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MBio Oct 2023Penicillin-binding proteins (PBPs) are essential for proper bacterial cell division and morphogenesis. The genome of encodes for two class B PBPs (PBP2x and 2b), which...
Penicillin-binding proteins (PBPs) are essential for proper bacterial cell division and morphogenesis. The genome of encodes for two class B PBPs (PBP2x and 2b), which are required for the assembly of the peptidoglycan framework and three class A PBPs (PBP1a, 1b and 2a), which remodel the peptidoglycan mesh during cell division. Therefore, their activities should be finely regulated in space and time to generate the pneumococcal ovoid cell shape. To date, two proteins, CozE and MacP, are known to regulate the function of PBP1a and PBP2a, respectively. In this study, we describe a novel regulator (CopD) that acts on both PBP1a and PBP2b. These findings provide valuable information for understanding bacterial cell division. Furthermore, knowing that ß-lactam antibiotic resistance often arises from PBP mutations, the characterization of such a regulator represents a promising opportunity to develop new strategies to resensitize resistant strains.
Topics: Streptococcus pneumoniae; Peptidoglycan; Penicillin-Binding Proteins; Lactams; Mutation; Bacterial Proteins; Microbial Sensitivity Tests; Peptidyl Transferases
PubMed: 37728370
DOI: 10.1128/mbio.01411-23 -
Communications Biology Mar 2024Peptidoglycan polymerases, enterobacterial common antigen polymerases, O-antigen ligases, and other bacterial polysaccharide polymerases (BP-Pols) are...
Peptidoglycan polymerases, enterobacterial common antigen polymerases, O-antigen ligases, and other bacterial polysaccharide polymerases (BP-Pols) are glycosyltransferases (GTs) that build bacterial surface polysaccharides. These integral membrane enzymes share the particularity of using diphospholipid-activated sugars and were previously missing in the carbohydrate-active enzymes database (CAZy; www.cazy.org ). While the first three classes formed well-defined families of similar proteins, the sequences of BP-Pols were so diverse that a single family could not be built. To address this, we developed a new clustering method using a combination of a sequence similarity network and hidden Markov model comparisons. Overall, we have defined 17 new GT families including 14 of BP-Pols. We find that the reaction stereochemistry appears to be conserved in each of the defined BP-Pol families, and that the BP-Pols within the families transfer similar sugars even across Gram-negative and Gram-positive bacteria. Comparison of the new GT families reveals three clans of distantly related families, which also conserve the reaction stereochemistry.
Topics: Glycosyltransferases; Sugars; Cluster Analysis; Peptidoglycan
PubMed: 38454040
DOI: 10.1038/s42003-024-05930-2 -
STAR Protocols Mar 2022Postbiotics cooperate to influence immune and metabolic outcomes in the host. Here we describe a protocol for assessment of blood glucose control following acute...
Postbiotics cooperate to influence immune and metabolic outcomes in the host. Here we describe a protocol for assessment of blood glucose control following acute administration of lipopolysaccharide (LPS) and peptidoglycan (PGN) in mice. This protocol can be adapted for testing a broad range of microbial molecules and ligands for host immune receptors. Experience with mouse handling is required. For complete details on the use and execution of this protocol, please refer to Anhê et al. (2021) and Cavallari et al. (2017).
Topics: Animals; Cell Wall; Lipopolysaccharides; Mice; Peptidoglycan
PubMed: 35072117
DOI: 10.1016/j.xpro.2021.101098 -
Microbiology Spectrum Feb 2023Vibrio cholerae can utilize a type VI secretion system (T6SS) to increase its intra- and interspecies competition. However, much still remains to be understood about the...
Vibrio cholerae can utilize a type VI secretion system (T6SS) to increase its intra- and interspecies competition. However, much still remains to be understood about the underlying mechanism of this intraspecies competition. In this study, we isolated an environmental V. cholerae strain E1 that lacked the typical virulence factors toxin-coregulated pilus and cholera toxin and that encoded a functional T6SS. We identified an evolved VgrG3 variant with a predicted C-terminal pesticin-like domain in V. cholerae E1, designated VgrG3. Using heterologous expression, protein secretion, and peptidoglycan-degrading assays, we demonstrated that VgrG3 is a T6SS-dependent effector harboring cell wall muramidase activity and that its toxicity can be neutralized by cognate immunity protein TsiV3. Site-directed mutagenesis proved that the aspartic acid residue at position 867 is crucial for VgrG3-mediated antibacterial activity. Bioinformatic analysis showed that genes encoding VgrG3-like homologs are distributed in species, are linked with T6SS structural genes and auxiliary genes, and the gene pair of V. cholerae probably evolved from Vibrio anguillarum and Vibrio fluvialis via homologous recombination. Through a time-lapse microscopy assay, we directly determined that cells accumulating VgrG3 disrupted bacterial division, while the cells continued to increase in size until the loss of membrane potential and cell wall breakage and finally burst. The results of the competitive killing assay showed that VgrG3 contributes to V. cholerae interspecies competition. Collectively, our study revealed a novel T6SS E-I pair representing a new T6SS toxin family which allows V. cholerae to gain dominance within polymicrobial communities by T6SS. The type VI secretion system used by a broad range of Gram-negative bacteria delivers toxic proteins to target adjacent eukaryotic and prokaryotic cells. Diversification of effector proteins determines the complex bacterium-bacterium interactions and impacts the health of hosts and environmental ecosystems in which bacteria reside. This work uncovered an evolved valine-glycine repeat protein G3, carrying a C-terminal pesticin-like domain (VgrG3), which has been suggested to harbor cell wall hydrolase activity and is able to affect cell division and the integrity of cell wall structure. Pesticin-like homologs constitute a family of T6SS-associated effectors targeting bacterial peptidoglycan which are distributed in species, and genetic loci of them are linked with T6SS structural genes and auxiliary genes. T6SS-delivered VgrG3 mediated broad-spectrum antibacterial activity for several microorganisms tested, indicating that VgrG3-mediated antimicrobial activity is capable of conferring bacteria a competitive advantage over competitors in the same niches.
Topics: Type VI Secretion Systems; Vibrio cholerae; Peptidoglycan; Ecosystem; Bacterial Proteins; Anti-Bacterial Agents; Cell Wall
PubMed: 36625646
DOI: 10.1128/spectrum.04267-22 -
The Journal of Biological Chemistry Mar 2020Bacteria account for 1000-fold more biomass than humans. They vary widely in shape and size. The morphological diversity of bacteria is due largely to the different... (Review)
Review
Bacteria account for 1000-fold more biomass than humans. They vary widely in shape and size. The morphological diversity of bacteria is due largely to the different peptidoglycan-based cell wall structures that encase bacterial cells. Although the basic structure of peptidoglycan is highly conserved, consisting of long glycan strands that are cross-linked by short peptide chains, the mature cell wall is chemically diverse. Peptidoglycan hydrolases and cell wall-tailoring enzymes that regulate glycan strand length, the degree of cross-linking, and the addition of other modifications to peptidoglycan are central in determining the final architecture of the bacterial cell wall. Historically, it has been difficult to biochemically characterize these enzymes that act on peptidoglycan because suitable peptidoglycan substrates were inaccessible. In this review, we discuss fundamental aspects of bacterial cell wall synthesis, describe the regulation and diverse biochemical and functional activities of peptidoglycan hydrolases, and highlight recently developed methods to make and label defined peptidoglycan substrates. We also review how access to these substrates has now enabled biochemical studies that deepen our understanding of how bacterial cell wall enzymes cooperate to build a mature cell wall. Such improved understanding is critical to the development of new antibiotics that disrupt cell wall biogenesis, a process essential to the survival of bacteria.
Topics: Bacteria; Bacterial Proteins; Cell Wall; N-Acetylmuramoyl-L-alanine Amidase; Peptidoglycan; Protein Structure, Tertiary; Staphylococcus aureus; Substrate Specificity
PubMed: 31974163
DOI: 10.1074/jbc.REV119.010155 -
ACS Chemical Biology Dec 2022Bacterial cell wall peptidoglycan is essential for viability, and its synthesis is targeted by antibiotics, including penicillin. To determine how peptidoglycan...
Bacterial cell wall peptidoglycan is essential for viability, and its synthesis is targeted by antibiotics, including penicillin. To determine how peptidoglycan homeostasis controls cell architecture, growth, and division, we have developed novel labeling approaches. These are compatible with super-resolution fluorescence microscopy to examine peptidoglycan synthesis, hydrolysis, and the localization of the enzymes required for its biosynthesis (penicillin binding proteins (PBPs)). Synthesis of a cephalosporin-based fluorescent probe revealed a pattern of PBPs at the septum during division, supporting a model of dispersed peptidoglycan synthesis. Metabolic and hydroxylamine-based probes respectively enabled the synthesis of glycan strands and associated reducing termini of the peptidoglycan to be mapped. Foci and arcs of reducing termini appear as a result of both synthesis of glycan strands and glucosaminidase activity of the major peptidoglycan hydrolase, SagB. Our studies provide molecular level details of how essential peptidoglycan dynamics are controlled during growth and division.
Topics: Humans; Staphylococcus aureus; Peptidoglycan; Cell Wall; Penicillin-Binding Proteins; Staphylococcal Infections; Microscopy, Fluorescence; Homeostasis; Bacterial Proteins
PubMed: 36414253
DOI: 10.1021/acschembio.2c00741 -
Biochemical Society Transactions Dec 2019Bacterial cell shape is a key trait governing the extracellular and intracellular factors of bacterial life. Rod-like cell shape appears to be original which implies... (Review)
Review
Bacterial cell shape is a key trait governing the extracellular and intracellular factors of bacterial life. Rod-like cell shape appears to be original which implies that the cell wall, division, and rod-like shape came together in ancient bacteria and that the myriad of shapes observed in extant bacteria have evolved from this ancestral shape. In order to understand its evolution, we must first understand how this trait is actively maintained through the construction and maintenance of the peptidoglycan cell wall. The proteins that are primarily responsible for cell shape are therefore the elements of the bacterial cytoskeleton, principally FtsZ, MreB, and the penicillin-binding proteins. MreB is particularly relevant in the transition between rod-like and spherical cell shape as it is often (but not always) lost early in the process. Here we will highlight what is known of this particular transition in cell shape and how it affects fitness before giving a brief perspective on what will be required in order to progress the field of cell shape evolution from a purely mechanistic discipline to one that has the perspective to both propose and to test reasonable hypotheses regarding the ecological drivers of cell shape change.
Topics: Bacteria; Bacterial Proteins; Biological Evolution; Cell Wall; Cytoskeleton; Peptidoglycan; RNA, Bacterial; RNA, Ribosomal, 16S
PubMed: 31829405
DOI: 10.1042/BST20180634 -
Current Opinion in Microbiology Apr 2021Synthesis of the bacterial cell envelope requires a regulated partitioning of resources from central metabolism. Here, we consider the key metabolic junctions that... (Review)
Review
Synthesis of the bacterial cell envelope requires a regulated partitioning of resources from central metabolism. Here, we consider the key metabolic junctions that provide the precursors needed to assemble the cell envelope. Peptidoglycan synthesis requires redirection of a glycolytic intermediate, fructose-6-phosphate, into aminosugar biosynthesis by the highly regulated branchpoint enzyme GlmS. MurA directs the downstream product, UDP-GlcNAc, specifically into peptidoglycan synthesis. Other shared resources required for cell envelope synthesis include the isoprenoid carrier lipid undecaprenyl phosphate and amino acids required for peptidoglycan cross-bridges. Assembly of the envelope requires a sharing of limited resources between competing cellular pathways and may additionally benefit from scavenging of metabolites released from neighboring cells or the formation of symbiotic relationships with a host.
Topics: Cell Membrane; Cell Wall; Peptidoglycan
PubMed: 33581378
DOI: 10.1016/j.mib.2021.01.015 -
Current Opinion in Pharmacology Oct 2019Mounting evidence indicates that gut microbiota exerts a broad range of effects on host physiology and development beyond the gastrointestinal tract, including the... (Review)
Review
Mounting evidence indicates that gut microbiota exerts a broad range of effects on host physiology and development beyond the gastrointestinal tract, including the modulation of brain development. However, the mechanisms mediating the interactions between the microbiota and the developing brain are still poorly understood. Pattern recognition receptors of the innate immune system that recognize microbial products, such as peptidoglycans have emerged as potential key regulators of gut microbiome-brain interactions. Peptidoglycan-sensing molecules are expressed in the placenta and brain during specific time windows of development. Moreover, peptidoglycans are ubiquitously present in circulation and can cross the blood brain barrier. This review brings together the current evidence supporting a broad function of peptidoglycans well beyond host's immunity, extending to neurodevelopment and behavior.
Topics: Animals; Bacteria; Brain; Humans; Microbiota; Peptidoglycan
PubMed: 31557694
DOI: 10.1016/j.coph.2019.08.003 -
Biochemistry Apr 2023Some bacteria survive in nutrient-poor environments and resist killing by antimicrobials by forming spores. The cortex layer of the peptidoglycan cell wall that...
Some bacteria survive in nutrient-poor environments and resist killing by antimicrobials by forming spores. The cortex layer of the peptidoglycan cell wall that surrounds mature spores contains a unique modification, muramic-δ-lactam, that is essential for spore germination and outgrowth. Two proteins, the amidase CwlD and the deacetylase PdaA, are required for muramic-δ-lactam synthesis in cells, but their combined ability to generate muramic-δ-lactam has not been directly demonstrated. Here we report an in vitro reconstitution of cortex peptidoglycan biosynthesis, and we show that CwlD and PdaA together are sufficient for muramic-δ-lactam formation. Our method enables characterization of the individual reaction steps, and we show for the first time that PdaA has transamidase activity, catalyzing both the deacetylation of -acetylmuramic acid and cyclization of the product to form muramic-δ-lactam. This activity is unique among peptidoglycan deacetylases and is notable because it may involve the direct ligation of a carboxylic acid with a primary amine. Our reconstitution products are nearly identical to the cortex peptidoglycan found in spores, and we expect that they will be useful substrates for future studies of enzymes that act on the spore cortex.
Topics: Spores, Bacterial; Peptidoglycan; Bacteria; Cell Wall; Lactams; Bacterial Proteins
PubMed: 37021938
DOI: 10.1021/acs.biochem.3c00100