Isothermal Titration Calorimetry Research Articles

Bispecific Thio‐linked Disaccharides as Inhibitors of Pseudomonas aeruginosa Lectins LecA (PA‐IL) and LecB (PA‐IIL): Dual‐Targeting Strategy

Pseudomonas aeruginosa is a prevalent opportunistic human pathogen, particularly associated with cystic fibrosis. Among its virulence factors are the LecA and LecB lectins. Both lectins play an important role in the adhesion to the host cells and display cytotoxic activity. In this study, we successfully synthesized hardly hydrolysable carbohydrate ligands targeting these pathogenic lectins, including two bispecific glycans. The interactions between LecA/LecB lectins and synthetic glycans were evaluated using hemagglutination (yeast agglutination) inhibition assays, comparing their efficacy with corresponding monosaccharides. Additionally, the binding affinities of bispecific glycans were assessed using isothermal titration calorimetry (ITC). Structural insight into the lectin‐ligand interaction was obtained by determining the crystal structures of LecA/LecB lectins in complex with one of the bispecific ligands using X ray crystallography. This comprehensive investigation into the inhibitory potential of synthetic glycosides against P. aeruginosa lectins sheds light on their potential application in antimicrobial therapy.

Specific binding between Arabidopsis thaliana phytochrome-interacting factor 3 (AtPIF3) bHLH and G-box originated prior to embryophyte emergence

The basic helix-loop-helix (bHLH) domain via critical amino acid residues on basic region binding to E-box (5′-CANNTG-3′) is known in embryophyte. However, the dictated E-box types selection by bHLH dimers and the significant impact of these critical amino acid residues along embryophyte evolution remain unclear. The Arabidopsis thaliana PIF3-bHLH (AtPIF3-bHLH) recombinant protein and a series of AtPIF3-bHLH mutants were synthesized and analyzed. The reduced DNA binding ability and affinity of AtPIF3-bHLH point-mutation proteins, observed via fluorescence-based electrophoretic mobility shift assay (fEMSA) and isothermal titration calorimetry (ITC), suggest the critical role of these DNA-recognition sites in maintaining the AtPIF3-bHLH–DNA interaction. The purifying selection signals and the DNA-recognition-site conservation at the species level suggest the invariant roles of these sites throughout embryophyte evolution. The G-box outcompeted other types of E-box for binding in our competitive fEMSAs. The dynamic hydrogen bond formed between AtPIF3-bHLH and the G-box core indicates flexible identification of the core region. These features highlight a fast fixation of the bHLH-G-box recognition mechanism through embryophyte evolution and serve as a blueprint for studying DNA recognition determinants of other TF families.

The chemoreceptor controlling the Wsp‐like transduction pathway in Halomonas titanicaeKHS3 binds and responds to purine derivatives

The chemosensory pathway HtChe2 from the marine bacterium Halomonas titanicae KHS3 controls the activity of a diguanylate cyclase. Constitutive activation of this pathway results in colony morphology alterations and an increased ability to form biofilm. Such characteristics resemble the behavior of the Wsp pathway of Pseudomonas. In this work, we investigate the specificity of Htc10, the only chemoreceptor coded within the HtChe2 gene cluster. The purine derivatives guanine and hypoxanthine were identified as ligands of the recombinantly produced Htc10 ligand‐binding domain, with dissociation constants in the micromolar range, and its structure was solved by X‐ray protein crystallography. The sensor domain of Htc10 adopts a double Cache folding, with ligands bound to the membrane‐distal pocket. A high‐resolution structure of the occupied guanine‐binding pocket allowed the identification of residues involved in ligand recognition. Such residues were validated by site‐directed mutagenesis and isothermal titration calorimetry analyses of the protein variants. Moreover, heterologous expression of Htc10 in a Pseudomonas putida mutant lacking the native Wsp chemoreceptor promoted biofilm formation, a phenotype that was further enhanced by Htc10‐specific ligands. To our knowledge, this is the first description of binding specificity of a chemoreceptor that controls the activity of an associated diguanylate cyclase, opening the way for dynamic studies of the signaling behavior of this kind of sensory complex.

Modulating the Supramolecular Assembly of α‐Cyclodextrin and Anderson‐type Polyoxometalate through Covalent Modifications

A series of unprecedented supramolecular complexes of covalently modified Anderson‐type polyoxometalates (POMs) and α‐cyclodextrins (α‐CDs) have been obtained and characterized in solid state by single‐crystal X‐ray diffraction, and in aqueous solution using various techniques including 1H DOSY, 2D NOESY 1H NMR, isothermal titration calorimetry (ITC), and electrospray ionization time‐of‐flight mass spectroscopy (ESI‐TOF‐MS). It has been demonstrated that the supramolecular assembly process can be modulated by different covalent modification modes of the Anderson POMs, giving rise to a new type of POM/α‐CD complexes featuring organic‐inorganic pseudo‐rotaxane structures, which are in good contrast to those of POM/γ‐CD with poly‐rotaxane type structures. Moreover, it is delighted to find that these pseudo‐rotaxanes of POM/α‐CD exhibit stable chirality in aqueous solution, which has not been accomplished in previously reported POM/γ‐CD assemblies.

Dissociation Constant (Kd) Measurement for Small-Molecule Binding Aptamers: Homogeneous Assay Methods and Critical Evaluations.

Since 1990, numerous aptamers have been isolated and discovered for use in various analytical, biomedical, and environmental applications. This trend continues to date. A critical step in the characterization of aptamer binding is to measure its binding affinity toward both target and non-target molecules. Dissociation constant (Kd) is the most commonly used value in characterizing aptamer binding. In this article, homogenous assays are reviewed for aptamers that can bind small-molecule targets. The reviewed methods include label-free methods, such as isothermal titration calorimetry, intrinsic fluorescence of target molecules, DNA staining dyes, and nuclease digestion assays, and labeled methods, such as the strand displacement reaction. Some methods are not recommended, such as those based on the aggregation of gold nanoparticles and the desorption of fluorophore-labeled DNA from nanomaterials. The difference between the measured apparent Kd and the true Kd of aptamer binding is stressed. In addition, avoiding the titration regime and paying attention to the time required to reach equilibrium are discussed. Finally, it is important to include mutated non-binding sequences as controls.

Gadolinium-Sensitive Artificial Nanochannel Membrane for Information Encryption.

Inspired from ion channels in the myelinated axon of Xenopus laevis found to be affected by gadolinium on axonal currents, we present a solid nanochannel membrane sensitive to gadolinium (Gd3+), which can be achieved via the use of the macrocyclic triacetic acid derivative in the host-guest chemistry approach. The macrocyclic nanochannel has good responsiveness toward Gd3+, even at the nanomolar concentration level, evidenced by discernible changes in rectification, ionic conductance, and XPS analyses. Notably, the Gd3+-sensitive nanochannel membrane can be switched by the addition of a diethylenetriaminepentaacetic acid (DTPA) derivative. Further studies have indicated that the gated behavior of Gd3+ in the nanochannel can be attributed to the strong binding strength between DO3A and Gd3+, which induces a surface charge reversal within the nanochannel. The mechanism has been confirmed through several experimental techniques, including isothermal titration calorimetry (ITC) experiments, fluorescence titration experiments, and finite element analysis. Based on its Gd3+ responsiveness of the constructed ion channel, we successfully developed an advanced multilevel information encryption application of the artificial solid nanochannel membrane. Furthermore, it is anticipated that a more effective encryption system will be built by utilizing the bionic ion channel system's ease of use and straightforward functionalization.

Construction of Somatostatin-Based Multiphase "Core-Shell" Coacervates as Photodynamic Biomimetic Organelles.

Biomimetic coacervates have recently attracted great interest in biomedical fields, especially for drug delivery and as protocells. However, these membraneless structures are easily coalesced and poorly targetable, limiting their real biomedical applications. Here multiphase "core-shell" coacervate (CSC) constructed by dsDNA and somatostatin (SST), a 14-mer cyclopeptide is designed. The CSC shows enhanced tumor targetability through SST binding to SST receptors on the tumor cells' surface. G4 quadruplex-hemin complex can be embedded in the CSC by interaction with SST, as demonstrated by molecular simulation and isothermal titration calorimetry. The G4-hemin embedded CSC can further recruit photosensitizers such as tetracarboxyphenyl porphyrin to form the CSC-GHT composite for photodynamic therapy (PDT). As photodynamic biomimetic organelles, CSC-GHT can convert oxygen to singlet oxygen (catalyzed by the catalase-mimetic activity of G4-hemin), resulting in enhanced PDT effect, which allows the inhibition of cellular migration in vitro and tumor growth in vivo. Owing to high stability, targetability, and biosafety, the proposed CSC can recruit various cargos from small dyes to large biomacromolecules (up to 430kDa), providing promising theranostic applications.

Allostery in homodimeric SARS-CoV-2 main protease

Many enzymes work as homodimers with two distant catalytic sites, but the reason for this choice is often not clear. For the main protease Mpro of SARS-CoV-2, dimerization is essential for function and plays a regulatory role during the coronaviral replication process. Here, to analyze a possible allosteric mechanism, we use X-ray crystallography, native mass spectrometry, isothermal titration calorimetry, and activity assays to study the interaction of Mpro with three peptide substrates. Crystal structures show how the plasticity of Mpro is exploited to face differences in the sequences of the natural substrates. Importantly, unlike in the free form, the Mpro dimer in complex with these peptides is asymmetric and the structures of the substrates nsp5/6 and nsp14/15 bound to a single subunit show allosteric communications between active sites. We identified arginines 4 and 298 as key elements in the transition from symmetric to asymmetric dimers. Kinetic data allowed the identification of positive cooperativity based on the increase in the processing efficiency (kinetic allostery) and not on the better binding of the substrates (thermodynamic allostery). At the physiological level, this allosteric behavior may be justified by the need to regulate the processing of viral polyproteins in time and space.

Charge Modulation at the Liquid Crystal Droplet-Aqueous Interface Enables Ultrasensitive, Nonspecific Protein Detection.

Thermotropic nematic liquid crystals (LC) have been utilized to sense/detect various analytes such as polymers, surfactants, lipids, etc. However, their use for protein detection depends on pre-adsorbed molecules, co-nematogens, or biomolecular agents for specificity.This approach impedes the platform's sensitivity with a detection limit for the folded proteins generally reported in the micromolar concentration range. Here, this work provides fundamental insights into the type of molecular interactions and their modulation that can drive ultrasensitive protein detection at an LC microdroplet/aqueous interface formed without adding an auxiliary co-nematogen. Using ultraviolet (UV) light treated 4-cyano-4'-pentylbiphenyl (5CB) LC and a flow-focused microfluidic device, we prepared different populations of monodisperse and highly negatively charged microdroplets in water. Adding an aqueous solution of various model proteins(α-synuclein, α-chymotrypsin, myoglobin, or bovine serum albumin, BSA) with different secondary structures and surface charges triggers a rapid radial- to bipolar-defect transition in these microdroplets. Isothermal titration calorimetry measurement and molecular dynamic simulation studies attribute this to the dominant electrostatic force-mediated adsorption of proteins at the LC/aqueous interface.Further, bioconjugation-based variation of protein surface charge allows tuning their detection limit.These findings can provide crucial physical cues for designing responsive LC systems and establishing a foundation for developing versatile, molecularly tailored, and highly specific biomolecular detection platforms.

Selection and characterization of novel aptamers specific for patulin and label-free split sensing

The selection of novel aptamers specific for patulin (PAT) and the construction of a label-free split aptasensor for the detection of PAT are described. The ssDNA library was immobilized on streptavidin resin, and the enriched libraries were sequenced via high-throughput sequencing. After sequence homology analyses, the candidate aptamers were identified using isothermal titration calorimetry (ITC). Split aptamers (S6-a-1/S6-b-2) were designed as the sandwich aptasensor for PAT detection. The results from the sequencing indicated that the DNA was enriched in the 18th round. The ITC analysis revealed that the aptamers of family 1 had high affinity for PAT, with kd values ranging from 0.45-3.64 µM. The candidate aptamers were shorter (46 bases) than previously reported PAT aptamers. The LOD of the split aptasensor based on PAT-6 was 0.25 µM, and the aptasensor was able to function properly in a 10 % apple juice matrix. Thus, a novel aptamer specific for PAT was reported, and a label-free split aptasensor with low cost and rapid response time for the detection of PAT residue was developed.

Lapachol, a natural food component, interacts with human serum albumin: Insights of its impact on the pharmacokinetics of clinically used drugs

Lapachol (LAP), a natural 1,4-naphthoquinone used in popular medicine of South American, is an antioxidant and antimicrobial compound used as a food additive; however, its interactive profile with the main protein carrier of compounds in the human bloodstream (human serum albumin, HSA) was not still characterized. Additionally, the impact of LAP in binding clinically drugs to albumin is still unknown. Thus, the present work describes the interaction HSA:LAP using different biophysical techniques, i.e., 1H saturation-transfer difference nuclear magnetic resonance (1H STD-NMR), isothermal titration calorimetry (ITC), steady-state and time-resolved fluorescence measurements combined with molecular docking calculations. LAP interacts with subdomain region IIA (site I), mainly driven by enthalpy effects, while subdomain region IB (site III) was identified as the second binding site, mainly driven by entropy effects. The binding is spontaneous, strong (binding constant average, Kaverage ≈ 4.45 × 105 M−1), and there is a positive cooperativity in the presence of ibuprofen, with the LAP structure fully buried into the protein cavities. Overall, LAP might impact the residence time (pharmacokinetic profile) of drugs that bind to subdomains regions IIA and IB of albumin, e.g., warfarin, phenylbutazone, diflunisal, naproxen, camptothecin, doxorubicin, daunorubicin, suramin, and tyrosine kinase inhibitors.

Structural characterization of noni (Morinda citrifolia L.) pectin and its inhibitory activity on pancreatic lipase

This study aimed to investigate the physicochemical properties and biological activities of noni pectin (NP) extracted using various solvents, three fractions of noni pectin were obtained using traditional chemical and deep eutectic solvents. NPs are composed of various ratios of galacturonic acid, arabinose, rhamnose, xylose, glucose, and galactose with different yields, molecular weights, esterification degrees (DE), and microstructures. Among the fractions, noni pectin extracted with Betaine-citric acid (BNP) exhibited higher molecular weight, degree for esterification and pancreatic lipase (PL) inhibitory activity than the other fractions. Enzyme kinetic analysis indicated that BNP inhibited PL via a noncompetitive mechanism. BNP significantly altered the secondary structure of PL and quenched PL fluorescence, suggesting a static quenching mechanism. Isothermal titration calorimetry (ITC) further demonstrated that the binding of BNP to PL was spontaneous and driven by enthalpy, primarily mediated by hydrogen bonding and van der Waals forces. Molecular docking simulations also confirmed strong noncovalent interactions (hydrogen bonding and van der Waals forces) between BNP and PL. This study validated the high efficiency of the deep eutectic solvent-extracted noni pectin fraction in pancreatic lipase inhibition.

The formation of protein coronas and the interaction of soy protein isolate and whey protein isolate with starch nanoparticles

The influence of food components on the physicochemical properties and biological responses of nanoparticles is pivotal. Since soy protein isolate (SPI) and whey protein isolate (WPI) are commonly used as plant and animal proteins in food, it is imperative to gain comprehensive knowledge of the interaction between SPI/WPI and nanoparticles. Accordingly, we examined the adsorption of SPI and WPI onto starch nanoparticles (SNPs) and their associated interactions. SNP aggregation was observed in the SPI/WPI, and coronas formed around the surfaces of the SNPs. The binding of the protein isolates reduced the zeta potential of the SNPs from −10.34 to −26.5 mV for the SPI and to −21.37 mV for the WPI. However, mixing the SNPs with the protein isolates did not alter the microenvironment of aromatic amino acids and had no substantial impact on the secondary structure of the proteins. Thermogravimetric analysis revealed that the formation of SPI/WPI coronas enhanced the thermal stability of the SNPs. Isothermal titration calorimetry indicated that the adsorption of the SPI and WPI onto the SNPs was driven primarily by weak van der Waals forces and hydrogen bonds. This study facilitates the predicting of organic nanoparticle behaviours in complex food matrix and human gastrointestinal environments.

Unveiling the interaction, cytotoxicity and antibacterial potential of pyridine derivatives: an experimental and theoretical approach with bovine serum albumin.

The binding interactions between bovine serum albumin (BSA) and three pyridine derivatives, i.e., 2-(5-bromopyridin-3-yl) acetic acid (L1), 3-bromo-5-nitropyridine (L2) and 2-chloro-4-nitropyridine (L3), have been carried out using UV-Vis and fluorescence spectroscopic methods. Fluorescence intensity quenching is observed by adding L2 and L3 to the BSA solution. The quenched fluorescence emission is due to the static nature. An isothermal titration calorimetry (ITC) experiment shows the binding ability of L1 with BSA. The binding constants are found to be 7.23 ± 0.32 × 105 M-1 for L1. The thermodynamic parameters were calculated from ITC measurements (i.e., ∆H = -2.78 ± 0.08 kcal/mol, ∆G = -5.65 ± 0.25 kcal/mol, and -T∆S = -2.87 ± 0.11 kcal/mol), which indicated that the protein-ligand complex formation between L1 and BSA is mainly due to the hydrogen bonds and van der Waals interactions. Cyclic voltammetry (CV) and structure activity and relationship (SAR) studies have been carried out to establish the relationship between ligands and proteins. Additionally, we conducted an antibacterial assay with gram-positive Staphylococcus aureus, Enterococcus faecalis, and negative bacterial strains Acinetobacter baumannii and Escherichia coli against L1, L2, and L3, aiming to address the challenges posed by the co-existence of multidrug-resistant bacteria. Finally, drosophila is used to test the cytotoxicity of ligands L1, L2, and L3's in vitro.

Arsenic (III) and (V) complexes from solvent free condition and their kinetic factors leading to arsenic nanoparticles

A clear melt from catechol and sodium salts of arsenic (III and V) were obtained individually from the grinding mixing protocol (GMP). The as-obtained molten mass from exothermic reaction between catechol and arsenic salts motivated isothermal titration calorimetry (ITC) advocating the propensity of complex catecholate formation. Individual ITC results substantiated structurally two different mono-anionic complexes, one for arsenic (III) and the other for arsenic (V) ions, of catechol in water at room temperature. Then, the compositions of both the mono-anionic complexes with stoichiometry 1:2 and 1:3 (w∕w) were confirmed for arsenic (III) and (V), respectively. It was shown here for the 1st time that both the complexes supplemented two neat precursors for useful arsenic nanoparticle (As NP) preparation from pH control redox reactions. Again, galvanic replacement reaction (GRR) between As NP and HAuCl4 evolved Au NPs of comparable size as that of As NPs. Thus, thermodynamic results, binding constant (Kb) values, redox kinetic studies and ion associate formation of the two catecholates clearly differentiated the stability (labile and inert character) of As(III) and As(V) catecholates. Conclusively, electrochemistry, galvanic replacement reaction, DLS, thermal analysis, and diverse spectrometric results provided 1st hand support for the complex catecholates of arsenic, a p-block metalloid as labile and inert characters respectively in two different (III) and (V) oxidation states.

The Phytophthora infestans effector Pi05910 suppresses and destabilizes host glycolate oxidase StGOX4 to promote plant susceptibility.

Phytophthora infestans is a notorious oomycete pathogen that causes potato late blight. It secretes numerous effector proteins to manipulate host immunity. Understanding mechanisms underlying their host cell manipulation is crucial for developing disease resistance strategies. Here, we report that the conserved RXLR effector Pi05910 of P. infestans is a genotype-specific avirulence elicitor on potato variety Longshu 12 and contributes virulence by suppressing and destabilizing host glycolate oxidase StGOX4. By performing co-immunoprecipitation, yeast-two-hybrid assays, luciferase complementation imaging, bimolecular fluorescence complementation and isothermal titration calorimetry assays, we identified and confirmed potato StGOX4 as a target of Pi05910. Further analysis revealed that StGOX4 and its homologue NbGOX4 are positive immune regulators against P. infestans, as indicated by infection assays on potato and Nicotiana benthamiana overexpressing StGOX4 and TRV-NbGOX4 plants. StGOX4-mediated disease resistance involves enhanced reactive oxygen species accumulation and activated the salicylic acid signalling pathway. Pi05910 binding inhibited enzymatic activity and destabilized StGOX4. Furthermore, mutagenesis analyses indicated that the 25th residue (tyrosine, Y25) of StGOX4 mediates Pi05910 binding and is required for its immune function. Our results revealed that the core RXLR effector of P. infestans Pi05910 suppresses plant immunity by targeting StGOX4, which results in decreased enzymatic activity and protein accumulation, leading to enhanced plant susceptibility.

Anti-cancer and anti-microbial drug encapsulated lipid vesicles as drug delivery systems: Calorimetric and spectroscopic study

Lipid-based drug delivery systems (DDS) have attracted considerable interest for their capacity to achieve controlled drug release and encapsulate a variety of drug molecules—hydrophobic, hydrophilic, and amphiphilic—enabling diverse therapeutic applications. The research focuses on the structural and functional aspects of lipid-based vesicles that encapsulate both anti-cancer and anti-microbial drugs. The study employs ultrasensitive isothermal titration calorimetry and differential scanning calorimetry to gain mechanistic insights into the partitioning of drugs in the lipid-based DDS, release mechanisms, and binding interactions with serum proteins after the release.The structural properties of 5-fluorouracil (5-FU) suggest that the drug can partition into the outer region of the bilayer, where it forms hydrogen bonds with the polar heads of the amphiphilic lipid chains. Kanamycin indicates strong partitioning into liposomes as it displays a binding constant of the order of 106. Erythromycin exhibits limited partitioning into the bilayer, as assessed by fitting ITC data using a sequential two-binding site model. The values of change in enthalpy and entropy reflect the interaction nature and desolvation process during the drug's partitioning into liposomes. Thermodynamic signatures and transition temperatures obtained from ITC and DSC studies indicate that no diffusion-based drug release occurs from DPPC and DSPC-based liposomes. The interaction heat between the released drug and carrier serum albumin protein is very low and shows no consistent pattern. This suggests that the drugs are stably encapsulated in the drug delivery system (DDS), with no significant leakage over time. Insights from such studies on the influence of drug molecule structure on its partitioning into various lipid vesicles and further release of the same drugs from it guide in criteria for developing improvised drug delivery systems.

The simultaneous application of fulvic acid and protein hydrolysate biostimulants enhances cucumber responses to Fe deficiency

Iron (Fe) is widely recognized as a critical factor in limiting crop production; however, eco-friendly strategies to address its deficiency are still required. The use of biostimulants has displayed promising results in mitigating Fe deficiency. Our hypothesis was that the combined application of two biostimulants with distinct molecular structures - fulvic acid (FA) and protein hydrolysate (PH) - could be more effective than the use of a single compound. The simultaneous presence of FA and PH (MIX) in a Fe-free nutrient solution led to a redistribution of endogenous Fe, resulting in a higher leaf SPAD index. Furthermore, the addition of FeCl3 as a Fe source (resupply) in MIX-treated plants enhanced the biostimulant effect, as evidenced by increased dry root and shoot weight and a more developed root system. In addition, the expression of Strategy-I-related genes, CsFRO1 and CsIRT1, remained elevated. These effects can be attributed to improved interaction between the roots and biostimulants through the formation of the FA-PH complex, as demonstrated by circular dichroism and isothermal titration calorimetry analyses.

Mg2+ binding to Coenzyme A

Magnesium (Mg2+), the second most abundant intracellular cation, plays a crucial role in cellular functions. In this study, we investigate the interaction between Mg2+ and coenzyme A (CoA), a thiol-containing cofactor central to cellular metabolism also involved in protein modifications. Isothermal titration calorimetry revealed a 1:1 binding stoichiometry between Mg2+ and free CoA under biologically relevant conditions. Association constants of (537 ± 20) M-1 and (312 ± 7) M-1 were determined at 25°C and pH 7.2 and 7.8, respectively, suggesting that a significant fraction of CoA is likely bound to Mg2+ both in the cytosol and in the mitochondrial matrix. Additionally, the process is entropically-driven, and our results support that the origin of the entropy gain is solvent-related. On the other hand, the combination of 1- and 2-dimensional nuclear magnetic resonance spectroscopy with molecular dynamics simulations and unsupervised learning demonstrate a direct coordination between Mg2+ and the phosphate groups of the 4-phosphopantothenate unit and bound to position 5’ of the adenosine ring. Interestingly, the phosphate in position 3' only indirectly contributes to Mg2+ coordination. Finally, we discuss how the binding of Mg2+ to CoA perturbates the chemical environment of different CoA atoms, regardless of their apparent proximity to the coordination site, through the modulation of the CoA conformational landscape. This insight holds implications for understanding the impact on both CoA and Mg2+ functions in physiological and pathological processes.

Cyclic di-GMP incorporates the transcriptional factor FleQ03 in Pseudomonas syringae MB03 to elicit biofilm-dependent resistance in response to Caenorhabditis elegans predation

Bacteria usually form biofilms as a defense mechanism against predation by bacterivorous nematodes. In this context, the second messenger c-di-GMP from the wild-type Pseudomonas syringae MB03 actuates the transcriptional factor FleQ03 to elicit biofilm-dependent nematicidal activity against Caenorhabditis elegans N2. P. syringae MB03 cells exhibited nematicidal activity and c-di-GMP content in P. syringae MB03 cells was increased after feeding to nematodes. Expression of a diguanylate cyclase (DGC) gene in P. syringae MB03 resulted in an increased c-di-GMP content, biofilm yield and nematicidal activity, whereas converse effects were obtained when expressing a phosphodiesterase (PDE) gene. Molecular docking and isothermal titration calorimetry assays verified the affinity activity between c-di-GMP and the FleQ03 protein. The disruption of the fleQ03 gene in P. syringae MB03, while increasing c-di-GMP content, significantly diminished both biofilm formation and nematicidal activity. Interestingly, P. syringae MB03 formed a full-body biofilm around the worms against predation, probably extending from the tail to the head, whereas it was not observed in the fleQ03 gene disrupted cells. Thus, we hypothesized that c-di-GMP incorporated FleQ03 to reinforce bacterial biofilm and biofilm-dependent pathogenicity in response to C. elegans predation, providing insights into a possible means of resisting bacterivorous nematodes by bacteria in natural ecosystems.