Structure of the assembly platform of the bacterial type II secretion system
Lead Research Organisation:
Queen Mary University of London
Department Name: Sch of Biological & Behavioural Sciences
Abstract
The bacterial type II secretion system delivers harmful proteins which can have devastating effects on animals, humans and plants. Human pathogens such as enterotoxigenic Escherichia coli, Vibrio cholerae and Pseudomonas aeruginosa cause children's diarrhea, cholera and respiratory infections, respectively, and there are many deaths annually. It has recenly been demonstrated that the type II secretion system is very important for nosocomial pathogen survival in humans where multi-drug resistance has developed. Moreover, impairment of the type II secretion system has been shown to allow the innate immune system to clear infection. Plant pathogens relying on the type II secretion system such as Dickeya, Pectobacterium and Xanthomonascause are responsible for devastating crop loss in the UK and Europe. The lytic enzymes secreted through this system destroy plant tissues and threaten food security. The bacterial type II secretion system spans the two membranes of the Gram-negative cell wall. Secretion is achieved by an inner-membrane complex assembling a short pilus to push the recruited effector from the periplasm (the space between the two membranes) through the gated pore in the outer-membrane. The energy needed for pilus assembly is provided by the cytoplasmic ATPase.
We have made excellent progress in imaging the type II secretion system. We have purified, imaged and generated 3D models of the inner-membrane assembly platform and associated ATPase. We have produced and imaged the entire envelope-spanning secretion system. In this proposal we shall extend the stability and resolution of these complexes so that we can build accurate molecular models of the inner-membrane assembly platform. We shall also image the assembly platform in the presence of the outer-membrane secretin to capture the arrangement of the periplasmic domains extending from the inner-membrane assembly platform. We shall achieve these results using cryo-electron microscopy. To demonstrate we can produce the deliverables stated in the proposal we have collected preliminary data on the inner-membrane assembly platform and produced a preliminary structure of the ATPase. We anticipate capturing the secretion system in different states and to be able to model the structural transitions between states. We are supported by our collaborator who can reverse engineer the type II secretion system in the bacterium to confirm the importance of the interactions we see in our experiments in the live bacterium.
As a result of this work, we will determine the structure of the inner-membrane assembly platform and its interaction with the cytoplasmic ATPase. We shall elucidate the structural transitions that can occur from the different complexes trapped or dissected from micrographs and shall provide a structural basis for revealing how the ATPase drives pilus assembly. We also aim to determine the organisation of domains in the periplasm, and this will help us to understand how specific proteins substrates are recruited to the secretion system and how signal is transduced between periplasm and the cytoplasm across the inner membrane assembly complex. Ultimately this work will help underpin the development of new antibiotics to combat infection.
We have made excellent progress in imaging the type II secretion system. We have purified, imaged and generated 3D models of the inner-membrane assembly platform and associated ATPase. We have produced and imaged the entire envelope-spanning secretion system. In this proposal we shall extend the stability and resolution of these complexes so that we can build accurate molecular models of the inner-membrane assembly platform. We shall also image the assembly platform in the presence of the outer-membrane secretin to capture the arrangement of the periplasmic domains extending from the inner-membrane assembly platform. We shall achieve these results using cryo-electron microscopy. To demonstrate we can produce the deliverables stated in the proposal we have collected preliminary data on the inner-membrane assembly platform and produced a preliminary structure of the ATPase. We anticipate capturing the secretion system in different states and to be able to model the structural transitions between states. We are supported by our collaborator who can reverse engineer the type II secretion system in the bacterium to confirm the importance of the interactions we see in our experiments in the live bacterium.
As a result of this work, we will determine the structure of the inner-membrane assembly platform and its interaction with the cytoplasmic ATPase. We shall elucidate the structural transitions that can occur from the different complexes trapped or dissected from micrographs and shall provide a structural basis for revealing how the ATPase drives pilus assembly. We also aim to determine the organisation of domains in the periplasm, and this will help us to understand how specific proteins substrates are recruited to the secretion system and how signal is transduced between periplasm and the cytoplasm across the inner membrane assembly complex. Ultimately this work will help underpin the development of new antibiotics to combat infection.
Technical Summary
We have assembled the reagents and expertise needed to determine the structure of the inner membrane assembly platform of the type II secretion system. The assembly platform (comprising proteins CEFLM) is at the centre of the secretion system and interacts with the outer membrane secretin D via the periplasmic domains of CLM and with the cytoplasmic ATPase E via the cytoplasmic domain of L. At the heart of the assembly platform is the potential rotor protein F which facilitates the assembly of the short periplasmic pilus comprising the major pilin G and initiation pilins IJK. We have recent NMR data on its interactions with other components.
Key to our success is stabilising the assembly platform by adding back components, by trapping individual states using specific cross-links and by segregating structural heterogeneity present in cryo-EM images using state-of-the-art software. We are also working from the top down using the full secretion system, for instance we recently produced the initiation complex in the E. coli IHE3034 system by deleting the major pilin subunit G and introducing the substrate SslE. In this proposed work, we expect to achieve near atomic resolution structures. We will use a combined approach using cross-linking mass spectrometry together with low/medium resolution single particle cryo-EM results if essential. We shall also check the biological relevance of our experimental and computational results by reverse engineering the bacterium.
We shall determine structures and model the structural transitions that can occur between the different complexes produced to provide a structural basis for elucidating how substrate recruitment in the periplasm is signalled to the cytoplasmic ATPase and how the energy form this ATPase is used to drive pseudo-pilus assembly, possibly via the rotor protein F. Ultimately this work will help underpin the development of molecules to help combat antimicrobial resistance and to protect crops.
Key to our success is stabilising the assembly platform by adding back components, by trapping individual states using specific cross-links and by segregating structural heterogeneity present in cryo-EM images using state-of-the-art software. We are also working from the top down using the full secretion system, for instance we recently produced the initiation complex in the E. coli IHE3034 system by deleting the major pilin subunit G and introducing the substrate SslE. In this proposed work, we expect to achieve near atomic resolution structures. We will use a combined approach using cross-linking mass spectrometry together with low/medium resolution single particle cryo-EM results if essential. We shall also check the biological relevance of our experimental and computational results by reverse engineering the bacterium.
We shall determine structures and model the structural transitions that can occur between the different complexes produced to provide a structural basis for elucidating how substrate recruitment in the periplasm is signalled to the cytoplasmic ATPase and how the energy form this ATPase is used to drive pseudo-pilus assembly, possibly via the rotor protein F. Ultimately this work will help underpin the development of molecules to help combat antimicrobial resistance and to protect crops.
