Search Results (130)

The rise of multidrug-resistant Pseudomonas aeruginosa underscores the need for novel therapeutic targets beyond conventional peptidoglycan biosynthesis. Some bacterial strains bypass MurA inhibition by fosfomycin via a cell wall salvage pathway. This study targeted P. aeruginosa AmgK (PaAmgK) and MurU (PaMurU) to identify inhibitors that could complement fosfomycin therapy. A malachite-green-based dual-enzyme assay enabled efficient activity measurements and high-throughput chemical screening. Screening 232 compounds identified Congo red and CTAB as potent PaMurU inhibitors. A targeted mass spectrometric analysis confirmed the selective inhibition of PaMurU relative to that of PaAmgK. Molecular docking simulations indicate that Congo red preferentially interacts with PaMurU through electrostatic contacts, primarily involving the residues Arg28 and Arg202. The binding of Congo red to PaMurU was corroborated further using SUPR-differential scanning fluorimetry (SUPR-DSF), which revealed ligand-induced thermal destabilization. Ongoing X-ray crystallographic studies, in conjunction with site-directed mutagenesis and enzyme kinetic analyses, aim to elucidate the binding mode at an atomic resolution. Full article

The by-products that may contain tetrodotoxin (TTX) produced during the processing of farmed pufferfish have caused food safety risks and environmental pollution. Peptidoglycan (PG) of lactic acid bacteria could adsorb TTX; however, its complex structure and poor solubility limited adsorption efficiency. In this study, hydroxyl modifications of three PGs (A3α, A1γ and A4α) were realized via TEMPO-mediated selective oxidation of the primary hydroxyl group. Compared with PGs, it was found that the carboxyl density of hydroxyl-modified PGs (HM-PGs) increased from 1.66 mmol/g to 3.33 mmol/g and the surface electronegativity increased from −36 mV to −59 mV. The adsorption capacity of HM-PGs to TTX reached 1.48 μg/mg, which was comparable to the adsorption of the conventional adsorbent chitosan for aflatoxin B1 (1.39 μg/mg). Moreover, HM-PGs decreased the toxicity of TTX from strong toxic to nearly non-toxic, with the toxicity reduction rate reached 99.85%. After treatment with HM-PGs, the mouse hippocampus and neuronal cell model confirmed that lower neural injury and sodium channel blocking effects were observed in the residual TTX, whose neurotoxicity was lower. Molecular docking simulation and physicochemical analysis revealed that the adsorption of TTX by HM-PGs was a complex adsorption mode driven by the synergy of physicochemical interaction. There were both physical adsorptions based on electrostatic and hydrophobic interactions and chemical binding with strong hydrogen bonding (1.46 Å) and Mayer bond order (0.1229). This study not only developed a new, efficient and safe tool for TTX removal, but also provided a theoretical basis for the development of biological toxin removal material. Full article

Zearalenone (ZEN), a non-steroidal estrogenic mycotoxin produced by Fusarium graminearum species, poses a significant threat to both human food safety and animal feed quality. In this study, we isolated a strain, designated as Bacillus licheniformis YJ25, from a contaminated moldy corn sample, demonstrating substantial effectiveness in removing ZEN. Our findings revealed that YJ25’s ZEN removal occurs primarily through cell wall adsorption, with enzymatic degradation representing a potential mechanism. In practical applications, enzymatic degradation may yield metabolites with heightened toxicity. However, liquid chromatography–mass spectrometry (LC–MS) analysis revealed that ZEN was not converted into α-/β-zearalenol (α-/β-ZEL) or α-/β-zearalanol (α-/β-ZAL) by YJ25, substantiating the safety profile of YJ25 in the removal of ZEN. Our mechanistic investigations revealed that the cell wall components peptidoglycan and teichoic acid serve as the primary binding sites for ZEN adsorption. Fourier-transform infrared spectroscopy (FTIR) analysis identified O-H, C-H, C=O, and C-O as the principal functional groups participating in the cell wall adsorption process. These investigations establish a scientific foundation for the prospective application of this strain as an efficient biological detoxification agent in food and feed safety management systems. Full article

The primary objective of this study was to identify and characterize pathogen defense proteins in the Nicotiana tabacum L. proteome, focusing on their structural, functional, and evolutionary properties, as well as their interactions with pathogen-derived molecules. Specifically, we aimed to comprehensively analyze the proteome to pinpoint potential uncharacterized defense-related protein that has emerging roles in immune responses and antioxidant activity across plants and animals. Through integrated computational approaches, we determined evolutionary relationships, and structural modeling of the selected protein was performed using different modeling software, followed by validation through multiple metrics, including stereochemical checks (Ramachandran plot), MolProbity analysis, and Z-scores. We further investigated the functional binding regions or interaction sites. We performed molecular docking to investigate the molecular interactions between selected proteins and pathogen-associated molecular patterns (PAMPs), specifically β-glucan and peptidoglycan (PGN), to elucidate their defensive mechanisms. Last, normal mode analysis (NMA), molecular dynamics simulation (MDS), and post-simulation analyses were employed to evaluate the stability and mobility of the protein–ligand complexes. Uncharacterized vitellogenin-like protein (VLP: ID A0A1S4CXB2) with the potential defense domain chosen because of its predicted immune-related features, stress response patterns, and unknown pathogen role at new immunity functions. Phylogenetic analysis revealed significant sequence homology with VLPs from other members of the Solanaceae family. Structural modeling showed a high-quality model, with docking studies indicating a stronger affinity for PGN (−10.16 kcal/mol) and β-glucan (−7.19 kcal/mol), highlighting its potential involvement in pathogen defense. NMA, MDS, and post-simulation analyses revealed that PGN exhibits more substantial binding stability and more extensive interactions with VLP than β-glucan. Our findings confirmed that VLPs in N. tabacum may function as pattern recognition receptors (PRRs), capable of recognizing and responding to pathogens by activating immune signaling pathways. Future experimental validation of these interactions could further elucidate the role of VLPs in plant defense and their potential application in biotechnological approaches for sustainable agriculture. Full article

Despite its ecological and economic importance, many aspects of Varroa destructor’s biology remain poorly understood, particularly its defense mechanisms against pathogens. The limited knowledge of Varroa’s immunity has hindered the development of RNA interference (RNAi)-based strategies targeting immune-related genes. In this study, we investigated the immune gene repertoire of V. destructor by querying its NCBI nr protein database and comparing it to model species of ticks (Ixodes scapularis) and mites (Galendromus occidentalis and Tetranychus urticae). Transcription of candidate immune genes was confirmed by analyzing a de novo assembled transcriptome of V. destructor. Our findings reveal that V. destructor shares key immunological traits with ticks, including lysozymes, chitinases, and thioester-containing proteins (TEPs), but also shares the absence of transmembrane peptidoglycan recognition proteins (PGRPs), Gram-negative binding proteins, and several lectin families involved in pathogen recognition. Additionally, Varroa mites, like ticks, lack homologs of crucial immune signaling components, such as the unpaired ligand (JAK/STAT), Eiger (JNK), and multiple elements of the IMD pathway. They also do not encode canonical antimicrobial peptides (AMPs) like defensins but possess putative homologs of ctenidins, AMPs previously identified in spiders and ticks, which may be adopted as a novel genetic readout for immune response in mites. Our findings lay the groundwork for future functional studies on mite immunity and open new avenues for RNAi-based biocontrol strategies targeting immune pathways to enhance Varroa management. Full article

In the food industry, food spoilage caused by spores is a pressing scientific challenge that needs to be addressed urgently, and spore germination is a key approach to solving this problem. Studies have shown that peptidoglycan-induced spore germination represents a novel mechanism of action, which can bind to the PASTA domain of the serine/threonine kinase PrkC. However, the signaling mechanism of peptidoglycan-induced spore germination remains unclear. This study focuses on Bacillus subtilis, using pull-down experiments to screen for proteins interacting with PrkC. There are 80 interaction proteins of PrkC that were identified in the spore. GO analysis reveals that PrkC-interacting proteins in the spore are mainly involved in metabolic processes, cell part and catalysis. KEGG results indicate that PrkC-interacting proteins in the spore are mainly involved in RNA degradation, quorum sensing, oxidative phosphorylation, etc. Additionally, proteins are categorized into six groups by function based on events that may be associated with post-germination triggered by peptidoglycan-induced activation of the PrkC signaling pathway, including “stimulate translation initiation” and “ATP synthesis and energy metabolism”. The experimental results provide a theoretical basis for further elucidating the signaling mechanism of PrkC, revealing the signaling pathway of peptidoglycan-induced spore germination, and identifying targeted inducers and repressors of spore germination. Full article

The initial phase of an insect’s innate immune response to foreign pathogens is triggered by the identification of exogenous invaders, a mechanism facilitated by pattern recognition receptors. Among these receptors, peptidoglycan recognition proteins (PGRPs), abundant in insects, are essential components of the innate immune system. The roles of PGRPs have been extensively elucidated in Drosophila melanogaster; however, the mechanism underlying the immune response of Aedes albopictus to pathogens is unclear. Herein, we successfully cloned the full-length cDNA of a PGRP gene from Ae. albopictus, designated as the AaPGRP-LB gene. The open reading frame of AaPGRP-LB encodes 203 amino acids, including a secretion signal peptide and a canonical PGRP conserved domain. Multisequence alignment revealed that AaPGRP-LB possesses the amino acid residues essential for zinc binding and amidase activity. Molecular docking studies demonstrated that AaPGRP-LB exhibits a strong binding affinity for DAP-type and LYS-type peptidoglycan. The mRNA expression level of the AaPGRP-LB gene significantly increased after oral infection with Escherichia coli or Staphylococcus aureus. The purified recombinant AaPGRP-LB (rAaPGRP-LB) exhibited strong agglutination properties and demonstrated significant antimicrobial efficacy against E. coli and S. aureus in the presence of zinc ions. This study highlights the critical role of AaPGRP-LB in the immune response of Ae. albopictus. These findings provide a foundation for future research on mosquito immune pathways for innovative vector control and disease prevention strategies. Full article

The lack of new antibacterial medicines and the rapid rise in bacterial resistance to antibiotics pose a major threat to individuals and healthcare systems. Despite the availability of various antibiotics, bacterial resistance has emerged for almost every antibiotic discovered to date. The increasing prevalence of multidrug-resistant bacterial strains has rendered some infections nearly untreatable, posing severe challenges to health care. Thus, the development of alternatives to conventional antibiotics is critical for the treatment of both humans and food-producing animals. Endolysins, which are peptidoglycan hydrolases encoded by bacteriophages, represent a promising new class of antimicrobials. Preliminary research suggests that endolysins are more effective against Gram-positive bacteria than Gram-negative bacteria when administered exogenously, although they can still damage the cell wall of Gram-negative bacteria. Numerous endolysins have a modular domain structure that divides their binding and catalytic activity into distinct subunits, which helps maximize their bioengineering and potential drug development. Endolysins and endolysin-derived antimicrobials offer several advantages as antibiotic substitutes. They have a unique mechanism of action and efficacy against bacterial persisters (without requiring an active host metabolism); subsequently, they target both Gram-positive and Gram-negative bacteria (including antibiotic-resistant strains), and mycobacteria. Furthermore, there has been limited evidence of endolysin being resistant. Because these enzymes target highly conserved links, resistance may develop more slowly compared to traditional antibiotics. This review provides an overview and insight of the potential applications of endolysins as novel antimicrobials. Full article

Background/Objectives: African swine fever (ASF), caused by African swine fever virus (ASFV), poses a significant threat to the global swine industry. This underscores the urgent need for safe and effective ASF vaccines. Methods: Here, we constructed five bacterium-like particles (BLPs) that each display one of the five ASFV antigens (F317L, H171R, D117L, B602L, and p54) based on the Gram-positive enhancer matrix-protein anchor (GEM-PA) system. GEM is a bacterial particle that contains only peptidoglycan, while PA is composed of three lysin motifs (Lysm) derived from the C-terminus of the AcmA protein, capable of non-covalently binding to GEM. By fusing the ASFV antigens with PA, the ASFV antigens can be firmly attached to the surface of GEM. Subsequently, the piglets were immunized via intramuscular injection with a mixture of BLPs-F317L, BLPs-H171R, BLPs-D117L, BLPs-B602L, and BLPs-p54. Results: The results showed that the piglets developed detectable serum IgG antibodies 2 weeks after the first immunization, and these high antibody levels were maintained 4 weeks after the booster immunization. Moreover, these piglets produced more IFN-γ-producing lymphocytes than the control piglets. Conclusions: The data indicate that the generated BLPs mixture can stimulate both humoral and cellular immune responses in piglets, these five ASFV proteins are promising antigens, and the BLPs generated represent candidate ASF vaccines. Full article

The crystal structure of the extracellular region of the second pneumococcal LCP, a polyisoprenyl-teichoic acid-peptidoglycan teichoic acid transferase Psr_Sp, was determined and refined to 2.15 Å resolution. Despite the low sequence homology with other LCP proteins, the Psr_Sp maintains the fold of the LCP domain, and the positions of the residues suggested to participate in the transferase function are conserved. The tunnel found in the Psr_Sp between the central β-sheet and three α-helices is wide enough to accommodate polyisoprenyl-teichoic acid. Comparison of the crystallographic temperature factors of LCP from distinct bacteria demonstrated that the four long loops located close to the teichoic acid and peptidoglycan binding sites have different relative mobilities. To compare the dynamics of the Psr_Sp in crystalline state and in solution, NMR spectra were recorded, and 88% of the residues were assigned in the ¹H-¹⁵N TROSY HSQC spectra. Perfect accordance in the secondary structure of the crystal structure of Psr_Sp with NMR data demonstrated correct assignment. Moreover, the relative mobility of the essential loops estimated from the crystallographic B-factor is in good agreement with order parameter S², predicted from chemical shift. We hypothesize that the dynamics of these loops are important for the substrate promiscuity of LCP proteins. Full article

Peptidoglycan recognition proteins (PGRPs) are a class of pattern recognition receptors (PRRs) that activate the innate immune system in response to microbial infection by detection of peptidoglycan, a distinct component of bacterial cell walls. Bioinformatic studies have revealed four PGRPs in the red imported fire ant Solenopsis invicta; nonetheless, the mechanism of the immune response of S. invicta induced by pathogens is still poorly understood. The peptidoglycan recognition protein full-length cDNA (designated as SiPGRP-S1/S2/S3/L) from S. invicta was used in this investigation. According to the sequencing analysis, there was a significant degree of homology between the anticipated amino acid sequence of SiPGRPs and other members of the PGRPs superfamily. Molecular docking studies demonstrated that SiPGRPs show strong binding affinity for a variety of PGN substrates. Additionally, tissue distribution analysis indicated that SiPGRPs are primarily expressed in several tissues of naïve larvae, including fat body, hemocytes, head, and thorax, as detected by quantitative real-time PCR (RT-qPCR). Microbial challenges resulted in variable changes in mRNA levels across different tissues. Furthermore, the antibacterial effects of antimicrobial peptides (AMPs) produced by major ants infected with Metarhizium anisopliae were assessed. These AMPs demonstrated inhibitory effects against M. anisopliae, Staphylococcus aureus, and Escherichia coli, with the most pronounced effect observed against E. coli. In conclusion, SiPGRPs act as pattern recognition receptors (PRRs) that identify pathogens and initiate the expression of AMPs in S. invicta, this mechanism contributes to the development of biopesticides designed for the targeted control of invasive agricultural pests. Full article

Endolysins of bacteriophages, which degrade the bacterial cell wall peptidoglycan, are applicable in many industries to deal with biofilms and bacterial infections. While multi-domain endolysins have both enzymatically active and cell wall-binding domains, single-domain endolysins consist only of an enzymatically active domain, and their mechanism of peptidoglycan binding remains unexplored, for this is a challenging task experimentally. This research aimed to explore the binding mechanism of endolysins using computational approaches, namely molecular docking and bioinformatical tools, and analyze the performance of these approaches. The docking engine Autodock Vina 1.1.2 and the 3D-RISM module of AmberTools 24 were studied in the current work and used for receptor–ligand affinity and binding energy calculations, respectively. Two possible mechanisms of single-domain endolysin–ligand binding were predicted by Autodock Vina and verified by the 3D-RISM. As a result, the previously obtained experimental results on peptidoglycan binding of the isolated gamma phage endolysin PlyG enzymatically active domain were supported by molecular docking. Both methods predicted that single-domain endolysins are able to bind peptidoglycan, with Autodock Vina being able to give accurate numerical estimates of protein–ligand affinities and 3D-RISM providing comparative values. Full article

Staphylococcus aureus is a Gram-positive bacterium known to cause mild to severe and potentially fatal infections such as endocarditis, sepsis, meningitis, pneumonia, and bacteremia, among others. The methicillin-resistant strain of Staphylococcus aureus (MRSA) arose because the bacterium acquired an additional penicillin-binding protein by lateral gene transfer, known as penicillin-binding protein 2a (PBP2a). It is responsible for cross-linking peptidoglycan chains in the formation of the bacterial cell wall, and it is a deathly pathogen because it can infect almost all sites in the body; thus, the development of novel PBP2a inhibitors and the treatment of infections caused by this bacterium is vital. In this work, a systematic study of molecular docking and molecular dynamics was carried out to determine the stability of a set of ligands, aza-heterocyclic compounds, against PBP2a, analyzing their RMSD, H-bonds interactions, and binding free energy. In addition, the pharmacokinetic properties are discussed, finding that our proposed ligand 5 is the most promising compound in terms of stability and energetic results. Full article

Acinetobacter baumannii (A. baumannii) has emerged as a prominent multidrug-resistant (MDR) pathogen, significantly complicating treatment strategies due to its formidable resistance mechanisms, particularly against carbapenems. Reduced membrane permeability, active antibiotic efflux, and enzymatic hydrolysis via different β-lactamases are the main resistance mechanisms displayed by A. baumannii, and they are all effective against successful treatment approaches. This means that alternate treatment approaches, such as combination therapy that incorporates beta-lactams, β-lactamase inhibitors, and novel antibiotics like cefiderocol, must be investigated immediately. Cefiderocol, a new catechol-substituted siderophore cephalosporin, improves antibacterial activity by allowing for better bacterial membrane penetration. Due to its unique structure, cefiderocol can more efficiently target and destroy resistant bacteria by using iron transport systems. Through its inhibition of peptidoglycan formation through binding to penicillin-binding proteins (PBPs), cefiderocol avoids conventional resistance pathways and induces bacterial cell lysis. The possibility of resistance development due to β-lactamase synthesis and mutations in PBPs, however, emphasizes the need for continued investigation into cefiderocol’s efficacy in combination treatment regimes. Cefiderocol’s siderophore mimic mechanism is especially important in iron-limited conditions because it can use ferric-siderophore transporters to enter cells. Additionally, its passive diffusion through bacterial porins increases its intracellular concentrations, making it a good option for treating carbapenem-resistant A. baumannii, especially in cases of severe infections and ventilator-associated diseases (IVACs). Cefiderocol may reduce MDR infection morbidity and mortality when combined with customized antimicrobial treatments, but further investigation is needed to improve patient outcomes and address A. baumannii resistance issues. Full article

Iprodione is a pesticide that belongs to the dicarboximide fungicide family. This pesticide was designed to combat various agronomical pests; however, its use has been restricted due to its environmental toxicity and risks to human health. In this study, we explored the proteomic changes in the Pseudomonas sp. C9 strain when exposed to iprodione, to gain insights into the affected metabolic pathways and enzymes involved in iprodione tolerance and biodegradation processes. As a result, we identified 1472 differentially expressed proteins in response to iprodione exposure, with 978 proteins showing significant variations. We observed that the C9 strain upregulated the expression of efflux pumps, enhancing its tolerance to iprodione and other harmful compounds. Peptidoglycan-binding proteins LysM, glutamine amidotransferase, and protein Ddl were similarly upregulated, indicating their potential role in altering and preserving bacterial cell wall structure, thereby enhancing tolerance. We also observed the presence of hydrolases and amidohydrolases, essential enzymes for iprodione biodegradation. Furthermore, the exclusive identification of ABC transporters and multidrug efflux complexes among proteins present only during iprodione exposure suggests potential counteraction against the inhibitory effects of iprodione on downregulated proteins. These findings provide new insights into iprodione tolerance and biodegradation by the Pseudomonas sp. C9 strain. Full article