Pseudomonas Aeruginosa  Responds to Altered Membrane Phospholipid Composition by Adjusting the Production of Two-Component Systems, Proteases and Iron Uptake Proteins

Caliskan, Muttalip; Poschmann, Gereon; Gudzuhn, Mirja; Waldera-Lupa, Daniel M.; Streit, Wolfgang R.; Jaeger, Karl‐Erich; Kovačić, Filip

doi:10.2139/ssrn.4303125

Cited by 3 publications

(3 citation statements)

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“…Comparative GC-MS profiling of FFAs secreted by P. aeruginosa Δ plaF and the wild-type cells revealed that PlaF can release medium-chain FFAs, including C10 and C14, from GPLs as in vitro substrates. This result corroborates our suggestion that PlaF exerts multiple physiological functions (48) through GPL degradation (6). We observed that the chain length of the FFAs has a minor impact on the tilting transition (6), indicating that other medium-chain FFAs may influence PlaF similarly as described here for C10.…”

Section: Discussionsupporting

confidence: 92%

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

Gentile

Modric

Thiele

et al. 2023

Preprint

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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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Section: Discussionsupporting

confidence: 92%

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

Gentile

Modric

Thiele

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Comparative GC-MS profiling of FFAs secreted by P. aeruginosa ΔplaF and the wild-type cells revealed that PlaF can release medium-chain FFAs, including C10 and C14, from GPLs as in vitro substrates. This result corroborates our suggestion that PlaF exerts multiple physiological functions 48 through GPL degradation. 6 We observed that the chain length of the FFAs has a minor impact on the tilting transition.…”

Section: Possible Physiological Relevance Of Ffa-mediated Plaf Inhibi...supporting

confidence: 92%

Molecular Mechanisms Underlying Medium-Chain Free Fatty Acid-Regulated Activity of the Phospholipase PlaF from Pseudomonas aeruginosa

Gentile,

Modric,

Thiele

et al. 2024

JACS Au

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PlaF is a membrane-bound phospholipase A 1 from Pseudomonas aeruginosa that is involved in remodeling membrane glycerophospholipids (GPLs) and modulating virulence-associated signaling and metabolic pathways. Previously, we identified the role of medium-chain free fatty acids (FFAs) in inhibiting PlaF activity and promoting homodimerization, yet the underlying molecular mechanism remained elusive. Here, we used unbiased and biased molecular dynamics simulations and free energy computations to assess how PlaF interacts with FFAs localized in the water milieu surrounding the bilayer or within the bilayer and how these interactions regulate PlaF activity. Mediumchain FFAs localized in the upper bilayer leaflet can stabilize inactive dimeric PlaF, likely through interactions with charged surface residues, as has been experimentally validated. Potential of mean force (PMF) computations indicate that membrane-bound FFAs may facilitate the activation of monomeric PlaF by lowering the activation barrier for changing into a tilted, active configuration. We estimated that the coupled equilibria of PlaF monomerization-dimerization and tilting at the physiological concentration of PlaF lead to the majority of PlaF forming inactive dimers when in a cell membrane loaded with decanoic acid (C10). This is in agreement with a suggested in vivo product feedback loop and gas chromatography−mass spectrometry profiling results, indicating that PlaF catalyzes the release of C10 from P. aeruginosa membranes. Additionally, we found that C10 in the water milieu can access the catalytic site of active monomeric PlaF, contributing to the competitive component of C10-mediated PlaF inhibition. Our study provides mechanistic insights into how medium-chain FFAs may regulate the activity of PlaF, a potential bacterial drug target.

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“…Others suppress drug efflux 7 . To explore whether ligation is negatively coupled with transport (Figures 5A-5F), PA14 iron 54 and both spermidine and putrescine uptake assays were initiated. 2,2'-bipyridyl depletion of intracellular iron facilitated measurement of exogenous Fe 2+ uptake through the chelation-dependent colorimetric change in ferrozine 55 .…”

Section: N-104 Ligation Suppresses Fe 2+ and Polyamine Uptakementioning

confidence: 99%

Targeting Iron - Respiratory Reciprocity Promotes Bacterial Death

Sharifian Gh.,

Norouzi,

Sorci

et al. 2024

Preprint

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Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through an unbiased resistance screen of 3,884 gene knockout strains, we uncovered a previously unknown non-lytic bactericidal mechanism that sequentially couples three transporters and downstream transcription to lethally suppress respiration of the highly virulent P. aeruginosa strain PA14 - one of three species on the WHO's 'Priority 1: Critical' list. By targeting outer membrane YaiW, cationic lacritin peptide 'N-104' translocates into the periplasm where it ligates outer loops 4 and 2 of the inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake. This broadly shuts down transcription of many biofilm-associated genes, including ferrous iron-dependent TauD and ExbB1. The mechanism is innate to the surface of the eye and is enhanced by synergistic coupling with thrombin peptide GKY20. This is the first example of an inhibitor of multiple bacterial transporters.

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Pseudomonas Aeruginosa Responds to Altered Membrane Phospholipid Composition by Adjusting the Production of Two-Component Systems, Proteases and Iron Uptake Proteins

Cited by 3 publications

References 0 publications

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

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

Molecular Mechanisms Underlying Medium-Chain Free Fatty Acid-Regulated Activity of the Phospholipase PlaF from Pseudomonas aeruginosa

Targeting Iron - Respiratory Reciprocity Promotes Bacterial Death

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