DS0401 -

The L,D-transpeptidase/peptidoglycan complex and its influence on the maturation of the mycobacterial cell wall: an NMR investigation – TransPepNMR

Submission summary

In the context of increasing antibiotic resistance, we propose to gain fundamental knowledge on the biogenesis of the bacterial cell wall in an integrative approach. This project aims at filling the gap between inhibition studies on isolated molecular targets and studies on complete cells. It will provide atomic-scale information on the interaction of biosynthetic enzymes with cell-wall polymers. For this purpose, we will develop a new approach based on biomolecular solid-state and DNP-enhanced NMR to tackle systems of increasing complexity. Additionally, state-of-the-art pulse-chase techniques and mass spectrometry will be implemented to follow the evolution of cell-wall polymers along the cell cycle. In particular, the role of peptidoglycan (PG) binding domains of L,D-transpeptidases (Ldts) in the polymerization and maturation of the mycobacterial cell wall will be investigated within a multidisciplinary consortium with expertise in solid-state NMR and DNP, microbiology, biochemistry, and structural biology. The choice of this particular bacterial species is motivated by the emergence of extensively drug resistant Mycobacterium tuberculosis and the relevance of Ldts as therapeutic targets in this pathogen.

To date, interaction of transpeptidases (TPs) with their substrates has mainly been investigated through short PG precursor analogues without addressing the complexity of the peptidoglycan network. Our recent analyses performed with the Bacillus subtilis Ldt demonstrated that substrate recognition involves dedicated domains at a distance of the active site. These domains remain to be identified and investigated in mycobacterial Ldts. The first objective of the present proposal thus aims at deciphering the structure and dynamics of the interaction between intact PG and M. tuberculosis Ldts by solid-state NMR. We will extract structural information on the complex using chemical shift perturbations. Accurate structure determination will require distance measurements through the combination of state-of-the-art NMR and isotopic labeling strategies. Outcomes of these in vitro innovative experiments will contribute to a better understanding of transpeptidation.

The second objective will determine the role of L,D- versus D,D-TPs in the formation of cross-links during the expansion and maturation of the mycobacterial PG network. Pulse-chase labeling and mass spectrometry techniques will be developed to differentiate neo-synthesized from pre-existing PG subunits and to quantify the respective contributions of the two TPs in expanding PG. The type and extent of chemical modifications involved in PG maturation will then be deduced. This study will be extended to the transition to the stationary phase, to “dormant” bacilli, and to ß-lactam stress. The first two objectives will further our understanding of peptidoglycan biosynthesis and maturation and potentially lead to the identification of novel strategies for the eradication of multidrug resistant M. tuberculosis.

The third objective largely consists in the development of a new integrative cell biology tool using low-temperature DNP-enhanced solid-state NMR. The potential of DNP will be explored to improve the sensitivity and the selectivity towards specific biopolymers of the mycobacterial cell envelope. In parallel, uniform and site-specific labeling strategies will be combined to other DNP-specific techniques to overcome current spectral resolution limitations due to the frozen state required for DNP experiments. Complementary spin- and isotopic-labeling of Ldts and PG will be designed to provide selective structural information, as a first step towards the investigation of interaction interfaces by DNP. This challenging objective is expected to lead to a major breakthrough in the field of DNP for the structural study of biomolecular assemblies.

Outcomes of the project are fundamental knowledge on mycobacterial cell-wall biosynthesis that are useful for drug development.

Project coordination

Jean-Pierre SIMORRE (Institut de Biologie Structurale)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

INAC/MEM Institut Nanoscience et Cryogénie
IBS CEA/CNRS/UGA Institut de Biologie Structurale
INSERM Institut National de la Santé et de la Recherche Médicale

Help of the ANR 547,233 euros
Beginning and duration of the scientific project: December 2016 - 48 Months

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