The Lipoprotein NlpE Is a Cpx Sensor That Serves as a Sentinel for Protein Sorting and Folding Defects in the Escherichia coli Envelope

Abstract

The envelope of Gram-negative bacteria is a complex compartment that is essential for viability. To ensure survival of the bacterial cells in fluctuating environments, several signal transduction systems, called envelope stress response systems (ESRSs), exist to monitor envelope biogenesis and homeostasis. The Cpx twocomponent system is an extensively studied ESRS in Escherichia coli that is active during exposure to a vast array of stresses and protects the envelope under those harmful circumstances. Overproduction of NlpE, a two-domain outer membrane lipoprotein of unclear function, has been used in numerous studies as a molecular trigger to turn on the system artificially. However, the mechanism of Cpx activation by NlpE, as well as its physiological relevance, awaited further investigation. In this paper, we provide novel insights into the role played by NlpE in the Cpx system. We found that, among all outer membrane lipoproteins in E. coli, NlpE is sufficient to induce Cpx when lipoprotein trafficking is perturbed. Under such conditions, fitness is increased by the presence of NlpE. Moreover, we show that NlpE, through its N-terminal domain, physically interacts with the Cpx sensor kinase CpxA. Our data suggest that NlpE also serves to activate the Cpx system during oxidative folding defects in the periplasm and that its C-terminal domain is involved in the sensing mechanism. Overall, our data demonstrate that NlpE acts as a sentinel for two important envelope biogenesis processes, namely, lipoprotein sorting and oxidative folding, and they further establish NlpE as a bona fide member of the Cpx twocomponent system. IMPORTANCE Bacteria rely on a sophisticated envelope to shield them against challenging environmental conditions and therefore need to ensure correct envelope assembly and integrity. A major signaling pathway that performs this role in Gramnegative species is the Cpx system. An outer membrane lipoprotein of unclear function, NlpE, has long been exploited as a research tool to study Cpx in E. coli, since it triggers this system when overproduced or mislocalized; however, the mechanism and physiological relevance of the NlpE-Cpx connection have awaited further investigation. We elucidate a new function for NlpE by showing that it physically interacts with the Cpx sensor CpxA and acts as a sentinel that specifically monitors two essential envelope biogenesis processes, namely, lipoprotein sorting and oxidative folding.

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 inEscherichia coliand 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.…”

How the assembly and protection of the bacterial cell envelope depend on cysteine residues

“…A second envelope stress sensor with disulfidebonded cysteines in its native conformation is the outer membrane lipoprotein NlpE (Fig. 4), which activates the Cpx stress response when lipoprotein trafficking to the outer membrane is perturbed (79,80). In this case, NlpE accumulates in the inner membrane where it can physically interact with the inner membrane sensor histidine kinase CpxA, triggering Cpx (79,80).…”

“…4), which activates the Cpx stress response when lipoprotein trafficking to the outer membrane is perturbed (79,80). In this case, NlpE accumulates in the inner membrane where it can physically interact with the inner membrane sensor histidine kinase CpxA, triggering Cpx (79,80). As a result, CpxA autophosphorylates and transfers its phosphoryl group to the cytoplasmic response regulator CpxR which then binds DNA to regulate the expression of a large set of genes (81,82).…”

“…NlpE consists of two distinct structural domains: an N-terminal domain, which interacts with CpxA (80) and is homologous to the lipocalin Blc, a bacterial lipoprotein that binds hydrophobic ligands, and a Cterminal domain, which adopts an oligonucleotide/oligosaccharide-binding fold (83). Each domain of NlpE contains a disulfide bond that is introduced by DsbA (80) (Fig.…”