Degradation of Components of the Lpt Transenvelope Machinery Reveals LPS-Dependent Lpt Complex Stability in Escherichia coli.

Alessandra M Martorana,Xiaofei Liang,Elisabete C C M Moura,Flavia Di Vincenzo,Paola Sperandeo,Alessandra Polissi,Pei Zhou,Eric Toone

doi:10.3389/fmolb.2021.758228

Alessandra M Martorana, Xiaofei Liang + Show 6 more

Open Access

https://doi.org/10.3389/fmolb.2021.758228

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Abstract

Lipopolysaccharide (LPS) is a peculiar component of the outer membrane (OM) of many Gram-negative bacteria that renders these bacteria highly impermeable to many toxic molecules, including antibiotics. LPS is assembled at the OM by a dedicated intermembrane transport system, the Lpt (LPS transport) machinery, composed of seven essential proteins located in the inner membrane (IM) (LptB2CFG), periplasm (LptA), and OM (LptDE). Defects in LPS transport compromise LPS insertion and assembly at the OM and result in an overall modification of the cell envelope and its permeability barrier properties. LptA is a key component of the Lpt machine. It connects the IM and OM sub-complexes by interacting with the IM protein LptC and the OM protein LptD, thus enabling the LPS transport across the periplasm. Defects in Lpt system assembly result in LptA degradation whose stability can be considered a marker of an improperly assembled Lpt system. Indeed, LptA recruitment by its IM and OM docking sites requires correct maturation of the LptB2CFG and LptDE sub-complexes, respectively. These quality control checkpoints are crucial to avoid LPS mistargeting. To further dissect the requirements for the complete Lpt transenvelope bridge assembly, we explored the importance of LPS presence by blocking its synthesis using an inhibitor compound. Here, we found that the interruption of LPS synthesis results in the degradation of both LptA and LptD, suggesting that, in the absence of the LPS substrate, the stability of the Lpt complex is compromised. Under these conditions, DegP, a major chaperone–protease in Escherichia coli, is responsible for LptD but not LptA degradation. Importantly, LptD and LptA stability is not affected by stressors disturbing the integrity of LPS or peptidoglycan layers, further supporting the notion that the LPS substrate is fundamental to keeping the Lpt transenvelope complex assembled and that LptA and LptD play a major role in the stability of the Lpt system.

Highlights

Gram-negative bacteria are surrounded by two distinct lipid bilayers: the inner membrane (IM) and the outer membrane (OM)
We treated the cells with LPC-058, a compound known to inhibit the activity of LpxC, which catalyzes the first essential step of LPS biosynthesis and we show that LptA and LptD steady-state levels decrease in the cell upon LPS biosynthesis blockage
In E. coli cells, LptA is degraded in the absence of the correct IM and OM docking sites, as depletion of LptC, LptD, or LptE (Chng et al, 2012) results in decreased LptA levels (Sperandeo et al, 2011)

Summary

Introduction

Gram-negative bacteria are surrounded by two distinct lipid bilayers: the inner membrane (IM) and the outer membrane (OM). The asymmetric configuration of the OM, with phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet, greatly hampers the permeation of many toxic molecules into the bacterium. The complex architecture of the Gram-negative bacterial cell envelope poses a great challenge to the development of novel antimicrobial molecules (Nikaido, 2003; Tacconelli et al, 2018). Targets in the LPS transport pathway have emerged providing innovative approaches for antibiotic development (Srinivas et al, 2010; Werneburg et al, 2012; Andolina et al, 2018; Vetterli et al, 2018; Moura et al, 2020; Zhang et al, 2019)

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