Dissecting Type Four Pili in Action – T4PNanoAction

We propose to characterize the structure of the piliation machinery using a combination of two innovative technological approaches. The first one is covalent cross-linking mass spectrometry. In particular, we propose to develop an in vivo approach, performed directly on living bacterial cells, which will allow the structure of the nanomachinery to be probed in its native environment. The second approach is cryo-Electron Microscopy. The aim is to achieve structures of the machinery at the highest resolution directly from intact cells. These experiments are ongoing.

We just finished to set-up an advanced in vivo cross-linking mass spectrometry platform to study the cellular interactome of living bacterial cells. It is based on in vivo labeling and involves a one-step enrichment by click chemistry on a solid support. Our approach shows an impressive efficiency on Neisseria meningitidis, leading to the identification of about 3300 cross-links for the LC-MS/MS analysis of a biological triplicate using a benchtop high-resolution Orbitrap mass spectrometer. Highly dynamic multiprotein complexes were successfully captured and characterized in all bacterial compartments, showing the great potential and precision of our proteome-wide approach. Our workflow paves new avenues for the large-scale and nonbiased analysis of protein–protein interactions. An optimization of the sample preparation for cryo-EM experiments is ongoing to obtain images at the highest resolution on a Titan microscope recently installed at the Institut Pasteur.

Our goal for the cross-linking MS part is to continue optimizing sample preparation to obtain more structural information on the piliation machinery in its native state. Different mutants in which the machinery is blocked at different stages of its formation will then be analyzed.
For the electron microscopy part, high resolution images will be acquired in the coming months.

Advanced In Vivo Cross-Linking Mass Spectrometry Platform to Characterize Proteome-Wide Protein Interactions. Martial Rey, Jonathan Dhenin, Youxin Kong, Lucienne Nouchikian, Isaac Filella, Magalie Duchateau, Mathieu Dupré, Riccardo Pellarin, Guillaume Duménil, and Julia Chamot-Rooke, Analytical Chemistry 2021.
doi.org/10.1021/acs.analchem.0c04430

Type IV Pili (T4P) are bacterial filamentous cell surface structures present in numerous species including the human pathogen Neisseria meningitidis where they play a central role in virulence by providing different functions such as adhesion to host cells. A multimeric molecular nanomachinery composed of 15 different proteins anchored in the bacterial inner and outer membranes constantly assembles and disassembles T4P at high speeds. The aim of this proposal is to establish the structure of the T4P nanomachinery in the different states associated with its dynamics and functional maturation, providing an unprecedented dynamic high-resolution view of these structures. This ambitious objective will be reached using the synergic combination of both innovative in vivo cross-linking mass spectrometry (XL-MS) and cryo-Electron Microscopy (cryo-EM) approaches. These two approaches are currently emerging as highly complementary. Indeed XL-MS data lead to information on cross-linked proteins structures and protein interaction networks that can be further used to improve the structural models obtained from cryo-EM experiments. This project is divided into four work-packages (WPs). In WP1, specific N. meningitidis mutants in which the machinery is either in an empty, assembly or disassembly state as well as mutants showing major phenotypes (partial or complete loss of certain functions) will be prepared for both XL-MS and cryo-EM experiments. WP2 will be dedicated to the development of an optimized in vivo XL-MS workflow on intact N. meningitidis cells, based on a protocol recently developed by the coordinator group. This protocol relies on the use of an original trifunctional cross-lining reagent designed by the project coordinator, which allows an efficient enrichment of cross-linked peptides. WP3 concerns cryo-EM analyses, in particular cryo-electron tomography (cryo-ET) and image processing. After an optimization of sample preparation, images will be acquired on WT, intact N. meningitidis cells and structures analyzed. Mutants for which the XL-MS data indicated significant differences with the WT in term of protein-protein interactions will be selected for further cryo-EM experiments. The outcome of WP3 is a density map obtained by cryo-ET for the T4P machinery of the WT strain. Within this envelope, several parts will be highly resolved and thus the attribution of the corresponding sub-units will be possible without any supplementary data. For the parts with lower resolution, both crystallographic data already available on some pil proteins (for N. meningitidis or closely-related species) will be used in conjunction with the cross-linking data (cross-linked peptides for the contact sites and dead-ends for solvent accessibility) obtained in WP2 to improve the structural models. All these data will be aggregated using appropriate tools such as IMP (Integrated Modeling Platform). This is the aim of WP4.
The consortium gathers three teams of the Institut Pasteur with complementary expertise in mass spectrometry, microbiology and cryo-electron microscopy. The combination of state-of-the-art approaches proposed in this project and the expertise of the consortium are unique. Exciting preliminary results have already been obtained by the consortium both for the in vivo cross-linking analysis of intact N. meningitidis cells and for cryo-EM. The information gained at different levels on the T4 piliation machinery (structural, functional) will be used for a better understanding of this complex system, highly involved in the virulence of many human pathogens. The high-resolution data obtained will provide a completely new way to address the T4P machinery in action offering new strategies for vaccine development and novel targets for drug design. In addition, the methods developed will also be broadly applicable to other bacterial pathogens.