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Journal of the American Chemical Society Jun 2015Natural modifications of peptidoglycan modulate the innate immune response. Peptidoglycan derivatives activate this response via the intracellular innate immune...
Natural modifications of peptidoglycan modulate the innate immune response. Peptidoglycan derivatives activate this response via the intracellular innate immune receptor, Nod2. To probe how these modifications alter the response, a novel and efficient carbohydrate synthesis was developed to allow for late-stage modification of the amine at the 2-position. Modification of the carbohydrate was found to be important for stabilizing Nod2 and generating the proper response. The native Nod2 ligands demonstrate a significant increase in the cellular stability of Nod2. Moreover, changing the identity of the natural ligands at the carbohydrate 2-position allows for the Nod2-dependent immune response to be either up-regulated or down-regulated. The ligand structure can be adjusted to tune the Nod2 response, suggesting that other innate immune receptors and their ligands could use a similar strategy.
Topics: Immunity, Innate; Nod2 Signaling Adaptor Protein; Peptidoglycan
PubMed: 26035228
DOI: 10.1021/jacs.5b01607 -
Angewandte Chemie (International Ed. in... Feb 2020Deepening our understanding of mammalian gut microbiota has been greatly hampered by the lack of a facile, real-time, and in vivo bacterial imaging method. To address...
Deepening our understanding of mammalian gut microbiota has been greatly hampered by the lack of a facile, real-time, and in vivo bacterial imaging method. To address this unmet need in microbial visualization, we herein report the development of a second near-infrared (NIR-II)-based method for in vivo imaging of gut bacteria. Using d-propargylglycine in gavage and then click reaction with an azide-containing NIR-II dye, gut microbiota of a donor mouse was strongly labeled with NIR-II fluorescence on their peptidoglycan. The bacteria could be readily visualized in recipient mouse gut with high spatial resolution and deep tissue penetration under NIR irradiation. The NIR-II-based metabolic labeling strategy reported herein, provides, to the best of our knowledge, the first protocol for facile in vivo visualization of gut microbiota within deep tissues, and offers an instrumental tool for deciphering the complex biology of these gut "dark matters".
Topics: Animals; Fluorescent Dyes; Gastrointestinal Microbiome; Infrared Rays; Mice; Molecular Structure; Optical Imaging; Peptidoglycan
PubMed: 31793153
DOI: 10.1002/anie.201910555 -
Combinatorial Chemistry & High... 2021Enzymatic degradation of peptidoglycan, a structural cell wall component of Gram-positive bacteria, has attracted considerable attention being a specific target for many...
INTRODUCTION
Enzymatic degradation of peptidoglycan, a structural cell wall component of Gram-positive bacteria, has attracted considerable attention being a specific target for many known antibiotics.
METHODS
Peptidoglycan hydrolases are involved in bacterial lysis through peptidoglycan degradation. β-N-acetyl-glucosaminidase, a peptidoglycan hydrolase, acts on O-glycosidic bonds formed by N-acetylglucosamine and N-acetyl muramic acid residues of peptidoglycan. Aim of present study was to study the action of β-N-acetylglucosaminidase, on methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-negative bacteria.
RESULTS
We investigated its dynamic behaviour using molecular dynamics simulation and observed that serine and alanine residues are involved in catalytic reaction in addition to aspartic acid, histidine, lysine and arginine residues. When simulated in its bound state, the RMSD values were found lesser than crystal form in the time stamp of 1000 picoseconds revealing its stability. Structure remained stably folded over 1000 picoseconds without undergoing any major change further confirming the stability of complex.
CONCLUSION
It can be concluded that enzymes belonging to this category can serve as a tool in eradicating Gram-positive pathogens and associated infections.
Topics: Acetylglucosaminidase; Carbohydrate Conformation; Humans; Models, Molecular; Peptidoglycan; Serratia marcescens
PubMed: 33155890
DOI: 10.2174/1386207323999201103204234 -
Analytical Chemistry Jun 2024Rapidly identifying and quantifying Gram-positive bacteria are crucial to diagnosing and treating bacterial lower respiratory tract infections (LRTIs). This work...
Rapidly identifying and quantifying Gram-positive bacteria are crucial to diagnosing and treating bacterial lower respiratory tract infections (LRTIs). This work presents a field-deployable biosensor for detecting Gram-positive bacteria from exhaled breath condensates (EBCs) based on peptidoglycan recognition using an aptamer. Dielectrophoretic force is employed to enrich the bacteria in 10 s without additional equipment or steps. Concurrently, the measurement of the sensor's interfacial capacitance is coupled to quantify the bacteria during the enrichment process. By incorporation of a semiconductor condenser, the whole detection process, including EBC collection, takes about 3 min. This biosensor has a detection limit of 10 CFU/mL, a linear range of up to 10 CFU/mL and a selectivity of 1479:1. It is cost-effective and disposable due to its low cost. The sensor provides a nonstaining, culture-free and PCR-independent solution for noninvasive and real-time diagnosis of Gram-positive bacterial LRTIs.
Topics: Biosensing Techniques; Peptidoglycan; Breath Tests; Gram-Positive Bacteria; Humans; Limit of Detection; Aptamers, Nucleotide
PubMed: 38730304
DOI: 10.1021/acs.analchem.4c00059 -
STAR Protocols Dec 2021Fluorescence microscopy is a method of choice for studying peptidoglycan assembly, but it presents two major challenges: the peptidoglycan must be labeled with a probe...
Fluorescence microscopy is a method of choice for studying peptidoglycan assembly, but it presents two major challenges: the peptidoglycan must be labeled with a probe that will not perturb the physiological process, and the spatial resolution must reach the nanometer scale to reveal fine details of the synthesis process. This protocol meets both challenges by combining biorthogonal metabolic labeling of peptidoglycan in with super-resolution fluorescence microscopy (dSTORM), also providing cues to adapt it to other bacteria. For complete details on the use and execution of this protocol, please refer to Trouve et al. (2021).
Topics: Animals; Female; Male; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Molecular Imaging; Peptidoglycan; Streptococcus pneumoniae
PubMed: 34977669
DOI: 10.1016/j.xpro.2021.101006 -
Journal of Natural Products Mar 2011Bacteria release immunostimulatory compounds to the environment, and one of the stimulants is the ligand of nucleotide-binding oligomerization domain protein 1 (Nod1),... (Review)
Review
Bacteria release immunostimulatory compounds to the environment, and one of the stimulants is the ligand of nucleotide-binding oligomerization domain protein 1 (Nod1), an intracellular protein involved in the recognition of the bacterial component peptidoglycans having a diaminopimelic acid (DAP) structure. The polymorphisms of Nod1 have been linked to several inflammatory diseases and allergies that are strongly affected by environmental factors. The present paper summarizes recent results on the isolation and structural elucidation of natural human Nod1 (hNod1) ligands from the Escherichia coli (E. coli) K-12 culture supernatant, the first chemical synthesis of these natural ligands and related PGN fragments structures, and the hNod1 stimulatory activities of the chemically synthesized DAP-type PGN fragments. For structural characterization studies, the 7-(diethylamino)coumarin-3-carbonyl (DEAC) labeling method was also used to enhance the sensitivity in mass spectrometry studies, in order to observe PGN fragments in a comprehensive manner. The results suggest that DAP-containing bacteria release certain hNod1 ligands to the environment and that these ligands accumulate in the environment and regulate the immune system through Nod1.
Topics: Bacteria; Diaminopimelic Acid; Escherichia coli; Humans; Nod1 Signaling Adaptor Protein; Peptidoglycan
PubMed: 21341753
DOI: 10.1021/np100795d -
Applied Biochemistry and Biotechnology Dec 2014Peptidoglycan is a highly complex and essential macromolecule of bacterial outer cell wall; it is a heteropolymer made up of linear glycan strands cross-linked by...
Peptidoglycan is a highly complex and essential macromolecule of bacterial outer cell wall; it is a heteropolymer made up of linear glycan strands cross-linked by peptides. Peptidoglycan has a particular composition which makes it a possible target for specific bacterial recognition. Aptamers are single-stranded DNA or RNA oligonucleotides that bind to target molecules with high affinity and specificity. Aptamers can be labeled with different radioisotopes and possess several properties that make them suitable for molecular imaging. The purpose of this study was to obtain aptamers for use as radiopharmaceutical in bacterial infection diagnosis. Two aptamers (Antibac1 and Antibac2) against peptidoglycan were selected through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology. The dissociation constant (Kd) for Antibac1 was 0.415 + 0.047 μM and for Antibac2 was 1.261 + 0.280 μM. These aptamers labeled with (32)P showed high affinity for Staphylococcus aureus cells. The binding to S. aureus and Escherichia coli in vitro were significantly higher than for Candida albicans and human fibroblasts, demonstrating their specificity for bacterial cells. These results point Antibac1 and Antibac2 as promising tools for bacterial infections identification.
Topics: Aptamers, Nucleotide; Candida albicans; Escherichia coli; Humans; Peptidoglycan; Staphylococcus aureus
PubMed: 25185503
DOI: 10.1007/s12010-014-1206-6 -
Research in Microbiology Jan 2014
Topics: Bacterial Physiological Phenomena; Cell Wall; Peptidoglycan
PubMed: 24239960
DOI: 10.1016/j.resmic.2013.11.002 -
Proceedings of the National Academy of... Apr 2014Fluorescent probes designed for activation by bioorthogonal chemistry have enabled the visualization of biomolecules in living systems. Such activatable probes with...
Fluorescent probes designed for activation by bioorthogonal chemistry have enabled the visualization of biomolecules in living systems. Such activatable probes with near-infrared (NIR) emission would be ideal for in vivo imaging but have proven difficult to engineer. We present the development of NIR fluorogenic azide probes based on the Si-rhodamine scaffold that undergo a fluorescence enhancement of up to 48-fold upon reaction with terminal or strained alkynes. We used the probes for mammalian cell surface imaging and, in conjunction with a new class of cyclooctyne D-amino acids, for visualization of bacterial peptidoglycan without the need to wash away unreacted probe.
Topics: Azides; Fluorescent Dyes; Molecular Imaging; Molecular Probe Techniques; Molecular Structure; Peptidoglycan
PubMed: 24706769
DOI: 10.1073/pnas.1322727111 -
Electrophoresis Oct 2019Peptidoglycan or murein is an essential polymer found in bacterial cell wall. It is a dynamic structure that is continuously remodeled or modified during bacterial cell... (Comparative Study)
Comparative Study
Peptidoglycan or murein is an essential polymer found in bacterial cell wall. It is a dynamic structure that is continuously remodeled or modified during bacterial cell growth or in presence of cell wall stresses. These modifications are still poorly understood mainly due to the peptidoglycan, which is rather non-soluble, and the difficulties to separate the hydrophilic glycopeptides (muropeptides) by reversed phase liquid chromatography, generated by the enzymatic digestion using mutanolysin, an N-acetyl-muramidase, cleaving the β bound between N-acetylglucosamine and N-acetylmuramic acid. Here, we report the use of CZE-MS for an easy and fast screening of muropeptides generated by the action of muramidase on the Bacillus licheniformis cell wall. Electron transfer and CID-MS were also used to unambiguously identify and localize the presence or the absence of amidation and acetylation moieties on muropeptide variants. The reference method to analyse muropeptides by reversed phase chromatography was also tested and the advantages and disadvantages of both methods were evaluated.
Topics: Bacillus licheniformis; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Electrophoresis, Capillary; Mass Spectrometry; Peptidoglycan
PubMed: 31169927
DOI: 10.1002/elps.201900147