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

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.

Keywords: Lpt system; LpxC inhibitor; bacterial cell envelope; lipopolysaccharide; outer membrane stability.

FIGURE 1

Structure of LPS and the Lpt complex. (A) The lipopolysaccharide (LPS) molecule consists of three parts: (i) the lipid A moiety, comprising a phosphorylated glucosamine disaccharide linked to saturated acyl chains that insert into the outer membrane (OM); (ii) the core oligosaccharide formed by non-repeating sugar units; and (iii) the O antigen (not depicted), a long oligosaccharide chain that varies highly between species and strains. (B) LPS is extracted from the inner membrane (IM) by the IM complex LptB₂FG and translocated through the hydrophobic cavity of the protein bridge formed by LptC, LptA, and the N-terminal region of LptD. The C-terminal domain of LptD forms the β-barrel that, together with LptE, inserts LPS into the OM.

FIGURE 2

Depletion of LptC does not affect the stability of the IM and OM Lpt sub-complexes. (A) Cells of BB3 were grown with 0.2% arabinose until OD₆₀₀ 0.2; the cells were then harvested, washed three times, and resuspended in an arabinose-supplemented (+ Ara) or arabinose-free (no Ara) medium. Samples for analysis of protein stability were collected at the time points indicated. (B) Whole-cell extracts were prepared and analyzed by western blot with anti-LptA, anti-LptD, anti-LptC, anti-LptB, and anti-LptE (as the loading control) antibodies. An equal number of cells (0.67 OD₆₀₀) were loaded into each lane. Growth curves and western blot analyses shown are representative of at least three independent experiments.

FIGURE 3

Inhibition of LPS synthesis affects LptA and LptD stability. (A) Escherichia coli wild-type cells were grown in the LB-Lennox medium. When cells reached OD₆₀₀ of 0.1, they were treated with 1xMIC of LPC-058 (LpxC inhibitor). Untreated cells were used as the control. Cell growth was monitored by OD₆₀₀ measurements. When cultures reached OD₆₀₀ of 1.0, they were diluted 10-fold, and samples for western blot analysis were taken after culture dilution at the indicated times. (B) Cells were treated or not treated with LPC-058 as indicated in panel (A) but not diluted. When the growth curves of treated and untreated cells separate (arrow), samples for western blot analysis were collected. Time points indicate the minutes after the growth curves separated. (C) LPS profiles of wild-type cells treated or not treated with LPC-058. Whole-cell extracts obtained at the same time point as in panel (B) were analyzed by western blot with anti-LPS and anti-LptE antibodies. An equal number of cells (0.67 OD₆₀₀ in panels (A) and (B) and 0.17 OD₆₀₀ in panel (C)) were loaded into each lane. Growth curves and western blot analyses shown are representative of at least three independent experiments.

FIGURE 4

Disrupting the LPS layer at the OM does not affect LptA and LptD levels. Escherichia coli wild-type cells were grown in the LB-Lennox medium. When cells reached OD₆₀₀ = 0.1, they were treated with EDTA, polymyxin B, or ammonium metavanadate at the concentration indicated. Untreated cells were used as the control. Cell growth was monitored by OD₆₀₀ measurements. When the growth curves of treated and untreated cells separate (arrow), samples for western blot analysis were collected. Time points indicate the minutes after the growth curves separated. An equal number of cells (0.67 OD₆₀₀) were loaded into each lane. Growth curves and western blot analyses shown are representative of at least three independent experiments.

FIGURE 5

Disturbing the PG layer does not affect LptA and LptD levels. Escherichia coli wild-type cells were grown in the LB-Lennox medium. When cells reached OD₆₀₀ = 0.1, they were treated with aztreonam or cefsulodin at the concentrations indicated. Untreated cells were used as the control. Cell growth was monitored by OD₆₀₀ measurements. When the growth curves of treated and untreated cells separate (arrow), samples for western blot analysis were collected. Time points indicate the minutes after the growth curves separated. An equal number of cells (0.67 OD₆₀₀) were loaded into each lane. Growth curves and western blot analyses shown are representative of at least three independent experiments.

FIGURE 6

DegP degrades LptD when the LPS biosynthesis is inhibited. Escherichia coli wild-type (wt), ΔdegP, and ΔbepA cells were grown in the LB-Lennox medium at 30°C. When cells reached OD₆₀₀ = 0.1, they were treated with 1xMIC of LPC-058 (LpxC inhibitor). Untreated cells were used as the control. (A) For the analysis of the steady-state level Lpt proteins, cell growth was monitored by OD₆₀₀ measurements. When the growth curves of treated and untreated cells separate (arrow), samples for western blot analysis were taken. Time points indicate the minutes after the growth curves separated. * indicates the degradation product of LptA. (B) OM fractions were purified from cells treated for 2 h with LPC-058. Equal amounts of proteins were loaded (upper panel). Crude extracts of the same cultures were analyzed by western blot, using anti-OmpA antibodies, and equal amounts of cells were loaded (lower panel, 0.67 OD₆₀₀). Growth curves and western blot analyses shown are representative of at least three independent experiments.