Dataset Information

Insight into the dual function of lipid phosphate phosphatase PgpB involved in two essential cell-envelope metabolic pathways in Escherichia coli.

ABSTRACT: Ubiquitous PAP2 lipid phosphatases are involved in a wide array of central physiological functions. PgpB from Escherichia coli constitutes the archetype of this subfamily of membrane proteins. It displays a dual function by catalyzing the biosynthesis of two essential lipids, the phosphatidylglycerol (PG) and the undecaprenyl phosphate (C55-P). C55-P constitutes a lipid carrier allowing the translocation of peptidoglycan subunits across the plasma membrane. PG and C55-P are synthesized in a redundant manner by PgpB and other PAP2 and/or unrelated membrane phosphatases. Here, we show that PgpB is the sole, among these multiple phosphatases, displaying this dual activity. The inactivation of PgpB does not confer any apparent growth defect, but its inactivation together with another PAP2 alters the cell envelope integrity increasing the susceptibility to small hydrophobic compounds. Evidence is also provided of an interplay between PAP2s and the peptidoglycan polymerase PBP1A. In contrast to PGP hydrolysis, which relies on a His/Asp/His catalytic triad of PgpB, the mechanism of C55-PP hydrolysis appeared as only requiring the His/Asp diad, which led us to hypothesize distinct processes. Moreover, thermal stability analyses highlighted a substantial structural change upon phosphate binding by PgpB, supporting an induced-fit model of action.

SUBMITTER: Tian X

PROVIDER: S-EPMC7413402 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Insight into the dual function of lipid phosphate phosphatase PgpB involved in two essential cell-envelope metabolic pathways in Escherichia coli.

Tian Xudong X Auger Rodolphe R Manat Guillaume G Kerff Frédéric F Mengin-Lecreulx Dominique D Touzé Thierry T

Scientific reports 20200806 1

Ubiquitous PAP2 lipid phosphatases are involved in a wide array of central physiological functions. PgpB from Escherichia coli constitutes the archetype of this subfamily of membrane proteins. It displays a dual function by catalyzing the biosynthesis of two essential lipids, the phosphatidylglycerol (PG) and the undecaprenyl phosphate (C55-P). C55-P constitutes a lipid carrier allowing the translocation of peptidoglycan subunits across the plasma membrane. PG and C55< ...[more]

PMID: 32764655

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Project description:LPP2 is one of three enzymes in the lipid phosphate phosphatase family (LPP1-3) that dephosphorylate extracellular and intracellular bioactive lipid phosphates and pyrophosphates. LPP2 increases cell growth and LPP2 expression is elevated in a variety of malignancies, implying that LPP2 is a pro-tumorigenic factor. Methods: LPP2 expression in human breast tumors and normal breast tissue was measured by qPCR. To understand the role of LPP2, we knocked out its expression in multiple cell lines using CRISPR/Cas9. Cell proliferation and migration were compared between wild type and LPP2 knockout cells. Cell cycle was measured by flow cytometry, and cell cycle proteins were determined by western blotting. Effects of LPP2 on tumor growth were investigated using syngeneic and xenograft mouse breast cancer models. Results: LPP2 mRNA levels were higher in ER/PR positive, ER/HER2 positive, and triple negative human breast tumors, relative to normal breast tissue. Higher levels of LPP2 in breast tumors, hepatocellular carcinoma, pancreatic adenocarcinoma, and melanomas were prognostic of poorer survival. LPP2 mRNA expression is also increased in Hs-578T, MDA-MB-231, MCF7 and MDA-MB-468 breast cancer cell lines, relative to non-malignant Hs-578Bst, MCF10A and MCF-12A cells. LPP2 knockout in breast cancer cells decreased cell growth by inhibiting G1/S transition, whereas, increasing LPP2 levels in Hs-578Bst and MCF10A cells promoted proliferation. The effects of LPP2 on cell cycle were associated with changes in cyclin A2, cyclin B1, and cell cycle inhibitors, p27 or p21. The level of c-Myc was downregulated by knocking out LPP2, and it was partly restored by re-expressing LPP2. The positive correlation between the expression of LPP2 and c-Myc exists in multiple cancer cell lines including breast, lung, upper aerodigestive tract and urinary tract cancer. LPP2 knockout in MDA-MB-231 or 4T1 cells suppressed tumor formation in mouse breast cancer models, and decreased the in vivo expression of Ki67 and c-Myc of the cancer cells. Conclusion: Targeting LPP2 could provide a new strategy for decreasing c-Myc expression and tumor growth.

Project description:Although distinct lipid phosphatases are thought to be required for processing lipid A (component of the outer leaflet of the outer membrane), glycerophospholipid (component of the inner membrane and the inner leaflet of the outer membrane), and undecaprenyl pyrophosphate (C55-PP; precursors of peptidoglycan and O antigens of lipopolysaccharide) in Gram-negative bacteria, we report that the lipid A 1-phosphatases, LpxEs, functionally connect multiple layers of cell envelope biogenesis in Gram-negative bacteria. We found that Aquifex aeolicus LpxE structurally resembles YodM in Bacillus subtilis, a phosphatase for phosphatidylglycerol phosphate (PGP) with a weak in vitro activity on C55-PP, and rescues Escherichia coli deficient in PGP and C55-PP phosphatase activities; deletion of lpxE in Francisella novicida reduces the MIC value of bacitracin, indicating a significant contribution of LpxE to the native bacterial C55-PP phosphatase activity. Suppression of plasmid-borne lpxE in F. novicida deficient in chromosomally encoded C55-PP phosphatase activities results in cell enlargement, loss of O-antigen repeats of lipopolysaccharide, and ultimately cell death. These discoveries implicate LpxE as the first example of a multifunctional regulatory enzyme that orchestrates lipid A modification, O-antigen production, and peptidoglycan biogenesis to remodel multiple layers of the Gram-negative bacterial envelope.IMPORTANCE Dephosphorylation of the lipid A 1-phosphate by LpxE in Gram-negative bacteria plays important roles in antibiotic resistance, bacterial virulence, and modulation of the host immune system. Our results demonstrate that in addition to removing the 1-phosphate from lipid A, LpxEs also dephosphorylate undecaprenyl pyrophosphate, an important metabolite for the synthesis of the essential envelope components, peptidoglycan and O-antigen. Therefore, LpxEs participate in multiple layers of biogenesis of the Gram-negative bacterial envelope and increase antibiotic resistance. This discovery marks an important step toward understanding the regulation and biogenesis of the Gram-negative bacterial envelope.

Dataset Information

Insight into the dual function of lipid phosphate phosphatase PgpB involved in two essential cell-envelope metabolic pathways in Escherichia coli.

Publications

Insight into the dual function of lipid phosphate phosphatase PgpB involved in two essential cell-envelope metabolic pathways in Escherichia coli.

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