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Scientific Reports Jul 2020Chlamydia trachomatis serovar L2 and Chlamydia muridarum, which do not express FtsZ, undergo polarized cell division. During division, peptidoglycan assembles at the...
Chlamydia trachomatis serovar L2 and Chlamydia muridarum, which do not express FtsZ, undergo polarized cell division. During division, peptidoglycan assembles at the pole of dividing Chlamydia trachomatis cells where daughter cell formation occurs, and peptidoglycan regulates at least two distinct steps in the polarized division of Chlamydia trachomatis and Chlamydia muridarum. Cells treated with inhibitors that prevent peptidoglycan synthesis or peptidoglycan crosslinking by penicillin-binding protein 2 (PBP2) are unable to initiate polarized division, while cells treated with inhibitors that prevent peptidoglycan crosslinking by penicillin-binding protein 3 (PBP3/FtsI) initiate polarized division, but the process arrests at an early stage of daughter cell growth. Consistent with their distinct roles in polarized division, peptidoglycan organization is different in cells treated with PBP2 and PBP3-specific inhibitors. Our analyses indicate that the sequential action of PBP2 and PBP3 drives changes in peptidoglycan organization that are essential for the polarized division of these obligate intracellular bacteria. Furthermore, the roles we have characterized for PBP2 and PBP3 in regulating specific steps in chlamydial cell division have not been described in other bacteria.
Topics: Bacterial Proteins; Cell Division; Chlamydia trachomatis; Penicillin-Binding Proteins; Peptidoglycan
PubMed: 32724139
DOI: 10.1038/s41598-020-69397-x -
PLoS Pathogens Jan 2022Salmonella enterica causes intracellular infections that can be limited to the intestine or spread to deeper tissues. In most cases, intracellular bacteria show moderate...
Salmonella enterica causes intracellular infections that can be limited to the intestine or spread to deeper tissues. In most cases, intracellular bacteria show moderate growth. How these bacteria face host defenses that recognize peptidoglycan, is poorly understood. Here, we report a high-resolution structural analysis of the minute amounts of peptidoglycan purified from S. enterica serovar Typhimurium (S. Typhimurium) infecting fibroblasts, a cell type in which this pathogen undergoes moderate growth and persists for days intracellularly. The peptidoglycan of these non-proliferating bacteria contains atypical crosslinked muropeptides with stem peptides trimmed at the L-alanine-D-glutamic acid-(γ) or D-glutamic acid-(γ)-meso-diaminopimelic acid motifs, both sensed by intracellular immune receptors. This peptidoglycan has a reduced glycan chain average length and ~30% increase in the L,D-crosslink, a type of bridge shared by all the atypical crosslinked muropeptides identified. The L,D-transpeptidases LdtD (YcbB) and LdtE (YnhG) are responsible for the formation of these L,D-bridges in the peptidoglycan of intracellular bacteria. We also identified in a fraction of muropeptides an unprecedented modification in the peptidoglycan of intracellular S. Typhimurium consisting of the amino alcohol alaninol replacing the terminal (fourth) D-alanine. Alaninol was still detectable in the peptidoglycan of a double mutant lacking LdtD and LdtE, thereby ruling out the contribution of these enzymes to this chemical modification. Remarkably, all multiple mutants tested lacking candidate enzymes that either trim stem peptides or form the L,D-bridges retain the capacity to modify the terminal D-alanine to alaninol and all attenuate NF-κB nuclear translocation. These data inferred a potential role of alaninol-containing muropeptides in attenuating pro-inflammatory signaling, which was confirmed with a synthetic tetrapeptide bearing such amino alcohol. We suggest that the modification of D-alanine to alaninol in the peptidoglycan of non-proliferating intracellular S. Typhimurium is an editing process exploited by this pathogen to evade immune recognition inside host cells.
Topics: Cell Line; Cell Wall; Humans; Immune Tolerance; Peptidoglycan; Salmonella Infections; Salmonella enterica
PubMed: 35077524
DOI: 10.1371/journal.ppat.1010241 -
The Journal of Biological Chemistry Jan 2020Peptidoglycan (PG) is a critical component of the bacterial cell wall and is composed of a repeating β-1,4-linked disaccharide of -acetylglucosamine and -acetylmuramic...
Peptidoglycan (PG) is a critical component of the bacterial cell wall and is composed of a repeating β-1,4-linked disaccharide of -acetylglucosamine and -acetylmuramic acid appended with a highly conserved stem peptide. In Gram-negative bacteria, PG is assembled in the cytoplasm and exported into the periplasm where it undergoes considerable maturation, modification, or degradation depending on the growth phase or presence of environmental stressors. These modifications serve important functions in diverse processes, including PG turnover, cell elongation/division, and antibiotic resistance. Conventional methods for analyzing PG composition are complex and time-consuming. We present here a streamlined MS-based method that combines differential analysis with statistical 1D annotation approaches to quantitatively compare PGs produced in planktonic- and biofilm-cultured We identified a core assembly of PG that is present in high abundance and that does not significantly differ between the two growth states. We also identified an adaptive PG assembly that is present in smaller amounts and fluctuates considerably between growth states in response to physiological changes. Biofilm-derived adaptive PG exhibited significant changes compared with planktonic-derived PG, including amino acid substitutions of the stem peptide and modifications that indicate changes in the activity of amidases, deacetylases, and lytic transglycosylases. The results of this work also provide first evidence of de--acetylated muropeptides from The method developed here offers a robust and reproducible workflow for accurately determining PG composition in samples that can be used to assess global PG fluctuations in response to changing growth conditions or external stimuli.
Topics: Biofilms; Cell Wall; Glycomics; Humans; Mass Spectrometry; Peptidoglycan; Plankton; Pseudomonas Infections; Pseudomonas aeruginosa
PubMed: 31771981
DOI: 10.1074/jbc.RA119.010505 -
Applied Microbiology and Biotechnology Jun 2022Clostridium difficile (C. difficile) is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease....
Clostridium difficile (C. difficile) is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Autolysin is a lytic enzyme that hydrolyzes peptidoglycans of the bacterial cell wall, with a catalytic domain and cell wall-binding domains, proven to be involved in bacterial cell wall remodeling and cell division. Although autolysins in C. difficile have been reported, the autolysins have failed to yield impressive results when used as exogenous lytic agents. In this study, we expressed and characterized the binding domains (Cwp19-BD and Acd-BD) and catalytic domains (Cwp19-CD, Acd-CD, and Cwl-CD) of C. difficile autolysins, and the domains with the best binding specificity and lytic activity were selected towards C. difficile to design a novel lytic protein Cwl-CWB2. Cwl-CWB2 showed good biosafety with significantly low hemolysis and without cytotoxicity. The results of fluorescence analysis and lytic assay demonstrated that Cwl-CWB2 has higher binding specificity and stronger lytic activity with a minimum inhibitory concentration at 13.39 ± 5.80 μg/mL against living C. difficile cells, which is significantly stronger than commercial lysozyme (3333.33 ± 1443.37 μg/mL) and other reported C. difficile autolysins. Besides, Cwl-CWB2 exhibited good stability as about 75% of the lytic activity was still retained when incubated at 37 °C for 96 h, which is considered to be a potential antimicrobial agent to combat C. difficile. KEY POINTS: • Several binding domains and catalytic domains, deriving from several Clostridium difficile autolysins, were expressed, purified, and functionally characterized. • A novel C. difficile lytic protein Cwl-CWB2 was designed from C. difficile autolysins. • The binding specificity and lytic activity of Cwl-CWB2 against C. difficile showed advantages compared with other reported C. difficile autolysins. • Cwl-CWB2 exhibited significantly low hemolysis and cytotoxicity against normal-derived colon mucosa 460 cell.
Topics: Cell Wall; Clostridioides difficile; Hemolysis; Humans; N-Acetylmuramoyl-L-alanine Amidase; Peptidoglycan
PubMed: 35699735
DOI: 10.1007/s00253-022-12010-0 -
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 -
Molecular Plant Pathology Jul 2018The essential stages of bacterial cell separation are described as the synthesis and hydrolysis of septal peptidoglycan (PG). The amidase, AmiC, which cleaves the...
Peptidoglycan hydrolysis mediated by the amidase AmiC and its LytM activator NlpD is critical for cell separation and virulence in the phytopathogen Xanthomonas campestris.
The essential stages of bacterial cell separation are described as the synthesis and hydrolysis of septal peptidoglycan (PG). The amidase, AmiC, which cleaves the peptide side-chains linked to the glycan strands, contributes critically to this process and has been studied extensively in model strains of Escherichia coli. However, insights into the contribution of this protein to other processes in the bacterial cell have been limited. Xanthomonas campestris pv. campestris (Xcc) is a phytopathogen that causes black rot disease in many economically important plants. We investigated how AmiC and LytM family regulators, NlpD and EnvC, contribute to virulence and cell separation in this organism. Biochemical analyses of purified AmiC demonstrated that it could hydrolyse PG and its activity could be potentiated by the presence of the regulator NlpD. We also established that deletion of the genes encoding amiC1 or nlpD led to a reduction in virulence as well as effects on colony-forming units and cell morphology. Moreover, further genetic and biochemical evidence showed that AmiC1 and NlpD affect the secretion of type III effector XC3176 and hypersensitive response (HR) induction in planta. These findings indicate that, in addition to their well-studied role(s) in cell separation, AmiC and NlpD make an important contribution to the type III secretion (T3S) and virulence regulation in this important plant pathogen.
Topics: Amidohydrolases; Hydrolysis; Peptidoglycan; Virulence; Xanthomonas campestris
PubMed: 29240286
DOI: 10.1111/mpp.12653 -
Proceedings of the National Academy of... Dec 2008The stress-bearing component of the bacterial cell wall--a multi-gigadalton bag-like molecule called the sacculus--is synthesized from peptidoglycan. Whereas the...
The stress-bearing component of the bacterial cell wall--a multi-gigadalton bag-like molecule called the sacculus--is synthesized from peptidoglycan. Whereas the chemical composition and the 3-dimensional structure of the peptidoglycan subunit (in at least one conformation) are known, the architecture of the assembled sacculus is not. Four decades' worth of biochemical and electron microscopy experiments have resulted in two leading 3-D peptidoglycan models: "Layered" and "Scaffold", in which the glycan strands are parallel and perpendicular to the cell surface, respectively. Here we resolved the basic architecture of purified, frozen-hydrated sacculi through electron cryotomography. In the Gram-negative sacculus, a single layer of glycans lie parallel to the cell surface, roughly perpendicular to the long axis of the cell, encircling the cell in a disorganized hoop-like fashion.
Topics: Caulobacter crescentus; Cell Wall; Escherichia coli; Models, Molecular; Peptidoglycan
PubMed: 19033194
DOI: 10.1073/pnas.0808035105 -
Journal of the American Chemical Society Oct 2017Bacteria have the natural ability to install protective postsynthetic modifications onto its bacterial peptidoglycan (PG), the coat woven into bacterial cell wall....
Bacteria have the natural ability to install protective postsynthetic modifications onto its bacterial peptidoglycan (PG), the coat woven into bacterial cell wall. Peptidoglycan O-acetyltransferase B (PatB) catalyzes the O-acetylation of PG in Gram (-) bacteria, which aids in bacterial survival, as it prevents autolysins such as lysozyme from cleaving the PG. We explored the mechanistic details of PatB's acetylation function and determined that PatB has substrate specificity for bioorthgonal short N-acetyl cysteamine (SNAc) donors. A variety of functionality including azides and alkynes were installed on tri-N-acetylglucosamine (NAG), a PG mimic, as well as PG isolated from various Gram (+) and Gram (-) bacterial species. The bioorthogonal modifications protect the isolated PG against lysozyme degradation in vitro. We further demonstrate that this postsynthetic modification of PG can be extended to use click chemistry to fluorescently label the mature PG in whole bacterial cells of Bacillus subtilis. Modifying PG postsynthetically can aid in the development of antibiotics and immune modulators by expanding the understanding of how PG is processed by lytic enzymes.
Topics: Acetyltransferases; Cysteamine; Gram-Negative Bacteria; Gram-Positive Bacteria; Molecular Structure; Peptidoglycan
PubMed: 28898061
DOI: 10.1021/jacs.7b06820 -
Microbiology Spectrum Oct 2022The cell wall peptidoglycan of bacteria is essential for their survival and shape development. The penicillin-binding proteins (PBPs) are responsible for the terminal...
The cell wall peptidoglycan of bacteria is essential for their survival and shape development. The penicillin-binding proteins (PBPs) are responsible for the terminal stage of peptidoglycan assembly. It has been shown that PBPC, a member of class A high-molecular-weight PBP, played an important role in morphology maintenance and stress response in Clavibacter michiganensis. Here, we reported the stress response strategies under viable but nonculturable (VBNC) state and revealed the regulation of peptidoglycan assembly by PBPC in C. michiganensis cells. Using atomic force microscopy imaging, we found that peptidoglycan of C. michiganensis cells displayed a relatively smooth and dense surface, whereas was characterized by a "ridge-and-groove" surface, which was more distinctive after Cu treatment. The peptidoglycan layer of wild type cells exhibited a significant increase in thickness and slight increase in cross-linkage following Cu treatment. Compared with wild type, the thickness and cross-linkage of peptidoglycan decreased during log phase in cells, but the peptidoglycan cross-linkage increased significantly under Cu stress, while the thickness did not change. It is noteworthy that the above changes in the peptidoglycan layer resulted in a significant increase in the accumulation of amylase and exopolysaccharide in . This study elucidates the role of PBPC in Gram-positive rod-shaped plant pathogenic bacterium in response to environmental stimuli by regulating the assembling of cell wall peptidoglycan, which is significant in understanding the survival of C. michiganensis under stress and the field epidemiology of tomato bacterial canker disease. Peptidoglycan of cell walls in bacteria is a cross-linked and meshlike scaffold that provides strength to withstand the external pressure. The increased cross-linkage in peptidoglycan and altered structure in VBNC cells endowed the cell wall more resistant to adversities. Here we systematically evaluated the stress response strategies in Gram-positive rod-shaped bacterium C. michiganensis log phase cells and revealed a significant increase of peptidoglycan thickness and slight increase of cross-linkage after Cu treatment. Most strikingly, knocking-out of PBPC leads to a significant increase in cross-linking of peptidoglycan in response to Cu treatment. Understanding the stress resistance mechanism and survival strategy of phytopathogenic bacteria is the basis of exploring bacterial physiology and disease epidemiology.
Topics: Peptidoglycan; Penicillin-Binding Proteins; Protein C; Cell Wall; Amylases
PubMed: 36040162
DOI: 10.1128/spectrum.01816-22 -
BMC Genomics May 2016The efficacy of antibiotics against bacterial infections is decreasing due to the development of resistance in bacteria, and thus, there is a need to search for...
BACKGROUND
The efficacy of antibiotics against bacterial infections is decreasing due to the development of resistance in bacteria, and thus, there is a need to search for potential alternatives to antibiotics. In this scenario, peptidoglycan hydrolases can be used as alternate antibacterial agents due to their unique property of cleaving peptidoglycan cell wall present in both gram-positive and gram-negative bacteria. Along with a role in maintaining overall peptidoglycan turnover in a cell and in daughter cell separation, peptidoglycan hydrolases also play crucial role in bacterial pathophysiology requiring development of a computational tool for the identification and classification of novel peptidoglycan hydrolases from genomic and metagenomic data.
RESULTS
In this study, the known peptidoglycan hydrolases were divided into multiple classes based on their site of action and were used for the development of a computational tool 'HyPe' for identification and classification of novel peptidoglycan hydrolases from genomic and metagenomic data. Various classification models were developed using amino acid and dipeptide composition features by training and optimization of Random Forest and Support Vector Machines. Random Forest multiclass model was selected for the development of HyPe tool as it showed up to 71.12 % sensitivity, 99.98 % specificity, 99.55 % accuracy and 0.80 MCC in four different classes of peptidoglycan hydrolases. The tool was validated on 24 independent genomic datasets and showed up to 100 % sensitivity and 0.94 MCC. The ability of HyPe to identify novel peptidoglycan hydrolases was also demonstrated on 24 metagenomic datasets.
CONCLUSIONS
The present tool helps in the identification and classification of novel peptidoglycan hydrolases from complete genomic or metagenomic ORFs. To our knowledge, this is the only tool available for the prediction of peptidoglycan hydrolases from genomic and metagenomic data.
AVAILABILITY
http://metagenomics.iiserb.ac.in/hype/ and http://metabiosys.iiserb.ac.in/hype/ .
Topics: Anti-Bacterial Agents; Cell Wall; Hydrolysis; Machine Learning; Metagenomics; Models, Statistical; N-Acetylmuramoyl-L-alanine Amidase; Open Reading Frames; Peptidoglycan; Reproducibility of Results; Web Browser
PubMed: 27229861
DOI: 10.1186/s12864-016-2753-8