 26695 is susceptible to two widely used CORMs, namely CORM-2 and CORM-3. Also, several H. pylori clinical isolates were killed by CORM-2, including those resistant to metronidazole. Moreover, sub-lethal doses of CORM-2 combined with metronidazole, amoxicillin and clarithromycin was found to potentiate the effect of the antibiotics. We further demonstrate that the mechanisms underpinning the antimicrobial effect of CORMs involve the inhibition of H. pylori respiration and urease activity. In vivo studies done in key cells of the innate immune system, such as macrophages, showed that CORM-2, either alone or when combined with metronidazole, strongly reduces the ability of H. pylori to infect animal cells. Hence, CORMs have the potential to kill antibiotic resistant strains of H. pylori.
Marta C. Justino, Chantal Ecobichon, André F. Fernandes, Ivo G. Boneca, and Lígia M. Saraiva
Mary Ann Liebert Inc
AIMS
The ability of pathogens to cope with the damaging effects of nitric oxide (NO), present in certain host niches and produced by phagocytes that support innate immunity, relies on multiple strategies that include the action of detoxifying enzymes. As for many other pathogens, these systems remained unknown for Helicobacter pylori. This work aimed at identifying and functionally characterizing an H. pylori system involved in NO protection.
RESULTS
In the present work, the hp0013 gene of H. pylori is shown to be related to NO resistance, as its inactivation increases the susceptibility of H. pylori to nitrosative stress, and significantly decreases the NADPH-dependent NO reduction activity of H. pylori cells. The recombinant HP0013 protein is able to complement an NO reductase-deficient Escherichia coli strain and exhibits significant NO reductase activity. Mutation of hp0013 renders H. pylori more vulnerable to nitric oxide synthase-dependent macrophage killing, and decreases the ability of the pathogen to colonize mice stomachs.
INNOVATION
Phylogenetic studies reveal that HP0013, which shares no significant amino acid sequence similarity to the other so far known microbial NO detoxifiers, belongs to a novel family of proteins with a widespread distribution in the microbial world.
CONCLUSION
H. pylori HP0013 represents an unprecedented enzymatic NO detoxifying system for the in vivo microbial protection against nitrosative stress.
Joana M. Baptista, Marta C. Justino, Ana M. P. Melo, Miguel Teixeira, and Lígia M. Saraiva
American Society for Microbiology
ABSTRACT Mammalian cells of innate immunity respond to pathogen invasion by activating proteins that generate a burst of oxidative and nitrosative stress. Pathogens defend themselves from the toxic compounds by triggering a variety of detoxifying enzymes. Escherichia coli flavorubredoxin is a nitric oxide reductase that is expressed under nitrosative stress conditions. We report that in contrast to nitrosative stress alone, exposure to both nitrosative and oxidative stresses abolishes the expression of flavorubredoxin. Electron paramagnetic resonance (EPR) experiments showed that under these conditions, the iron center of the flavorubredoxin transcription activator NorR loses the ability to bind nitric oxide. Accordingly, triggering of the NorR ATPase activity, a requisite for flavorubredoxin activation, was impaired by treatment of the protein with the double stress. Studies of macrophages revealed that the contribution of flavorubredoxin to the survival of E. coli depends on the stage of macrophage infection and that the lack of protection observed at the early phase is related to inhibition of NorR activity by the oxidative burst. We propose that the time-dependent activation of flavorubredoxin contributes to the adaptation of E. coli to the different fluxes of hydrogen peroxide and nitric oxide to which the bacterium is subjected during the course of macrophage infection.
Claire E. Vine, Marta C. Justino, Lígia M. Saraiva, and Jeffrey Cole
Elsevier BV
Marta C. Justino, Joana M. Baptista, and Lígia M. Saraiva
Springer Science and Business Media LLC
Smilja Todorovic, Marta C. Justino, Gerd Wellenreuther, Peter Hildebrandt, Daniel H. Murgida, Wolfram Meyer-Klaucke, and Lígia M. Saraiva
Springer Science and Business Media LLC
Tim W. Overton, Marta C. Justino, Ying Li, Joana M. Baptista, Ana M. P. Melo, Jeffrey A. Cole, and Lígia M. Saraiva
American Society for Microbiology
ABSTRACT Expression of two genes of unknown function, Staphylococcus aureus scdA and Neisseria gonorrhoeae dnrN , is induced by exposure to oxidative or nitrosative stress. We show that DnrN and ScdA are di-iron proteins that protect their hosts from damage caused by exposure to nitric oxide and to hydrogen peroxide. Loss of FNR-dependent activation of aniA expression and NsrR-dependent repression of norB and dnrN expression on exposure to NO was restored in the gonococcal parent strain but not in a dnrN mutant, suggesting that DnrN is necessary for the repair of NO damage to the gonococcal transcription factors, FNR and NsrR. Restoration of aconitase activity destroyed by exposure of S. aureus to NO or H 2 O 2 required a functional scdA gene. Electron paramagnetic resonance spectra of recombinant ScdA purified from Escherichia coli confirmed the presence of a di-iron center. The recombinant scdA plasmid, but not recombinant plasmids encoding the complete Escherichia coli sufABCDSE or iscRSUAhscBAfdx operons, complemented repair defects of an E. coli ytfE mutant. Analysis of the protein sequence database revealed the importance of the two proteins based on the widespread distribution of highly conserved homologues in both gram-positive and gram-negative bacteria that are human pathogens. We provide in vivo and in vitro evidence that Fe-S clusters damaged by exposure to NO and H 2 O 2 can be repaired by this new protein family, for which we propose the name r epair of i ron c enters, or RIC, proteins.
João B. Vicente, Marta C. Justino, Vera L. Gonçalves, Lígia M. Saraiva, and Miguel Teixeira
Elsevier
Marta C. Justino, Cláudia C. Almeida, Miguel Teixeira, and Lígia M. Saraiva
Elsevier BV
DNA microarray experiments showed that the expression of the Escherichia coli ytfE gene is highly increased upon exposure to nitric oxide. We also reported that deletion of ytfE significantly alters the phenotype of E. coli, generating a strain with enhanced susceptibility to nitrosative stress and defective in the activity of several iron-sulfur-containing proteins. In this work, it is shown that the E. coli ytfE confers protection against oxidative stress. Furthermore, we found that the damage of the [4Fe-4S]2+ clusters of aconitase B and fumarase A caused by exposure to hydrogen peroxide and nitric oxide stress occurs at higher rates in the absence of ytfE. The ytfE null mutation also abolished the recovery of aconitase and fumarase activities, which is observed in wild type E. coli once the stress is scavenged. Notably, upon the addition of purified holo-YtfE protein to the mutant cell extracts, the enzymatic activities of fumarase and aconitase are fully recovered and at rates similar to the wild type strain. We concluded that YtfE is critical for the repair of iron-sulfur clusters damaged by oxidative and nitrosative stress conditions.
Marta C. Justino, Cláudia C. Almeida, Vera L. Gonçalves, Miguel Teixeira, and LÃgia M. Saraiva
Oxford University Press (OUP)
Our previous analysis of the transcriptome of Escherichia coli under nitrosative stress showed that the ytfE gene was one of the highest induced genes. Furthermore, the E. coli strain mutated on the ytfE gene was found to be more sensitive to nitric oxide than the wild-type strain. In the present work, we show that the mutation of the ytfE gene in E. coli yielded a strain that grows poorly under anaerobic respiratory conditions and that has an increased sensitivity to iron starvation. Furthermore, all examined iron-sulphur proteins have decreased activity levels in the strain lacking ytfE. Altogether, the results suggest a role for ytfE in iron-sulphur cluster biogenesis. YtfE was overexpressed in E. coli and it is shown to contain a di-iron centre of the histidine-carboxylate family.
Marta C. Justino, Vera M.M. Gonçalves, and Lígia M. Saraiva
Elsevier BV
Marta C. Justino, João B. Vicente, Miguel Teixeira, and Lígia M. Saraiva
Elsevier BV
Nitric oxide produced by activated macrophages plays a key role as one of the immune system's weapons against pathogens. Because the lifetime of nitric oxide is short in aerobic conditions, whereas in anaerobic conditions the cytotoxic effects of nitric oxide are greatly increased as in the infection/inflammation processes, it is important to establish which systems are able to detoxify nitric oxide under anaerobic conditions. In the present work a new set of Escherichia coli K-12 genes conferring anaerobic resistance to nitric oxide is presented, namely the gene product of YtfE and a potential transcriptional regulator of the helix-turn-helix LysR-type (YidZ). The crucial role of flavohemoglobin for anaerobic nitric oxide protection is also demonstrated. Furthermore, nitric oxide is shown to cause a significant alteration of the global E. coli gene transcription profile that includes the increase of the transcript level of genes encoding for detoxification enzymes, iron-sulfur cluster assembly systems, DNA-repairing enzymes, and stress response regulators.