Why are the most lethal pathogens the simplest? Lack of D-amino acid usage, coherent fractal morphology, and Hz-level biological oscillations prevalent in beneficial pathogens.

The growing number of bacterial infections and the rise of antibiotic resistance require approaches for antimicrobial development. Peptidoglycan, essential for maintaining the integrity and shape of the bacterial cell wall, is regulated by the coordinated activity of peptidoglycan synthesis and remodeling enzymes. While peptidoglycan synthesis enzymes have served as antibiotic targets for decades, peptidoglycan hydrolases have remained largely underexplored. Here, we review recent advances in the development of small-molecule inhibitors of peptidoglycan hydrolases as antimicrobial targets.

Removal of nitrate by aerobic denitrification granular sludge under strongly alkaline and low carbon to nitrogen ratio conditions: Performance and mechanism.

A rapid culture-free method is needed to improve diagnostic efficiency and guide timely antimicrobial therapy for urinary tract infections (UTIs). Previously, we utilized the lipidomics-based fast lipid analysis technique (FLAT) to screen for uropathogens by identifying distinctive microbial membrane lipid profiles, specifically lipid A in gram-negative and cardiolipin in gram-positive bacteria. This culture-free assay demonstrated high sensitivity (94%) in detecting gram-negative bacteria but poor sensitivity (51%) for gram-positive bacteria. In this study, we pretreated urine pellets with lysozyme prior to FLAT analysis to break down the peptidoglycan layer bacteria, thereby promoting the efficient release of cardiolipin. The limit of detection (LOD) for 4 gram-positive bacteria and Escherichia coli was evaluated using contrived samples with known CFU/mL values and varying concentrations of lysozyme. Subsequently, we validated the optimized method in a clinical cohort of 76 urine samples known to contain gram-positive bacteria as confirmed by urine culture. Optimal sensitivity was achieved by treating 1 mL of urine pellets with 100 µg lysozyme and incubating for 60 minutes, resulting in a 100-fold increase in cardiolipin LOD and a 95% detection rate for gram-positive bacteria. Signal-to-noise ratio for lipid A was also improved. Polymicrobial urine cultures with gram-negative and gram-positive species were identified in 2 patients. The lysozyme-enhanced FLAT assay enables rapid and culture-free detection of both gram-negative and gram-positive uropathogens directly from urine. The unified workflow decreases the analytical turnaround time by at least 90% making it well-suited for high-throughput clinical laboratories.

Traffic-related air pollution in utero modifies cytokine responses to stimuli of umbilical cord blood cells: a cohort study.

To establish infection, Salmonella confronts a dynamic barrage of host-induced stresses. The peptidoglycan layer is essential for maintaining bacterial cell integrity and counteracting these environmental stress. Its synthesis relies on the lipid carrier undecaprenyl phosphate, which is generated by the enzyme undecaprenyl pyrophosphate phosphatase (UppP). While UppP is linked to virulence in other pathogens, its role in Salmonella remains unclear. We show that an uppP mutant in S. Typhimurium exhibits altered cell morphology, reduced stiffness, and impaired survival in RAW 264.7 macrophages. The mutant is also attenuated in systemic infection in C57BL/6 mice. These defects are associated with increased sensitivity to nitrosative stress. Notably, iNOS inhibition or deficiency restores intracellular survival of the uppP mutant in both RAW 264.7 macrophages and the mouse model, implicating UppP in resistance to nitrosative stress. Our findings reveal a critical role for UppP in promoting Salmonella survival within macrophages and contributing to systemic pathogenesis.

Ultrasound-assisted extraction of peptidoglycan from Lactiplantibacillus plantarum: Structural characterization, anti-browning enhancement, and immunomodulatory effects.

The bacterial cell wall is a covalently linked meshwork of peptidoglycan (PG) that helps maintain cell shape and prevents osmotic lysis. This structure must be flexible enough to accommodate transenvelope protein complexes, but strong enough to withstand high intracellular pressure. To elongate and divide, cells must remodel the cell wall through the concerted action of PG synthesis and degradation. Endopeptidases, a class of PG-degrading enzymes, facilitate cell growth by hydrolysing PG crosslinks. Vibrio cholerae encodes several functionally redundant endopeptidases, two of which are nearly identical: ShyA and ShyC. To investigate the differential roles of these enzymes, we assessed the growth and morphology of shyA and shyC mutants. We found that native levels of ShyA, but not ShyC, facilitate adaptation to low-osmolarity medium. Cells lacking shyA exhibited a longer lag phase and aberrant morphology during adaptation. Lastly, our experiments revealed that cells lacking ShyA's LysM domain exhibited more severe defects than cells lacking shyA altogether, implicating the LysM domain in the proper regulation of ShyA activity.

Staphylococcus aureus, a spherical Gram-positive bacterium commonly found to coinhabit humans, can also cause minor skin infections to life-threatening conditions such as pneumonia in individuals with weakened immune systems. The bacterium has developed resistance against conventional antibiotics. This underscores the urgent need for novel therapeutic strategies that act on the cell structure and therefore integrity of the bacterium. Wall teichoic acids (WTAs) are essential anionic glycopolymers covalently anchored to the peptidoglycan layer of Gram-positive bacteria, including S. aureus and are crucial for bacterial survival. The biosynthesis of WTA occurs by a multi-step process in the cytoplasm and proceeds through membrane translocation and incorporation into the cell wall. The earliest and most essential step in this pathway is catalyzed by TarA, which transfers N-acetylglucosamine (GlcNAc) to undecaprenyl phosphate, forming the WTA precursor lipid I. TarA catalyzes the reaction that serves as the first committed step in WTA biosynthesis, without which the entire WTA polymer cannot be constructed or transported. The TarA protein domain has emerged as a promising target for drug development due to its pivotal role in cell wall biosynthesis. We obtained the S. aureus TarA three-dimensional structure from AlphaFold2, performed virtual screening on diverse compound libraries so as to establish their binding to the target protein, which led to the identification of hit compounds with good binding affinity towards TarA domain and involvement of key amino acid residue interactions. This was followed by molecular docking studies, assessment of drug likeness properties of hit compounds and molecular dynamics (MD) simulations of S. aureus TarA-hit molecule complexes using Amber18 bio-simulations package. MD trajectory analysis; root mean square deviation, root mean square fluctuation, hydrogen bonding analysis, solvent accessible surface area, principal component analysis, secondary structure analysis, clustering analysis, free energy landscape, interactive hydrogen bond matrix, binding free energies of the simulated complexes and steered MD simulations were studied. This study resulted in the identification of new hit molecules with a potential to reduce the risk of the S. aureus infections.

Cohen syndrome is an autosomal recessive genetic disease caused by mutations in the vacuolar protein sorting homolog B (VPS13B) gene that leads to a variety of complications including periodontitis. However, the molecular mechanism underlying periodontal inflammation caused by VPS13B dysfunction in human gingival epithelial cells remains unclear. A previous report noted that coxsackievirus and adenovirus receptor (CXADR) and junctional adhesion molecule 1 (JAM1) are involved in barrier functions against penetration by lipopolysaccharide (LPS) and peptidoglycan (PGN) into gingival tissues. The present study was conducted to examine the effects and significance of VPS13B on gingival barrier function. It was confirmed that loss of VPS13B resulted in decreased cell surface localization of CXADR, but not of JAM1. Additionally, abundant lysosomal localization of CXADR was detected in VPS13B-knockout cells followed treatment with bafilomycin A1, an inhibitor of lysosomal degradation. Other findings indicated that cell-surface localization of the CXADR-chimeric protein, in which C-terminus was exchanged with JAM1, was not disturbed by VPS13B knockout. Finally, VPS13B knockout led to greater permeability of gingival epithelial cell layers and tissues to LPS and PGN, which was restored by increased expression of CXADR-JAM1c-term. Together, these results show that VPS13B is involved in intracellular trafficking of CXADR as well as the barrier function of human gingival epithelial tissues, and thus indicate the molecular basis for periodontal complications in patients affected by Cohen syndrome.

Gold nanoparticles (AuNPs) have attracted significant attention due to their broad range of applications in the health and environmental fields and the fact that gold is an inert metal with low physiological toxicity, making it a favorable candidate for biomedical use. In line with the growing interest in green nanotechnology, plant-based nanoparticle synthesis has emerged as a sustainable, low-cost, and environmentally friendly alternative to conventional methods. Among these, AuNPs synthesized using plant extracts have been extensively studied, particularly concerning their antimicrobial properties. In this sense, this study aimed to perform a bibliometric review of the antimicrobial potential of plant-based AuNPs focusing on articles published between 2014 and 2025. Searches were conducted at the Web of Science, PubMed, and Scopus databases, totaling 190 eligible studies. India is the leading country in terms of number of publications (36.84%), with leaves as the most employed plant part (42.6%). The reported AuNPs synthesized from various plant parts, including seeds, fruits, as well as leaves, exhibited significant antimicrobial activities. Although some AuNPs, such as those synthesized from seeds, were more effective against Gram-positive bacteria, others, such as those from fruits, showed greater activity against Gram-negative strains, and several leaf-based ones demonstrated broad-spectrum efficacy against both groups. These antimicrobial performance variations are primarily attributed to the presence of specific organic compounds like flavonoids, terpenoids, and phenolics unique to each plant species used in the synthesis process. Escherichia coli and Staphylococcus aureus were the most frequently tested bacterial strains, with main AuNPs effects comprising damage to their thiol and peptidoglycan cell walls. One hundred thirty three (Emmanuel et al. in Microb Pathog 113:295-302, 2017) articles expanded their evaluations to include fungi, protozoa and cytotoxic effects, with many reports encouraging results against antibiotic-resistant bacterial strains, reinforcing the potential of plant-based AuNPs as innovative antimicrobial therapy alternatives.

Envelope biogenesis in Staphylococcus aureus is concentrated at the septum and includes peptidoglycan synthesis, lipo- and wall-teichoic acid production, and the targeted secretion of YSIRK/GXXS signal peptide-bearing proteins. How S. aureus confines these processes to the dividing crosswall remains unclear. EzrA, a scaffolding protein structurally related to eukaryotic spectrins, has been implicated in linking cell division to envelope synthesis, yet its precise role is poorly understood. Here, we re-examine the function of EzrA for its contribution to envelope biogenesis and homeostasis. We observe that ezrA null mutants synthesize excess peptidoglycan that is incorporated in a dispersed pattern, no longer strictly confined to the septum. A similar loss of spatial restriction was observed for protein A, a surface protein whose YSIRK/GXXS signal peptide directs septal secretion and anchoring. In wild-type cells, newly synthesized peptidoglycan co-localized with nascent protein A anchoring sites at the septum, whereas this spatial coupling was disrupted in the absence of EzrA. In addition, loss of EzrA resulted in impaired nucleoid occlusion with septal guillotining of the chromosome. Together, these findings support a model in which EzrA acts as a molecular organizer of cell division, coordinating septal biosynthesis and envelope assembly while ensuring proper nucleoid occlusion.

Methicillin resistant Staphylococcus aureus (MRSA) bacteremia has a high rate of morbidity and mortality. Multiple clinical studies have demonstrated improved outcomes when MRSA bacteremia is treated with dual antibiotic therapy that includes a β-lactam antibiotic such as cefazolin. This is a paradox as MRSA should be inherently resistant to this class of antibiotics. We report on a serendipitous observation of a phenotype where MRSA became sensitive to cefazolin when cultured in a physiologic relevant media of fetal bovine serum as well as in synovial fluid. This could be observed across multiple clinical isolates. Expected resistance was maintained when cultured in Muller Hinton Broth (MHB). MRSA β-lactam antibiotic resistance is mediated by PBP2a, a penicillin-binding protein encoded by mecA. We hypothesized that this phenotype of antibiotic sensitivity in physiologic medium was based, in part, on levels of PBP2a expression and post-translational modifications of peptidoglycan wall teichoic acid (WTA). We, therefore, conducted quantitative RT-PCR analysis and Western blotting which demonstrated limited mecA expression PBP2a protein level when cultured in FBS as compared to the clinical microbiology standard MHB, respectively. Whole genome sequencing of loss of function mutants generated through serial passaging in FBS revealed that the clp family of proteins and rpo genes were involved in β-lactam resistance. Cell wall peptidoglycan analysis suggested that WTA glycosylation was altered between β-lactam resistant and sensitive MRSA phenotypes. Together, this suggests that clpP, rpoB, and WTA glycosylation are involved with the β-lactam sensitivity phenotype in MRSA and can be new potential targets for MRSA treatment.

Antimicrobial resistance poses major therapeutic challenges, particularly for multidrug-resistant mycobacterial infections caused by Mycobacterium tuberculosis (Mtb) and non-tuberculous mycobacteria (NTM). l,d-Transpeptidases (Ldts) are attractive drug targets due to their essential role in peptidoglycan cell wall crosslinking, yet existing assays suffer from low throughput and limited sensitivity. We report a versatile, bead-based platform for high-throughput analysis of Ldt activity and inhibitor discovery. We incubated peptidoglycan stem peptides, either naturally harvested or synthetically immobilized on abiotic surfaces, with Ldts and a fluorescent acyl acceptor to quantitatively monitor crosslinking. After optimizing assay parameters, we profiled six Mycobacterium smegmatis Ldt paralogs, including the first characterization of a class 6 Ldt with chemically defined substrate sequences. Utilizing a series of acyl acceptors, we demonstrated modifications within the acyl acceptor that are tolerated by mycobacterial Ldts. Screening of β-lactam antibiotics revealed potent inhibition by (carba)penems, while cephalosporins, monobactams and penams showed negligible activity. The assay achieved excellent performance metrics and was successfully adapted to ELISA and 96-well formats, providing a powerful tool for discovering Ldt-targeted therapeutics against tuberculosis and related infections.

Background/Objectives: Granulicatella adiacens infective endocarditis is conventionally managed with penicillin, ampicillin, or ceftriaxone in combination with gentamicin, although double beta-lactam regiments have been proposed a safer alternative to reduce aminoglycoside-associated nephrotoxicity. To date, the High-Molecular-Mass Penicillin-Binding Proteins (HMM-PBPs) of G. adiacens and their affinities for beta-lactam antibiotics have not been previously characterized. This study investigated the HMM-PBP profile of G. adiacens, with particular interest on sequence alterations and beta-lactam binding properties, both as single agents and in combination. Methods: Beta-lactam activity, synergistic interactions and PBP binding affinities were evaluated in a clinical isolate (IS 48) and compared with those in the reference strain ATCC 49175. Binding of PBPs to ampicillin, ceftriaxone, and ceftobiprole, alone or in combination, was investigated by Bocillin-FL labeling. PBP homology and conserved active-sites motifs were assessed by sequence alignment, and pbp gene mutations were identified by whole-genome sequencing. Results: The clinical isolate was non-susceptible to ampicillin, resistant to ceftriaxone and exhibited higher minimum inhibitory concentrations (MICs) for ceftobiprole relative to the fully susceptible ATCC reference strain. Five HMM PBPs with high enterococcal homology, were identified. In the IS 48 isolate, the class A PBP showed distinct amino acid substitutions in proximity to the catalytic centers. Despite these alterations, PBP1A and PBP2A were strongly inhibited by the tested beta-lactams, whereas PBP2 and PBP2B demonstrated low acylation rates. Combination of ampicillin with either ceftobiprole or ceftriaxone resulted in enhanced acylation of the three bifunctional HMM PBPs compared with monotreatment. IC50 values were consistently higher for the IS 48 clinical isolate, suggesting decreased target availability and/or reduced beta-lactam affinity under clinical conditions. Conclusions: The resistance phenotype of G. adiacens clinical isolate appears to be primarily associated with altered PBP beta-lactam interactions. Nonetheless, beta-lactam combination regimes remain effective by achieving substantial inhibition of key HMM-PBPs involved in peptidoglycan synthesis, thereby supporting the rationale for dual beta-lactam therapy in this setting.

During a survey for natural-rubber-degrading actinobacteria associated with soils from Hevea brasiliensis plantations in Thailand, two strains, ABSL1-1T and ABSL49-1T, were isolated using mineral salts medium with natural rubber as the sole carbon source. Polyphasic taxonomy placed both strains within the genus Gordonia. Strain ABSL1-1T showed the highest 16S rRNA gene sequence similarity to Gordonia otitidis NBRC 100426T (98.5%) and Gordonia soli NBRC 108243T (98.3%), while ABSL49-1T showed the highest similarity to Gordonia polyisoprenivorans DSM 44302T (98.4%). The digital DNA-DNA hybridization (dDDH) and average nucleotide identity based on blast values between ABSL1-1T and closely related type strains were 20.1-20.9%, and 74.1-76.5%, respectively, while those for ABSL49-1T and closely related type strains were 20.6-22.9%, and 74.3-79.1%, respectively. The cell-wall peptidoglycan of both strains contained meso-diaminopimelic acid and the whole-cell sugars comprised ribose, arabinose, galactose and glucose. Both strains contained MK-9(H2) as the major menaquinone and phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside were detected as the polar lipids. The predominant fatty acids of ABSL1-1T were C16:0, and C18:1 ω9c, while those for ABSL49-1T were C16:0, C18:1 ω9c, summed feature 3 (C16:1 ω7c/C16:1 ω6c) and C18:0 10-methyl. The G+C contents of the genomic DNA of strains ABSL1-1T and ABSL49-1T were 67.0 mol% and 66.0 mol%, respectively. Based on the results of a polyphasic taxonomic analysis, strains ABSL1-1ᵀ and ABSL49-1ᵀ represent the type strains of two novel species of the genus Gordonia, for which the names Gordonia heveisoli sp. nov. (type strain ABSL1-1ᵀ=TBRC 15892ᵀ=NBRC 116252ᵀ) and Gordonia gummivorans sp. nov. (type strain ABSL49-1ᵀ=TBRC 15624ᵀ=NBRC 115559ᵀ) are proposed.

Modulation of peptidoglycan crosslink network in Escherichia coli enhances water-responsive actuation.

Genome-wide identification of scavenger receptor family genes in Octopus sinensis and their immune response to PGN, poly I:C, and Vibrio parahaemolyticus.

Excessive osteoclast activity contributes to osteoporosis, a condition marked by reduced bone density. While certain probiotics have shown potential in enhancing bone mass, the specific bioactive components and their therapeutic efficacy remain unclear. In this study, we isolated peptidoglycans (PGNs) from eleven Lactobacillus species and evaluated their effects on ovariectomized (OVX) mouse models of osteoporosis. Oral administration of PGNs, particularly from Lactiplantibacillus plantarum, Lacticaseibacillus casei, and Lactobacillus ruminis, significantly restored trabecular bone volume in OVX mice. Among them, L. plantarum PGN (Lp.PGN) demonstrated the most potent effect, enhancing bone volume when administered intraperitoneally or intravenously. Histological analysis revealed increased Runx2-positive osteoblasts and reduced TRAP-positive osteoclasts in Lp.PGN-treated mice. Calcein double labeling confirmed enhanced new bone formation. In vitro, Lp.PGN promoted osteoblast mineralization and suppressed osteoclast differentiation in co-culture systems. Notably, Lp.PGN failed to improve bone mass in NOD2-deficient OVX mice, indicating that NOD2 signaling is essential for its osteoprotective effects. Additionally, Lp.PGN reduced pro-inflammatory cytokines and the RANKL/OPG ratio, and modulated gut microbiota composition by decreasing Proteobacteria and increasing Firmicutes, resembling the profile of sham-operated controls. In conclusion, NOD2-activating PGN attenuates bone loss under osteoporotic conditions by increasing osteoblast differentiation and suppressing osteoclast differentiation. Lp.PGN could play an important role in enhancing bone mass through NOD2 signaling, and NOD2 ligands could be considered as postbiotic-based therapeutic agents for the treatment of bone diseases.

Comparative analysis reveals the diverse immune responses of Pacific white shrimp Litopenaeus vannamei post different pathogen-associated molecular patterns stimulation.