Lipoprotein Lpp regulates the mechanical properties of the E. coli cell envelope

Marion Mathelié-Guinlet,Abir T Asmar,Yves F Dufrêne,Jean-François Collet

doi:10.1038/s41467-020-15489-1

Abstract

The mechanical properties of the cell envelope in Gram-negative bacteria are controlled by the peptidoglycan, the outer membrane, and the proteins interacting with both layers. In Escherichia coli, the lipoprotein Lpp provides the only covalent crosslink between the outer membrane and the peptidoglycan. Here, we use single-cell atomic force microscopy and genetically engineered strains to study the contribution of Lpp to cell envelope mechanics. We show that Lpp contributes to cell envelope stiffness in two ways: by covalently connecting the outer membrane to the peptidoglycan, and by controlling the width of the periplasmic space. Furthermore, mutations affecting Lpp function substantially increase bacterial susceptibility to the antibiotic vancomycin, indicating that Lpp-dependent effects can affect antibacterial drug efficacy.

Highlights

The mechanical properties of the cell envelope in Gram-negative bacteria are controlled by the peptidoglycan, the outer membrane, and the proteins interacting with both layers
Atomic force microscopy (AFM) was used to capture the structural and mechanical properties of E. coli, with the aim to answer the following questions: does Lpp play a role in regulating cell envelope mechanics; is the mechanical function of Lpp primarily controlled through the physical connection between outer membrane (OM) and PG, and/or through modulation of the periplasmic width; does Lpp-dependent cell mechanics influence the sensitivity to antibiotics? To this end, we compared the behavior of wild-type (WT) bacteria with that of bacterial strains with Lpp functional mutations, i.e., Lpp a b
Note that considering the variability inherent to cells in the exponential phase, fuzzy features were sometimes observed on all strains, that we attribute to the interaction between the AFM tip and LPS

Summary

Introduction

The mechanical properties of the cell envelope in Gram-negative bacteria are controlled by the peptidoglycan, the outer membrane, and the proteins interacting with both layers. In Gramnegative bacteria, such as Escherichia coli, the cell envelope is made up of an inner membrane (IM; a classical phospholipid bilayer around the cytoplasm) and an outer membrane (OM; an asymmetric structure with phospholipids in the inner leaflet and lipopolysaccharides in the outer leaflet) separated by an aqueous environment, the periplasm (Fig. 1a)[1] This later compartment contains peptidoglycan (PG), a polymer of glycan strands crosslinked by short peptides that provides shape and rigidity to the cell[2]. AFM was used to capture the structural and mechanical properties of E. coli, with the aim to answer the following questions: does Lpp play a role in regulating cell envelope mechanics; is the mechanical function of Lpp primarily controlled through the physical connection between OM and PG, and/or through modulation of the periplasmic width; does Lpp-dependent cell mechanics influence the sensitivity to antibiotics? The results show that the Lpp-dependent OM–PG connection plays a key role in controlling the stiffness of the cell envelope and its sensitivity to drugs, with the OM-to-IM separation distance and the Lpp-PG covalent link being of particular importance

Methods

Results

Conclusion