Structural, mechanistic, and physiological insights into phospholipase A-mediated membrane phospholipid degradation in Pseudomonas aeruginosa

Abstract

Cells steadily adapt their membrane glycerophospholipid (GPL) composition to changing environmental and developmental conditions. While the regulation of membrane homeostasis via GPL synthesis in bacteria has been studied in detail, the mechanisms underlying the controlled degradation of endogenous GPLs remain unknown. Thus far, the function of intracellular phospholipases A (PLAs) in GPL remodeling (Lands cycle) in bacteria is not clearly established. Here, we identified the first cytoplasmic membrane-bound phospholipase A1 (PlaF) from Pseudomonas aeruginosa, which might be involved in the Lands cycle. PlaF is an important virulence factor, as the P. aeruginosa ΔplaF mutant showed strongly attenuated virulence in Galleria mellonella and macrophages. We present a 2.0-Å-resolution crystal structure of PlaF, the first structure that reveals homodimerization of a single-pass transmembrane (TM) full-length protein. PlaF dimerization, mediated solely through the intermolecular interactions of TM and juxtamembrane regions, inhibits its activity. The dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network, which might explain the product (fatty acid) feedback inhibition observed with the purified PlaF protein. We used molecular dynamics simulations and configurational free energy computations to suggest a model of PlaF activation through a coupled monomerization and tilting of the monomer in the membrane, which constrains the active site cavity into contact with the GPL substrates. Thus, these data show the importance of the PlaF mediated GPL remodeling pathway for virulence and could pave the way for the development of novel therapeutics targeting PlaF.

Substrate Access Mechanism in a Novel Membrane-Bound Phospholipase A of Pseudomonas aeruginosa Concordant with Specificity and Regioselectivity

“…The crystal structure of PlaF is available from the Protein Data Bank (PDB) 79 (PDB id: 6I8W). 80 The last five residues of the C-terminus were missing in the structure and, hence, were added using the MODELLER. 81 The starting configuration of PlaF for MD simulations was prepared by embedding t-PlaF A into a lipid bilayer membrane consisting of 75% DLPE and 25% DLPG.…”

Virulence adaptation ofPseudomonas aeruginosaphospholipase mutant with altered membrane phospholipid composition

“…PL remodelling mechanism suggested for PlaF, is similar to the recently discovered P. aeruginosa pathway involving a phospholipase C and glycosyltransferase-catalysed replacement of glycerophospholipids of outer membrane with glycolipids in response to phosphate limitation [40]. The function of PlaF in in vivo PL modulation was strengthened by findings that PlaF shows in vitro catalytic activity towards physiological substrates and that PlaF activity is regulated by a negative feedback loop based on an association with a reaction product, fatty acids (FAs) [38].…”

“…Recently, we discovered two intracellular PLAs, PlaF and PlaB, in P. aeruginosa with the ability to hydrolyse endogenous membrane PLs [37, 38]. In vivo PlaF degradation of six PLs differing in head groups and acyl chains was suggested to be responsible for alterations of their membrane PL composition [38] through a pathway analogous to the eukaryotic Lands cycle PL remodelling pathway [39].…”

“…Recently, we discovered two intracellular PLAs, PlaF and PlaB, in P. aeruginosa with the ability to hydrolyse endogenous membrane PLs [37, 38]. In vivo PlaF degradation of six PLs differing in head groups and acyl chains was suggested to be responsible for alterations of their membrane PL composition [38] through a pathway analogous to the eukaryotic Lands cycle PL remodelling pathway [39]. PL remodelling mechanism suggested for PlaF, is similar to the recently discovered P. aeruginosa pathway involving a phospholipase C and glycosyltransferase-catalysed replacement of glycerophospholipids of outer membrane with glycolipids in response to phosphate limitation [40].…”

“…A crystal structure and biochemical and computational results provided a mechanism of how dimer to monomer transition of PlaF and subsequent rotation of PlaF at the membrane surface leads to recognition of PL substrate, its extraction from the membrane through the tunnel to the active site where it is hydrolysed and the products are likely released to the membrane [38, 41, 42]. We suggest that PlaF-dependent modulation of the membrane PL composition is responsible for the strongly attenuated virulence of P. aeruginosa ΔplaF in a Galleria mellonella virulence assay and macrophage cytotoxicity assay.…”