FROM DYNAMICS & STRUCTURAL DIVERSITY OF T6SS MEMBRANE COMPLEXES TO THE DEVELOPMENT OF VIRULENCE BLOCKERS – T6MeD-OC

The T6MeD-OC project brings together an interdisciplinary consortium, combining clinical, fundamental, molecular and structural microbiology with medicinal chemistry. In the first part of the project, we have developed an integrated pipeline to screen banks of chemical molecules and peptides that inhibit the assembly of the T6SS nanomachine. This pipeline includes in vitro methods using purified proteins, in vivo screens directly on different bacterial pathogens as well as experiments in a virulence model Galleria mellonella, using clinical strains.
In the second part of the project, we are using biochemistry of membrane proteins, biophysics approaches, cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) to unravel (1) the high resolution structure of the canonical T6SS membrane complex (“cT6-MC”) and (2) to study the functioning and structural diversity of non-conventional T6SS membrane complexes (“ncT6-MC”) from Acinetobacter baumannii and Bacteroides fragilis.

First part: In collaboration with the group of Dr. Xavier Morelli (CRCM, Marseille), our team has developed a multi-scale pipeline for the high-to-medium throughput screening of T6SS-inhibitors in Pseudomonas aeruginosa. This pipeline represents a continuum from in vitro to in vivo assays to select and monitor the effect of potential anti-T6SS small molecules or peptides. Our anti-T6SS pipeline is based on the targeting of a specific protein-protein interface (PPI) crucial for the assembly of the MC: the TssJ-TssM PPI. Our dedicated anti-T6SS pipeline is based on 3 assays monitoring the formation of the H2 TssJ-TssM complex (in vitro and in vivo) and 2 assays monitoring the functionality of the P. aeruginosa H2-T6SS. Our screen has identified 4 candidate drugs and “first-in-class” inhibitors of the P. aeruginosa T6SS.
In collaboration with the group of Dr. Riccardo Pellarin (Institut Pasteur, Paris), we have designed a biomimetic peptide inhibitor of the assembly of the EAEC T6SS baseplate complex and we have shown its potential use to block the virulence of other pathogenic species (manuscript in preparation).
Second part: In collaboration with the group of Dr. Jean-Pierre Duneau (LISM, Marseille), and Dr. Benjamin D Ross (Dartmouth Geisel School of Medicine, US), we have started to investigate the structural diversity of ncT6-MC. In the WHO priority ESKAPE pathogen Acinetobacter baumannii, we have identified a missing partner in the abMC and are starting the unravel the high-resolution structure of the entire MC using cryo-EM (manuscript in preparation). In the human microbiote bacteria Bacteroides fragilis, we have for the first time identified and determined the composition of the bfMC. Works are underway in my group and in the group of BD Ross to determine the high-resolution structure of the bfMC and to reconstitute its phylogeny history.

If our optimized pipeline allows us to discover new T6SS inhibitors and improve the molecules already identified, we will conduct tests in the short term in mammalian models of acute or chronic infection. In the longer term, this project will improve the treatment of respiratory infectious diseases from which patients suffer from cystic fibrosis. These anti-virulence molecules can act as a replacement or in combination with an antibiotic therapy which, today, is no longer effective in combating multi-resistant strains of P. aeruginosa. By tackling the first steps which lead to colonization of the lungs of patients by P. aeruginosa, this new type of treatment which will result from our research should limit the proliferation of resistant species and therefore allow an effective and lasting treatment of infected patients.

The determination of the high-resolution structure of the ncT6-MC in Acinetobacter baumannii and Bacteroides fragilis, will demonstrate for the first time specific architectural adaptations of the T6SS nanomachine as well as a new phylogenetic history. Ultimately, these structures will pave the way towards the development of species-specific virulence blockers that will help to fight against antibiotic resistant bacterial infections.

• Manuscript in preparation:

Cherrak Y, Filella-Merce I, Schimdt V, Sgoluppi V, Chaiaheloudjou R, Pellarin R and Durand E*.
From the structure of the TssKFG wedge complex to the identification of novel targets for anti-T6SS drug.

Cherrak Y, Kandolo O, Le Guenno H, Kosta A, Espinosa L, Roche P, Betzi S, Morelli X and Durand E*. Composition and architecture of the non-canonical T6SS membrane complex in Acinetobacter baumannii.


• Patent in preparation:

Schimdt V, Legoff M, Roche P, Hoffer L, Betzi S, Morelli X* and Durand E* (to be define lately with the valorization cell, i.e CNRS INNOVATION). First-in-class anti-T6SS drug targeting the virulence of Pseudomonas aeruginosa.

Bacterial resistance to antibiotics is a serious health threat and search for new anti-virulence molecules is a worldwide priority. Targeting virulence factors, which are not essential for bacterial life, is expected to prevent the rapid development of resistance mechanisms. This requires in depth molecular understanding of the virulence factors used against the human host. The type VI secretion system (T6SS) is an important virulence factor shared by human pathogens, such as Acinetobacter baumannii, Francisella tularensis, Pseudomonas aeruginosa or Enteroaggregative Escherichia coli. The T6SS is a macromolecular machine composed of three architectural complexes – the membrane complex (MC), the baseplate and the tail – that coordinately participate to the injection of toxins into target cells. The MC is a central and conserved part of the T6SS that is relevant for a drug target. But, understanding the extent of the MC diversity (differences between pathogens and human resident microbiota) in terms of structure and composition and demonstrating its dynamic architecture (close-to-open transition, protein domains forming the outer-membrane channel) are all primordial steps for the rational choice of inhibitor targets. This project will decipher the structural diversity and dynamic of the T6SS MC from various important human pathogens and from a representative of the human microbiota. We will bring unprecedented level of fine details concerning the molecular adaptation of the MC across human pathogens and understand the functional consequences of such changes. We will thus highlight hot spots for inhibitor targets and develop tools for screening small molecule inhibitors of the T6SS, both in vitro and in vivo, targeting different steps in the dynamic assembly of the MC. This project will be pioneering in identifying T6SS inhibitors. We plan to address these questions based on a multi-disciplinary consortium, with expertise in biochemistry and structural biology of membrane protein complexes, molecular microbiology, molecular dynamics, HTS screening of chemical library, drug design and bacterial virulence models for important human pathogens. This work will pave the way towards the identification of new therapeutic targets and to the development of antimicrobial molecules to treat infectious diseases.