Novel intracellular phospholipase B from<i>Pseudomonas aeruginosa</i>with activity towards endogenous phospholipids affects biofilm assembly

Aj, Weiler; Spitz, Olivia; Gudzuhn, Mirja; Sn, Schott-Verdugo; Kamel, Michael; Thiele, Björn; Sand, Wolfgang; Kedrov, Alexej; Schmitt, Lutz; Gohlke, Holger; Kovačić, Filip

doi:10.1101/2021.06.15.448513

Cited by 3 publications

(2 citation statements)

References 75 publications

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“…MALS was performed as per the previous report (Weiler et al ., 2021). Superdex 200 Increase 10/300 GL column (GE Healthcare) was pre-equilibrated overnight at 0.1 ml/minute flow ratewith buffer E (20 mM HEPES pH 8.0, 200 mM NaCl, 1 mM β-ME).…”

Section: Methodsmentioning

confidence: 99%

A MademoiseLLE domain binding platform links the key RNA transporter to endosomes

Devan

Schott‐Verdugo

Müntjes

et al. 2021

Preprint

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Spatiotemporal expression is mostly achieved by transport and translation of mRNAs at defined subcellular sites. An emerging mechanism mediating mRNA trafficking is microtubule-dependent co-transport of mRNAs on shuttling endosomes. Although progress has been made in identifying various components of the endosomal mRNA transport machinery, a mechanistic understanding of how these RNA-binding proteins are connected to endosomes is still lacking. Here, we demonstrate that a flexible MademoiseLLE (MLLE) domain platform within Rrm4 of Ustilago maydis is crucial for endosomal attachment. Our structure/function analysis uncovered three MLLE domains at the C-terminus of Rrm4 with a functionally defined hierarchy. MLLE3 recognizes two PAM2-like sequences of the adaptor protein Upa1 and is essential for endosomal shuttling of Rrm4. MLLE1 and MLLE2 are most likely accessory domains that exhibit a variant binding mode for interaction with currently unknown partners. Thus, endosomal attachment of the mRNA transporter is orchestrated by a sophisticated MLLE domain binding platform.

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Section: Methodsmentioning

confidence: 99%

A MademoiseLLE domain binding platform links the key RNA transporter to endosomes

Devan

Schott‐Verdugo

Müntjes

et al. 2021

Preprint

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“…Gas Chromatography-mass Spectrometric (GC-MS) Analysis of FAs Extracted from P. aeruginosa P. aeruginosa PA01 (wild-type, WT) and the ΔplaF mutant were cultivated overnight (37 C, LB medium, agitation), and cells were separated from the supernatant, followed by chloroform/methanol extraction of FFAs, derivatization of FFAs with N-methyl-N-(trimethylsilyl) trifluoroacetamide, and quantification by GC-MS as described in ref. (50).…”

Section: 1mentioning

confidence: 99%

Molecular Mechanisms Underlying Medium-Chain Free Fatty Acid-regulated Activity of the Phospholipase PlaF fromPseudomonas aeruginosa

Gentile

Modric

Thiele

et al. 2023

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PlaF is a membrane-bound phospholipase A1 from Pseudomonas aeruginosa that is involved in the remodeling of the membrane phospholipid composition. It modulates virulence-associated signaling and metabolic networks, although it remains unclear whether P. aeruginosa's physiological changes are elicited by altered phospholipid composition or by free fatty acids (FFAs) released from phospholipids. Previously, we identified that undecanoic acid bound to the active site of PlaF in the crystal structure inhibits PlaF activity and triggers the formation of PlaF dimers. However, the detailed molecular mechanism governing the inhibition and dimerization of PlaF by medium-chain length FFAs has remained elusive. In this study, we used unbiased and biased molecular dynamics simulations and free energy computations to assess how FFAs localized in different cell compartments can interact with active and inactive configurations of PlaF and how this may regulate PlaF activity. Our results suggest that medium-chain length FFAs can inhibit PlaF both competitively and non-competetively, in line with previous experiments. They stabilize inactive dimeric PlaF when localized in the upper membrane leaflet and enter a tunnel connected to the catalytic triad when localized in the periplasmic space. We predict mutations for either case to probe these effects experimentally. Our findings support the suggestion from in vivo results shown here and in vitro results presented previously that PlaF activity may be regulated through a product-feedback mechanism. They also open up new perspectives on how to inhibit PlaF, which has been suggested as a promising target for developing new antibiotics against P. aeruginosa.

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Evidence for a bacterial Lands cycle phospholipase A: Structural and mechanistic insights into membrane phospholipid remodeling

Bleffert

Granzin

Caliskan

et al. 2021

Preprint

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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 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. A dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network which likely explains 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 GPL remodeling pathway for virulence and pave the way for the development of a novel therapeutic class of antibiotics targeting PlaF-mediated membrane GPL remodeling.

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Novel intracellular phospholipase B fromPseudomonas aeruginosawith activity towards endogenous phospholipids affects biofilm assembly

Cited by 3 publications

References 75 publications

A MademoiseLLE domain binding platform links the key RNA transporter to endosomes

A MademoiseLLE domain binding platform links the key RNA transporter to endosomes

Molecular Mechanisms Underlying Medium-Chain Free Fatty Acid-regulated Activity of the Phospholipase PlaF fromPseudomonas aeruginosa

Evidence for a bacterial Lands cycle phospholipase A: Structural and mechanistic insights into membrane phospholipid remodeling

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