Molecular interactions with the bacterial cell wall

Antibiotic resistance

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Jean-Pierre Simorre (CNRS researcher)
Catherine Bougault (MdC)
Isabel Ayala (CNRS Engineer)
Laguri Cédric (CNRS Researcher)
Tiago Baeta (PhD Student)
Meriem Maalej (PhD STudent)
Karine Giandoreggio-Barranco (AI CDD)

Solid state NMR investigation of LipoPolySaccharides

Lipopolysaccharides (LPS) are complex glycolipids forming the outside layer of gram-negative bacteria. Their hydrophobic and heterogeneous nature greatly hampers their structural study in an environment similar to the bacterial surface. We have studied LPS purified from E. coli and pathogenic P. aeruginosa assembled in solution as vesicles or elongated micelles. Solid-state NMR with Magic-angle spinning permitted the identification of NMR signals arising from regions with different flexibilities in the LPS, from the lipid components to the O-antigen polysaccharides. Atomic scale data on the LPS enabled to study the interaction of gentamicin antibiotic bound to P. aeruginosa LPS for which we could confirm that a specific oligosaccharide is involved in the antibiotic binding. The possibility to study LPS alone and bound to a ligand when it is assembled in membrane like structures opens great prospects for the investigation of proteins and antibiotics that specifically target such an important molecule at the surface of gram-negative

LipoPolySaccharides Transport system in E. coli periplasm

Transport of lipopolysaccharides (LPS) to the surface of the outer membrane is essential for viability of Gram-negative bacteria. Periplasmic LptC and LptA proteins of the LPS transport system (Lpt) are responsible for LPS transfer between the Lpt inner and outer membrane complexes. Here, using a monomeric E. coli LptA mutant, we first show in vivo that a stable LptA oligomeric form is not strictly essential for bacteria. The LptC-LptA complex was characterized by a combination of SAXS and NMR methods and a low resolution model of the complex was determined. We were then able to observe interaction of LPS with LptC, the monomeric LptA mutant as well as with the LptC-LptA complex. A LptC-LPS complex was built based on NMR data in which the lipid moiety of the LPS is buried at the interface of the two β-jellyrolls of the LptC dimer. The selectivity of LPS for this intermolecular surface and the observation of such cavities at homo- or heteromolecular interfaces in LptC and LptA suggests that intermolecular sites are essential for binding LPS during its transport. link

Decrypting a cellular guidance system to fight against pneumococcal infections

Accurate placement of the bacterial division site is a prerequisite for the generation of two viable and identical daughter cells. In Streptococcus pneumoniae (pneumococcus), the positive regulatory mechanism involving the membrane protein MapZ positions precisely the conserved cell division protein FtsZ at the cell centre. Reasearchers of the Institut de Biologie et de Chimie des Protéines in Lyon et of the NMR group of the IBS characterized the structure of the extracellular domain of MapZ. This structure–function analysis of MapZ, published in Nature Communications, provides the first molecular characterization of a positive regulatory process of bacterial cell division which may be helpfull to develop new antibiotics.

Structure–function analysis of the extracellular domain of the pneumococcal cell division site positioning protein MapZ. link

Molecular interactions with the bacterial cell wall by liquid state, standard and DNP solid state NMR

The bacterial cell wall is essential for the survival of bacteria. It gives the bacterial cell its shape and protects it against osmotic pressure, while allowing cell growth and division. It is made up of peptidoglycan (PG), a biopolymer forming a multi-gigadalton bag-like structure, and additionally in Gram-positive bacteria, of covalently linked anionic polymers called wall teichoic acids (WTA). TAs are thought to play important roles in ion trafficking, host-cell adhesion, inflammation and immune activation.

inhibition of the peptidoglycan enzymes by antibiotics

The machinery involved in the synthesis of this envelop is crucial and is one of the main antibiotic target. Different protein as transpeptidase, transpeptidase activator or hydrolase are recruited to maintain the morphogenesis of the peptidoglycan during the bacterial cell cycle. Based on few examples involved in the machinery of synthesis of the peptidoglycan, we will demonstrates that a combination of liquid and solid-state NMR can be a powerful tool to screen for cell-wall interacting proteins in vitro and on cell..
In particular, structure of the L,D-transpeptidases that results in b-lactam resistance in M. tuberculosis, has been studied in presence of the bacterial cell wall and in presence of antibiotic. The NMR study reveals new insights into the inhibition

On cell NMR by Solid state NMR

In parallel, we have investigated the potential of Dynamic Nuclear Polarization (DNP) to investigate cell surface directly in intact cells. Our results show that increase in sensitivity can be obtained together with the possibility of enhancing specifically cell-wall signals. It opens new avenues for the use of DNP-enhanced solid-state NMR as an on-cell investigation tool. link