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Disarming Pseudomonas aeruginosa Virulence by the Inhibitory Action of 1,10-Phenanthroline-5,6-Dione-Based Compounds: Elastase B (LasB) as a Chemotherapeutic Target

Elastase B (lasB) is a multifunctional metalloenzyme secreted by the gram-negative pathogen Pseudomonas aeruginosa, and this enzyme orchestrates several physiopathological events during bacteria-host interplays. LasB is considered to be a potential target for the development of an innovative chemoth...

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Autores principales: Galdino, Anna Clara M., Viganor, Lívia, de Castro, Alexandre A., da Cunha, Elaine F. F., Mello, Thaís P., Mattos, Larissa M., Pereira, Marcos D., Hunt, Mary C., O’Shaughnessy, Megan, Howe, Orla, Devereux, Michael, McCann, Malachy, Ramalho, Teodorico C., Branquinha, Marta H., Santos, André L. S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688126/
https://www.ncbi.nlm.nih.gov/pubmed/31428062
http://dx.doi.org/10.3389/fmicb.2019.01701
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author Galdino, Anna Clara M.
Viganor, Lívia
de Castro, Alexandre A.
da Cunha, Elaine F. F.
Mello, Thaís P.
Mattos, Larissa M.
Pereira, Marcos D.
Hunt, Mary C.
O’Shaughnessy, Megan
Howe, Orla
Devereux, Michael
McCann, Malachy
Ramalho, Teodorico C.
Branquinha, Marta H.
Santos, André L. S.
author_facet Galdino, Anna Clara M.
Viganor, Lívia
de Castro, Alexandre A.
da Cunha, Elaine F. F.
Mello, Thaís P.
Mattos, Larissa M.
Pereira, Marcos D.
Hunt, Mary C.
O’Shaughnessy, Megan
Howe, Orla
Devereux, Michael
McCann, Malachy
Ramalho, Teodorico C.
Branquinha, Marta H.
Santos, André L. S.
author_sort Galdino, Anna Clara M.
collection PubMed
description Elastase B (lasB) is a multifunctional metalloenzyme secreted by the gram-negative pathogen Pseudomonas aeruginosa, and this enzyme orchestrates several physiopathological events during bacteria-host interplays. LasB is considered to be a potential target for the development of an innovative chemotherapeutic approach, especially against multidrug-resistant strains. Recently, our group showed that 1,10-phenanthroline-5,6-dione (phendione), [Ag(phendione)(2)]ClO(4) (Ag-phendione) and [Cu(phendione)(3)](ClO(4))(2).4H(2)O (Cu-phendione) had anti-P. aeruginosa action against both planktonic- and biofilm-growing cells. In the present work, we have evaluated the effects of these compounds on the (i) interaction with the lasB active site using in silico approaches, (ii) lasB proteolytic activity by using a specific fluorogenic peptide substrate, (iii) lasB gene expression by real time-polymerase chain reaction, (iv) lasB protein secretion by immunoblotting, (v) ability to block the damages induced by lasB on a monolayer of lung epithelial cells, and (vi) survivability of Galleria mellonella larvae after being challenged with purified lasB and lasB-rich bacterial secretions. Molecular docking analyses revealed that phendione and its Ag(+) and Cu(2+) complexes were able to interact with the amino acids forming the active site of lasB, particularly Cu-phendione which exhibited the most favorable interaction energy parameters. Additionally, the test compounds were effective inhibitors of lasB activity, blocking the in vitro cleavage of the peptide substrate, aminobenzyl-Ala-Gly-Leu-Ala-p-nitrobenzylamide, with Cu-phendione having the best inhibitory action (K(i) = 90 nM). Treating living bacteria with a sub-inhibitory concentration (½ × MIC value) of the test compounds caused a significant reduction in the expression of the lasB gene as well as its mature protein production/secretion. Further, Ag-phendione and Cu-phendione offered protective action for lung epithelial cells, reducing the A549 monolayer damage by approximately 32 and 42%, respectively. Interestingly, Cu-phendione mitigated the toxic effect of both purified lasB molecules and lasB-containing bacterial secretions in the in vivo model, increasing the survival time of G. mellonella larvae. Collectively, these data reinforce the concept of lasB being a veritable therapeutic target and phendione-based compounds (mainly Cu-phendione) being prospective anti-virulence drugs against P. aeruginosa.
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spelling pubmed-66881262019-08-19 Disarming Pseudomonas aeruginosa Virulence by the Inhibitory Action of 1,10-Phenanthroline-5,6-Dione-Based Compounds: Elastase B (LasB) as a Chemotherapeutic Target Galdino, Anna Clara M. Viganor, Lívia de Castro, Alexandre A. da Cunha, Elaine F. F. Mello, Thaís P. Mattos, Larissa M. Pereira, Marcos D. Hunt, Mary C. O’Shaughnessy, Megan Howe, Orla Devereux, Michael McCann, Malachy Ramalho, Teodorico C. Branquinha, Marta H. Santos, André L. S. Front Microbiol Microbiology Elastase B (lasB) is a multifunctional metalloenzyme secreted by the gram-negative pathogen Pseudomonas aeruginosa, and this enzyme orchestrates several physiopathological events during bacteria-host interplays. LasB is considered to be a potential target for the development of an innovative chemotherapeutic approach, especially against multidrug-resistant strains. Recently, our group showed that 1,10-phenanthroline-5,6-dione (phendione), [Ag(phendione)(2)]ClO(4) (Ag-phendione) and [Cu(phendione)(3)](ClO(4))(2).4H(2)O (Cu-phendione) had anti-P. aeruginosa action against both planktonic- and biofilm-growing cells. In the present work, we have evaluated the effects of these compounds on the (i) interaction with the lasB active site using in silico approaches, (ii) lasB proteolytic activity by using a specific fluorogenic peptide substrate, (iii) lasB gene expression by real time-polymerase chain reaction, (iv) lasB protein secretion by immunoblotting, (v) ability to block the damages induced by lasB on a monolayer of lung epithelial cells, and (vi) survivability of Galleria mellonella larvae after being challenged with purified lasB and lasB-rich bacterial secretions. Molecular docking analyses revealed that phendione and its Ag(+) and Cu(2+) complexes were able to interact with the amino acids forming the active site of lasB, particularly Cu-phendione which exhibited the most favorable interaction energy parameters. Additionally, the test compounds were effective inhibitors of lasB activity, blocking the in vitro cleavage of the peptide substrate, aminobenzyl-Ala-Gly-Leu-Ala-p-nitrobenzylamide, with Cu-phendione having the best inhibitory action (K(i) = 90 nM). Treating living bacteria with a sub-inhibitory concentration (½ × MIC value) of the test compounds caused a significant reduction in the expression of the lasB gene as well as its mature protein production/secretion. Further, Ag-phendione and Cu-phendione offered protective action for lung epithelial cells, reducing the A549 monolayer damage by approximately 32 and 42%, respectively. Interestingly, Cu-phendione mitigated the toxic effect of both purified lasB molecules and lasB-containing bacterial secretions in the in vivo model, increasing the survival time of G. mellonella larvae. Collectively, these data reinforce the concept of lasB being a veritable therapeutic target and phendione-based compounds (mainly Cu-phendione) being prospective anti-virulence drugs against P. aeruginosa. Frontiers Media S.A. 2019-08-02 /pmc/articles/PMC6688126/ /pubmed/31428062 http://dx.doi.org/10.3389/fmicb.2019.01701 Text en Copyright © 2019 Galdino, Viganor, de Castro, da Cunha, Mello, Mattos, Pereira, Hunt, O’Shaughnessy, Howe, Devereux, McCann, Ramalho, Branquinha and Santos. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Galdino, Anna Clara M.
Viganor, Lívia
de Castro, Alexandre A.
da Cunha, Elaine F. F.
Mello, Thaís P.
Mattos, Larissa M.
Pereira, Marcos D.
Hunt, Mary C.
O’Shaughnessy, Megan
Howe, Orla
Devereux, Michael
McCann, Malachy
Ramalho, Teodorico C.
Branquinha, Marta H.
Santos, André L. S.
Disarming Pseudomonas aeruginosa Virulence by the Inhibitory Action of 1,10-Phenanthroline-5,6-Dione-Based Compounds: Elastase B (LasB) as a Chemotherapeutic Target
title Disarming Pseudomonas aeruginosa Virulence by the Inhibitory Action of 1,10-Phenanthroline-5,6-Dione-Based Compounds: Elastase B (LasB) as a Chemotherapeutic Target
title_full Disarming Pseudomonas aeruginosa Virulence by the Inhibitory Action of 1,10-Phenanthroline-5,6-Dione-Based Compounds: Elastase B (LasB) as a Chemotherapeutic Target
title_fullStr Disarming Pseudomonas aeruginosa Virulence by the Inhibitory Action of 1,10-Phenanthroline-5,6-Dione-Based Compounds: Elastase B (LasB) as a Chemotherapeutic Target
title_full_unstemmed Disarming Pseudomonas aeruginosa Virulence by the Inhibitory Action of 1,10-Phenanthroline-5,6-Dione-Based Compounds: Elastase B (LasB) as a Chemotherapeutic Target
title_short Disarming Pseudomonas aeruginosa Virulence by the Inhibitory Action of 1,10-Phenanthroline-5,6-Dione-Based Compounds: Elastase B (LasB) as a Chemotherapeutic Target
title_sort disarming pseudomonas aeruginosa virulence by the inhibitory action of 1,10-phenanthroline-5,6-dione-based compounds: elastase b (lasb) as a chemotherapeutic target
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688126/
https://www.ncbi.nlm.nih.gov/pubmed/31428062
http://dx.doi.org/10.3389/fmicb.2019.01701
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