Biochemical characterization of the Helicobacter pylori Cag Type 4 Secretion System protein CagN and its interaction
Simon H Bats1, Célia Bergé2, Nina Coombs3
1Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, 30625 Hannover, Germany; Max von Pettenkofer Institute, Ludwig Maximilians Universität LMU München, Pettenkoferstraße 9a, 80336 München, Germany.
Abstract:
Highly virulent Helicobacter pylori strains contain the cag pathogenicity island (cagPAI). It codes for about 30 proteins forming a type IV secretion system (T4SS) which translocates the pro-inflammatory protein CagA into epithelial host cells. While CagA and various other Cag proteins have been extensively studied, several cagPAI proteins are poorly characterized or of unknown function. CagN (HP0538) is of unknown function but highly conserved in the cagPAI suggesting an important role. cagM (HP0537) is the first gene of the cagMN operon and its product is part of the CagT4SS core complex. Both proteins do not have detectable homologs in other type IV secretion systems. We have characterized the biochemical and structural properties of CagN and CagM and their interaction. We demonstrate by circular dichroism, Multi-Angle Light Scattering (MALS) and small angle X-ray scattering (SAXS) that CagN is a folded, predominantly monomeric protein with an elongated shape in solution. CagM is folded and forms predominantly dimers that are also elongated in solution. We found by various in vivo and in vitro methods that CagN and CagM directly interact with each other. CagM self-interacts stably with a low nanomolar KD and can form stable multimers. Finally, in vivo experiments show that deletion of CagM reduces the amounts of CagN and other outer CagPAI proteins in H. pylori cells.
Insights
The study reveals that Helicobacter pylori CagN and CagM proteins directly interact, with CagM influencing CagN stability within the cag pathogenicity island (cagPAI) type IV secretion system.
Area of Science:
- Microbiology
- Molecular Biology
- Structural Biology
Background:
- Highly virulent Helicobacter pylori strains possess the cag pathogenicity island (cagPAI), encoding a type IV secretion system (T4SS).
- The T4SS translocates the CagA protein into host cells, but several cagPAI proteins, including CagN and CagM, remain poorly characterized.
- CagN and CagM are conserved within the cagPAI and lack homologs in other T4SS, suggesting unique functions.
Purpose of the Study:
- To biochemically and structurally characterize CagN and CagM proteins from Helicobacter pylori.
- To investigate the interaction between CagN and CagM.
- To elucidate the role of CagM in the stability of CagN and other cagPAI proteins.
Main Methods:
- Circular dichroism (CD) spectroscopy to assess protein folding.
- Multi-Angle Light Scattering (MALS) and Small Angle X-ray Scattering (SAXS) for solution structure analysis.
- In vivo and in vitro assays to determine protein-protein interactions and stability.
Main Results:
- CagN is a folded, predominantly monomeric protein with an elongated solution structure.
- CagM is folded, forms stable dimers and multimers, and exhibits an elongated solution structure.
- CagN and CagM directly interact, with CagM self-interacting with high affinity (low nanomolar KD).
- Deletion of CagM in H. pylori leads to reduced levels of CagN and other outer cagPAI proteins.
Conclusions:
- CagN and CagM are stably interacting proteins crucial for the integrity of the cagPAI T4SS.
- CagM plays a significant role in stabilizing CagN and potentially other outer cagPAI components.
- These findings provide new insights into the poorly understood components of the H. pylori T4SS.
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