A Stress Response Monitoring Lipoprotein Trafficking to the Outer Membrane

Abstract

Gram-negative bacteria produce lipid-anchored lipoproteins that are trafficked to their outer membrane (OM). These lipoproteins are essential components in each of the molecular machines that build the OM, including the Bam machine that assembles β-barrel proteins and the Lpt pathway that transports lipopolysaccharide. Stress responses are known to monitor Bam and Lpt function, yet no stress system has been found that oversees the fundamental process of lipoprotein trafficking. We used genetic and chemical biology approaches to induce several different lipoprotein trafficking stresses in Escherichia coli. Our results identified the Cpx two-component system as a stress response for monitoring trafficking. Cpx is activated by trafficking defects and is required to protect the cell against the consequence of the resulting stress. The OM-targeted lipoprotein NlpE acts as a sensor that allows Cpx to gauge trafficking efficiency. We reveal that NlpE signals to Cpx while it is transiting the inner membrane (IM) en route to the OM and that only a small highly conserved N-terminal domain is required for signaling. We propose that defective trafficking causes NlpE to accumulate in the IM, activating Cpx to mount a transcriptional response that protects cells. Furthermore, we reconcile this new role of NlpE in signaling trafficking defects with its previously proposed role in sensing copper (Cu) stress by demonstrating that Cu impairs acylation of lipoproteins and, consequently, their trafficking to the OM. IMPORTANCE The outer membrane built by Gram-negative bacteria such as Escherichia coli forms a barrier that prevents antibiotics from entering the cell, limiting clinical options at a time of prevalent antibiotic resistance. Stress responses ensure that barrier integrity is continuously maintained. We have identified the Cpx signal transduction system as a stress response that monitors the trafficking of lipid-anchored lipoproteins to the outer membrane. These lipoproteins are needed by every machine that builds the outer membrane. Cpx monitors just one lipoprotein, NlpE, to detect the efficiency of lipoprotein trafficking in the cell. NlpE and Cpx were previously shown to play a role in resistance to copper. We show that copper blocks lipoprotein trafficking, reconciling old and new observations. Copper is an important element in innate immunity against pathogens, and our findings suggest that NlpE and Cpx help E. coli survive the assault of copper on a key outer membrane assembly pathway.

Variant Signal Peptides of Vaccine Antigen, FHbp, Impair Processing Affecting Surface Localization and Antibody-Mediated Killing in Most Meningococcal Isolates

“…The absence of lipid by Lgt activity would explain the subsequent failure of LspA to cleave the SP of these isolates (Figure 5A). A recent study in E. coli showed that prevention of diacylglyceryl modification at the conserved cysteine residue of Lpp, by Lgt depletion, prevented Lsp from processing the SP as inferred from its electrophoretic mobility (May et al, 2019). This supports earlier studies that demonstrated that substitution of the Cys residue of Lpp prevented diacylglyceryl modification with accumulation of Lpp exclusively of slower mobility and of the size expected for the unprocessed preprolipoprotein (Inouye et al, 1983).…”

Metabolism of long-chain fatty acids affects disulfide bond formation in Escherichia coli and activates envelope stress response pathways as a combat strategy

“…An outer membrane lipoprotein, NlpE, accumulates at the inner membrane due to defects in lipoprotein trafficking and physically interacts with CpxA. Since NlpE is also a DsbA substrate, its mutant lacking the C-terminal disulfide bond turns on Cpx and Cpx activation in Δ dsbA is NlpE-dependent, this lipoprotein has been proposed to sense redox imbalance in the envelope [ 50 , 51 ]. Another mechanism of Cpx activation involves titration of CpxP, a periplasmic inhibitor of CpxA.…”

Metabolism of long-chain fatty acids affects disulfide bond formation in Escherichia coli and activates envelope stress response pathways as a combat strategy

“…An outer membrane lipoprotein, NlpE, accumulates at the inner membrane due to defects in lipoprotein trafficking and physically interacts with CpxA. Since NlpE is also a DsbA substrate, its mutant lacking the C-terminal disulfide bond turns on Cpx and Cpx activation in Δ dsbA is NlpE-dependent, this lipoprotein has been proposed to sense redox imbalance in the envelope [49, 50]. Another mechanism of Cpx activation involves titration of CpxP, a periplasmic inhibitor of CpxA.…”