Cargando…

Fragment-based screening identifies inhibitors of ATPase activity and of hexamer formation of Cagα from the Helicobacter pylori type IV secretion system

Type IV secretion systems are multiprotein complexes that mediate the translocation of macromolecules across the bacterial cell envelope. In Helicobacter pylori a type IV secretion system encoded by the cag pathogenicity island encodes 27 proteins and most are essential for virulence. We here presen...

Descripción completa

Detalles Bibliográficos
Autores principales: Arya, Tarun, Oudouhou, Flore, Casu, Bastien, Bessette, Benoit, Sygusch, Jurgen, Baron, Christian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482174/
https://www.ncbi.nlm.nih.gov/pubmed/31019200
http://dx.doi.org/10.1038/s41598-019-42876-6
_version_ 1783413837908672512
author Arya, Tarun
Oudouhou, Flore
Casu, Bastien
Bessette, Benoit
Sygusch, Jurgen
Baron, Christian
author_facet Arya, Tarun
Oudouhou, Flore
Casu, Bastien
Bessette, Benoit
Sygusch, Jurgen
Baron, Christian
author_sort Arya, Tarun
collection PubMed
description Type IV secretion systems are multiprotein complexes that mediate the translocation of macromolecules across the bacterial cell envelope. In Helicobacter pylori a type IV secretion system encoded by the cag pathogenicity island encodes 27 proteins and most are essential for virulence. We here present the identification and characterization of inhibitors of Cagα, a hexameric ATPase and member of the family of VirB11-like proteins that is essential for translocation of the CagA cytotoxin into mammalian cells. We conducted fragment-based screening using a differential scanning fluorimetry assay and identified 16 molecules that stabilize the protein suggesting that they bind Cagα. Several molecules affect binding of ADP and four of them inhibit the ATPase activity. Analysis of enzyme kinetics suggests that their mode of action is non-competitive, suggesting that they do not bind to the active site. Cross-linking suggests that the active molecules change protein conformation and gel filtration and transmission electron microscopy show that molecule 1G2 dissociates the Cagα hexamer. Addition of the molecule 1G2 inhibits the induction of interleukin-8 production in gastric cancer cells after co-incubation with H. pylori suggesting that it inhibits Cagα in vivo. Our results reveal a novel mechanism for the inhibition of the ATPase activity of VirB11-like proteins.
format Online
Article
Text
id pubmed-6482174
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher Nature Publishing Group UK
record_format MEDLINE/PubMed
spelling pubmed-64821742019-05-03 Fragment-based screening identifies inhibitors of ATPase activity and of hexamer formation of Cagα from the Helicobacter pylori type IV secretion system Arya, Tarun Oudouhou, Flore Casu, Bastien Bessette, Benoit Sygusch, Jurgen Baron, Christian Sci Rep Article Type IV secretion systems are multiprotein complexes that mediate the translocation of macromolecules across the bacterial cell envelope. In Helicobacter pylori a type IV secretion system encoded by the cag pathogenicity island encodes 27 proteins and most are essential for virulence. We here present the identification and characterization of inhibitors of Cagα, a hexameric ATPase and member of the family of VirB11-like proteins that is essential for translocation of the CagA cytotoxin into mammalian cells. We conducted fragment-based screening using a differential scanning fluorimetry assay and identified 16 molecules that stabilize the protein suggesting that they bind Cagα. Several molecules affect binding of ADP and four of them inhibit the ATPase activity. Analysis of enzyme kinetics suggests that their mode of action is non-competitive, suggesting that they do not bind to the active site. Cross-linking suggests that the active molecules change protein conformation and gel filtration and transmission electron microscopy show that molecule 1G2 dissociates the Cagα hexamer. Addition of the molecule 1G2 inhibits the induction of interleukin-8 production in gastric cancer cells after co-incubation with H. pylori suggesting that it inhibits Cagα in vivo. Our results reveal a novel mechanism for the inhibition of the ATPase activity of VirB11-like proteins. Nature Publishing Group UK 2019-04-24 /pmc/articles/PMC6482174/ /pubmed/31019200 http://dx.doi.org/10.1038/s41598-019-42876-6 Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Arya, Tarun
Oudouhou, Flore
Casu, Bastien
Bessette, Benoit
Sygusch, Jurgen
Baron, Christian
Fragment-based screening identifies inhibitors of ATPase activity and of hexamer formation of Cagα from the Helicobacter pylori type IV secretion system
title Fragment-based screening identifies inhibitors of ATPase activity and of hexamer formation of Cagα from the Helicobacter pylori type IV secretion system
title_full Fragment-based screening identifies inhibitors of ATPase activity and of hexamer formation of Cagα from the Helicobacter pylori type IV secretion system
title_fullStr Fragment-based screening identifies inhibitors of ATPase activity and of hexamer formation of Cagα from the Helicobacter pylori type IV secretion system
title_full_unstemmed Fragment-based screening identifies inhibitors of ATPase activity and of hexamer formation of Cagα from the Helicobacter pylori type IV secretion system
title_short Fragment-based screening identifies inhibitors of ATPase activity and of hexamer formation of Cagα from the Helicobacter pylori type IV secretion system
title_sort fragment-based screening identifies inhibitors of atpase activity and of hexamer formation of cagα from the helicobacter pylori type iv secretion system
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482174/
https://www.ncbi.nlm.nih.gov/pubmed/31019200
http://dx.doi.org/10.1038/s41598-019-42876-6
work_keys_str_mv AT aryatarun fragmentbasedscreeningidentifiesinhibitorsofatpaseactivityandofhexamerformationofcagafromthehelicobacterpyloritypeivsecretionsystem
AT oudouhouflore fragmentbasedscreeningidentifiesinhibitorsofatpaseactivityandofhexamerformationofcagafromthehelicobacterpyloritypeivsecretionsystem
AT casubastien fragmentbasedscreeningidentifiesinhibitorsofatpaseactivityandofhexamerformationofcagafromthehelicobacterpyloritypeivsecretionsystem
AT bessettebenoit fragmentbasedscreeningidentifiesinhibitorsofatpaseactivityandofhexamerformationofcagafromthehelicobacterpyloritypeivsecretionsystem
AT syguschjurgen fragmentbasedscreeningidentifiesinhibitorsofatpaseactivityandofhexamerformationofcagafromthehelicobacterpyloritypeivsecretionsystem
AT baronchristian fragmentbasedscreeningidentifiesinhibitorsofatpaseactivityandofhexamerformationofcagafromthehelicobacterpyloritypeivsecretionsystem