Thanatin Impairs Lipopolysaccharide Transport Complex Assembly by Targeting LptC-LptA Interaction and Decreasing LptA Stability.

Elisabete C C M Moura,Alessandra Polissi,Emanuela Erba,Cedric Laguri,Alessandra Romanelli,Alessandra M Martorana,Paola Sperandeo,Tiago Baeta,Jean-Pierre Simorre

doi:10.3389/fmicb.2020.00909

Abstract

The outer membrane (OM) of Gram-negative bacteria is a highly selective permeability barrier due to its asymmetric structure with lipopolysaccharide (LPS) in the outer leaflet. In Escherichia coli, LPS is transported to the cell surface by the LPS transport (Lpt) system composed of seven essential proteins forming a transenvelope bridge. Transport is powered by the ABC transporter LptB2FGC, which extracts LPS from the inner membrane (IM) and transfers it, through LptC protein, to the periplasmic protein LptA. Then, LptA delivers LPS to the OM LptDE translocon for final assembly at the cell surface. The Lpt protein machinery operates as a single device, since depletion of any component leads to the accumulation of a modified LPS decorated with repeating units of colanic acid at the IM outer leaflet. Moreover, correct machine assembly is essential for LPS transit and disruption of the Lpt complex results in LptA degradation. Due to its vital role in cell physiology, the Lpt system represents a good target for antimicrobial drugs. Thanatin is a naturally occurring antimicrobial peptide reported to cause defects in membrane assembly and demonstrated in vitro to bind to the N-terminal β-strand of LptA. Since this region is involved in both LptA dimerization and interaction with LptC, we wanted to elucidate the mechanism of inhibition of thanatin and discriminate whether its antibacterial effect is exerted by the disruption of the interaction of LptA with itself or with LptC. For this purpose, we here implemented the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system to probe in vivo the Lpt interactome in the periplasm. With this system, we found that thanatin targets both LptC–LptA and LptA–LptA interactions, with a greater inhibitory effect on the former. We confirmed in vitro the disruption of LptC–LptA interaction using two different biophysical techniques. Finally, we observed that in cells treated with thanatin, LptA undergoes degradation and LPS decorated with colanic acid accumulates. These data further support inhibition or disruption of Lpt complex assembly as the main killing mechanism of thanatin against Gram-negative bacteria.

Highlights

The emergence and spread of multidrug resistant pathogens pose an alarming threat to human and animal health worldwide
Samples were taken at different time points within 2 h from MG1655 cultures grown in the presence or absence of thanatin at 5.25 μg/mL (1.5 × Minimal Inhibitory Concentration (MIC)) and analyzed by western blotting using anti-LptA antibodies
The abundance of LptD did not change over time, indicating that the steady-state level of this outer membrane (OM) component is not affected by thanatin treatment

Summary

Introduction

The emergence and spread of multidrug resistant pathogens pose an alarming threat to human and animal health worldwide. The old classes of antibiotics are becoming ineffective at killing an increasing number of pathogens and the decline in the discovery and development of new drugs, experienced in recent years, is seriously eroding the ability of clinicians to control infectious diseases, making the identification of new antimicrobial compounds with novel mechanisms of action an urgent need (Tacconelli et al, 2018) This situation is even more worrisome for Gram-negative pathogens since they are endowed with an asymmetric outer membrane (OM), surrounding the inner membrane (IM) and delimiting a peptidoglycancontaining periplasmic space, that protects them from harmful hydrophobic compounds such as antibiotics (Nikaido, 2003). The biosynthesis of the lipid A-core domain takes place at the cytoplasmic side of the IM, whereas the assembly of mature LPS occurs at the periplasmic side of the IM, after flipping of the lipid A-core across the IM by the essential transporter MsbA (Polissi and Georgopoulos, 1996; Raetz and Whitfield, 2002; Doerrler et al, 2004)

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