Biology subjects

Kovacic, F.

Publications and source records attributed to Kovacic, F..

10 recordsLinked to original sources

bioRxiv2025

A Novel Approach to Combat Pseudomonas aeruginosa: Repurposing Pharmaceuticals for Inhibition of Phospholipase A

Phospholipases A (PLAs) play critical roles in cellular physiology, making human PLAs established drug targets. On the other hand, the potential of bacterial PLAs as targets for antimicrobial drug development remains underexplored. In this study, we curated a library of 23 approved and investigational pharmaceuticals, some of which inhibit human PLA-like enzymes, through a combination of ligand structure-based searches and textual mining in literature and compound databases. Experimental screening identified that compounds GW4869, darapladib, and rilapladib significantly inhibit Pseudomonas aeruginosa growth by more than 50 %. While these compounds did not reduce biofilm formation, GW4869 increased the proportion of dead cells in established biofilms, suggesting its role in compromising biofilm cell viability. Biochemical assays revealed that all three compounds inhibited the enzymatic activity of PlaF, a PLA virulence factor of P. aeruginosa, by decreasing the affinity of a model substrate. Molecular dynamics simulations and binding free energy analyses indicate that GW4869 binds to the substrate-binding and product-release tunnels of PlaF, suggesting GW4869 as a non-covalent competitive inhibitor. Notably, the mutant strain P. aeruginosa {Delta}plaF proved to be GW4869 resistant and did not display differential growth upon GW4869 treatment, further indicating PlaF as the primary GW4869 target. Furthermore, GW4869 and rilapladib significantly enhanced the efficacy of the last-resort antibiotic imipenem in combination treatments. These findings highlight the potential of GW4869, darapladib, and rilapladib to act as repurposed inhibitors of PlaF or PLA-dependent mechanisms in P. aeruginosa and underscore the promise of combination therapies against intracellular PLAs to combat antimicrobial resistance.

molecular biology↗

bioRxiv2024

α-Aminooxyacetic acid derivatives acting as pro-drugs against Mycobacterium tuberculosis

Tuberculosis (TB), a significant cause of mortality globally, continues to claim 1.5 million lives each year. Despite recent advances in TB management, the emergence of multidrug-resistant strains of TB is exacerbating the treatment of TB. Therefore, there is an immediate necessity to uncover new anti-TB compounds with unprecedented targets. This study introduces novel antimycobacterial molecules that are based on -aminooxyacetic acid core structures. The lead compounds KSK-104 and KSK-106 displayed potent sub-micromolar antibacterial activity against Mycobacterium tuberculosis H37Rv and XDR clinical isolates, while exhibiting virtually no cytotoxicity against various human cells. Complementation experiments following whole genome sequencing of spontaneously resistant mutants generated against these bactericidal compounds suggested that they are pro-drugs that are intracellularly hydrolyzed by one or both of two specific amidohydrolases, Rv0552 and AmiC. Furthermore, proteomic and transcriptomic analyses of stressed cells and genetic interaction mapping employing transposon insertion sequencing suggest a "dirty drug" mechanism that involves the simultaneous attack of the various drug cleavage products on multiple intracellular targets. Our results suggest a primary role of the pyridoxal 5-phosphate (PLP) synthesis and salvage pathway and/or PLP-dependent enzymes, the oxidative stress network, and the largely uncharacterized Rv3092c-Rv3095 gene cluster in the mode of action.

microbiology↗

bioRxiv2024

The Bacterial Quorum-Sensing Signal 2-Aminoacetophenone Rewires Immune Cell Bioenergetics through the PGC-1α/ERRα Axis to Mediate Tolerance to Infection

How bacterial pathogens exploit host metabolism to promote immune tolerance and persist in infected hosts remains elusive. To achieve this, we show that Pseudomonas aeruginosa (PA), a recalcitrant pathogen, utilizes the quorum sensing (QS) signal 2-aminoacetophenone (2-AA). Here, we unveil how 2-AA-driven immune tolerization causes distinct metabolic perturbations in macrophages mitochondrial respiration and bioenergetics. We present evidence indicating that these effects stem from decreased pyruvate transport into mitochondria. This reduction is attributed to decreased expression of the mitochondrial pyruvate carrier (MPC1), which is mediated by diminished expression and nuclear presence of its transcriptional regulator, estrogen-related nuclear receptor alpha (ERR). Consequently, ERR exhibits weakened binding to the MPC1 promoter. This outcome arises from the impaired interaction between ERR and the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1). Ultimately, this cascade results in diminished pyruvate influx into mitochondria and, consequently reduced ATP production in tolerized macrophages. Exogenously added ATP in infected macrophages restores the transcript levels of MPC1 and ERR- and enhances cytokine production and intracellular bacterial clearance. Consistent with the in vitro findings, murine infection studies corroborate the 2-AA-mediated long-lasting decrease in ATP and acetyl-CoA and its association with PA persistence, further supporting this QS signaling molecule as the culprit of the host bioenergetic alterations and PA persistence. These findings unveil 2-AA as a modulator of cellular immunometabolism and reveal an unprecedented mechanism of host tolerance to infection involving the PGC-1/ERR axis in its influence on MPC1/OXPHOS-dependent energy production and PA clearance. These paradigmatic findings pave the way for developing treatments to bolster host resilience to pathogen-induced damage. Given that QS is a common characteristic of prokaryotes, it is likely that 2-AA-like molecules with similar functions may be present in other pathogens.

immunology↗

bioRxiv2023

Molecular Mechanisms Underlying Medium-Chain Length Free Fatty Acid-regulated Activity of the Phospholipase PlaF from P. aeruginosa

PlaF is a membrane-bound phospholipase A1 from P. aeruginosa that is involved in remodeling membrane glycerophospholipids (GPLs) and modulation of virulence-associated signaling and metabolic pathways. Previously, we identified the role of medium-chain free fatty acids (FFA) 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. Medium-chain FFAs localized in the upper bilayer leaflet can stabilize inactive dimeric PlaF, likely through interactions with charged surface residues as 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 of 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 GC-MS 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 FFA may regulate the activity of PlaF, a potential bacterial drug target.

biophysics↗

bioRxiv2022

Virulence adaptation of Pseudomonas aeruginosa phospholipase mutant with altered membrane phospholipid composition

Membrane protein and phospholipid (PL) composition changes in response to environmental cues and during infections. Covalent modification and remodelling of the acyl chain length of PLs is an important bacterial adaptation mechanism. However, little is known about which bacterial pathways are regulated in response to altered PL composition. Here, we showed that P. aeruginosa phospholipase A, PlaF, which modulates membrane PL composition, is important for biofilm biogenesis, and we performed whole-cell quantitative proteomics of P. aeruginosa wild-type and {Delta}plaF biofilms to identify pathways regulated by PlaF. The results revealed profound alterations in the abundance of several two-component systems (TCSs), including accumulation of PprAB, which controls the transition to biofilm. Furthermore, a unique phosphorylation pattern of transcriptional regulators, transporters and metabolic enzymes, as well as differential production of seven proteases, in {Delta}plaF, indicate that PlaF-mediated virulence adaptation involves complex transcriptional and posttranscriptional regulation. Moreover, proteomics revealed the depletion of pyoverdine-mediated iron uptake pathway proteins in {Delta}plaF, which agrees with the decreased concentrations of extracellular pyoverdine and intracellular iron and is likely responsible for its prolonged lag growth phase, presumably due to reduced iron uptake. Conversely, the accumulation of proteins from alternative iron-uptake systems in {Delta}plaF suggests that PlaF may function as a switch between different iron-acquisition pathways. The observation that {Delta}plaF accumulates PL-acyl chain modifying and PL synthesis enzymes reveals novel insights into the role of PlaF for membrane PL homeostasis. Although the precise mechanism by which PlaF simultaneously affects multiple pathways remains to be elucidated, we suggest that PlaF-catalyses the degradation of PLs which then serve as a signal that is amplified by proteins of two-component, phosphorylation and proteolytic degradation systems to elicit the global adaptive response in P. aeruginosa.

molecular biology↗

bioRxiv2022

The periplasmic chaperone Skp prevents misfolding of the secretory lipase A from Pseudomonas aeruginosa

Pseudomonas aeruginosa is a wide-spread opportunistic human pathogen and a high-risk factor for immunodeficient people and patients with cystic fibrosis. The extracellular lipase A belongs to the virulence factors of P. aeruginosa. The lipase undergoes folding and activation in the periplasm prior the secretion. Here, we demonstrate that the ubiquitous periplasmic chaperone Skp of P. aeruginosa, but not SurA, FkpA, PpiD or YfgM, efficiently prevents misfolding of the aggregation-prone lipase A and facilitates its activation by a specific foldase LipH. Small-angle X-ray scattering visualizes the trimeric architecture of P. aeruginosa Skp and identifies two primary conformations of the chaperone, a compact and a widely open. We describe two binding modes of Skp to the lipase, with affinities of 20 nM and 2 M, which correspond to 1:1 and 1:2 stoichiometry of the lipase:Skp complex. Two Skp trimers are required to stabilize the lipase via the apolar interactions, which are not affected by high salt concentrations typical for the sputum of cystic fibrosis patients. The chaperoning effect of Skp points to its potent role in maturation and secretion of the lipase in Pseudomonas species.

biochemistry↗

bioRxiv2021

Substrate access mechanism in a novel membrane-bound phospholipase A of Pseudomonas aeruginosa concordant with specificity and regioselectivity

PlaF is a cytoplasmic membrane-bound phospholipase A1 from Pseudomonas aeruginosa that alters the membrane glycerophospholipid (GPL) composition and fosters the virulence of this human pathogen. PlaF activity is regulated by a dimer-to-monomer transition followed by tilting of the monomer in the membrane. However, how substrates reach the active site and how the characteristics of the active site tunnels determine the activity, specificity, and regioselectivity of PlaF for natural GPL substrates has remained elusive. Here, we combined unbiased and biased all-atom molecular dynamics (MD) simulations and configurational free energy computations to identify access pathways of GPL substrates to the catalytic center of PlaF. Our results map out a distinct tunnel through which substrates access the catalytic center. PlaF variants with bulky tryptophan residues in this tunnel revealed decreased catalysis rates due to tunnel blockage. The MD simulations suggest that GPLs preferably enter the active site with the sn-1 acyl chain first, which agrees with the experimentally demonstrated PLA1 activity of PlaF. We propose that the acyl chain-length specificity of PlaF is determined by the structural features of the access tunnel, which results in favorable free energy of binding of medium-chain GPLs. The suggested egress route conveys fatty acid products to the dimerization interface and, thus, contributes to understanding the product feedback regulation of PlaF by fatty acid-triggered dimerization. These findings open up opportunities for developing potential PlaF inhibitors, which may act as antibiotics against P. aeruginosa.

biochemistry↗

bioRxiv2021

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

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 {Delta}plaF mutant showed strongly attenuated virulence in Galleria mellonella and macrophages. We present a 2.0-[A]-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. SynopsisMembrane homeostasis can be regulated by phospholipase-controlled deacylation of endogenous glycerophospholipids (GPLs) followed by reacylation of products, known as the Lands cycle in eukaryotes. Here we show that the human pathogen Pseudomonas aeruginosa uses intracellular phospholipase A1 (PlaF) to modulate membrane GPL composition, which is the first example in bacteria. This newly identified PLA1 indirectly regulates the bacterial virulence properties by hydrolyzing a specific set of membrane GPLs. The crystal structure of full-length PlaF dimers bound to natural ligands, MD simulations, and biochemical approaches provide insights into the molecular mechanism of dimerization-mediated inactivation of this single-pass transmembrane PLA1. Our findings shed light on a mechanism by which bacterial intracellular PLAs might regulate membrane homeostasis what showcases these enzymes as a promising target for a new class of antibiotics.

molecular biology↗

bioRxiv2021

Novel intracellular phospholipase B from Pseudomonas aeruginosa with activity towards endogenous phospholipids affects biofilm assembly

Pseudomonas aeruginosa is a severe threat to immunocompromised patients due to its numerous virulence factors and multiresistance against antibiotics. This bacterium produces and secretes various toxins with hydrolytic activities including phospholipases A, C and D. However, the function of intracellular phospholipases for bacterial virulence has still not been established. Here we demonstrate that the hypothetical gene pa2927 of P. aeruginosa encodes a novel phospholipase B named PaPlaB. PaPlaB isolated from detergent-solubilized membranes of E. coli rapidly degraded various GPLs including endogenous GPLs isolated from P. aeruginosa cells. Cellular localization studies suggest that PaPlaB is peripherally bound to the inner and outer membrane of E. coli, yet the active form was predominantly associated with the cytoplasmic membrane. In vitro activity of purified and detergent-stabilized PaPlaB increases at lower protein concentrations. The size distribution profile of PaPlaB oligomers revealed that decreasing protein concentration triggers oligomer dissociation. These results indicate that homooligomerisation regulates PaPlaB activity by a yet unknown mechanism, which might be required for preventing bacteria from self-disrupting the membrane. We demonstrated that PaPlaB is an important determinant of the biofilm lifestyle of P. aeruginosa, as shown by biofilm quantification assay and confocal laser scanning microscopic analysis of biofilm architecture. This novel intracellular phospholipase B with a putative virulence role contributes to our understanding of membrane GPL degrading enzymes and may provide a target for new therapeutics against P. aeruginosa biofilms.

biochemistry↗

bioRxiv2021

Predicting drug targets by homology modelling of Pseudomonas aeruginosa proteins of unknown function

Efficacies of antibiotics to treat bacterial infections rapidly decline due to antibiotic resistance. This stimulated the development of novel antibiotics, but most attempts failed. As a response, the idea of mining uncharacterised genes of pathogens to identify potential targets for entirely new classes of antibiotics raised. Without knowing the biochemical function of a protein it is difficult to validate its potential for drug targeting; therefore progress in the functional characterisation of bacterial proteins of an unknown function must be accelerated. Here we present a paradigm for comprehensively predicting biochemical functions of a large set of proteins encoded by hypothetical genes in human pathogens, to identify candidate drug targets. A high-throughput approach based on homology modelling with ten templates per target protein was applied on the set of 2103 P. aeruginosa proteins encoded by hypothetical genes. Obtained >21000 homology modelling results and available biological and biochemical information about several thousand templates was scrutinised to predict the function of reliably modelled proteins of unknown function. This approach resulted in assigning, one or often multiple, putative functions to hundreds of enzymes, ligand-binding proteins and transporters. New biochemical functions were predicted for 41 proteins whose essential or virulence-related roles in P. aeruginosa were already experimentally demonstrated. Eleven of them were shortlisted as promising drug targets which participate in essential pathways (maintaining genome and cell wall integrity), virulence-related processes (adhesion, cell motility, host recognition) or antibiotic resistance, which are general drug targets. These proteins are conserved among other WHO priority pathogens but not in humans, therefore they represent high-potential targets for pre-clinical studies. These and many more biochemical functions assigned to uncharacterised proteins of P. aeruginosa, available as PaPUF database may guide the design of experimental screening of inhibitors which is a crucial step toward validation of the most potential targets for the development of novel drugs against P. aeruginosa and other high-priority pathogens.

bioinformatics↗