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Nature Sep 2022Bacterial cell wall components provide various unique molecular structures that are detected by pattern recognition receptors (PRRs) of the innate immune system as...
Bacterial cell wall components provide various unique molecular structures that are detected by pattern recognition receptors (PRRs) of the innate immune system as non-self. Most bacterial species form a cell wall that consists of peptidoglycan (PGN), a polymeric structure comprising alternating amino sugars that form strands cross-linked by short peptides. Muramyl dipeptide (MDP) has been well documented as a minimal immunogenic component of peptidoglycan. MDP is sensed by the cytosolic nucleotide-binding oligomerization domain-containing protein 2 (NOD2). Upon engagement, it triggers pro-inflammatory gene expression, and this functionality is of critical importance in maintaining a healthy intestinal barrier function. Here, using a forward genetic screen to identify factors required for MDP detection, we identified N-acetylglucosamine kinase (NAGK) as being essential for the immunostimulatory activity of MDP. NAGK is broadly expressed in immune cells and has previously been described to contribute to the hexosamine biosynthetic salvage pathway. Mechanistically, NAGK functions upstream of NOD2 by directly phosphorylating the N-acetylmuramic acid moiety of MDP at the hydroxyl group of its C6 position, yielding 6-O-phospho-MDP. NAGK-phosphorylated MDP-but not unmodified MDP-constitutes an agonist for NOD2. Macrophages from mice deficient in NAGK are completely deficient in MDP sensing. These results reveal a link between amino sugar metabolism and innate immunity to bacterial cell walls.
Topics: Acetylmuramyl-Alanyl-Isoglutamine; Animals; Bacteria; Cell Wall; Hexosamines; Immunity, Innate; Macrophages; Mice; Nod2 Signaling Adaptor Protein; Peptidoglycan; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor)
PubMed: 36002575
DOI: 10.1038/s41586-022-05125-x -
Journal of Bacteriology Sep 2020Uropathogenic (UPEC) is the leading cause of human urinary tract infections (UTIs), and many patients experience recurrent infection after successful antibiotic...
Uropathogenic (UPEC) is the leading cause of human urinary tract infections (UTIs), and many patients experience recurrent infection after successful antibiotic treatment. The source of recurrent infections may be persistent bacterial reservoirs that are in a quiescent state and thus are not susceptible to antibiotics. Here, we show that multiple UPEC strains require a quorum to proliferate with glucose as the carbon source. At low cell density, the bacteria remain viable but enter a quiescent, nonproliferative state. Of the clinical UPEC isolates tested to date, 35% (51/145) enter this quiescent state, including isolates from the recently emerged, multidrug-resistant pandemic lineage ST131 (i.e., strain JJ1886) and isolates from the classic endemic lineage ST73 (i.e., strain CFT073). Moreover, quorum-dependent UPEC quiescence is prevented and reversed by small-molecule proliferants that stimulate colony formation. These proliferation cues include d-amino acid-containing peptidoglycan (PG) tetra- and pentapeptides, as well as high local concentrations of l-lysine and l-methionine. Peptidoglycan fragments originate from the peptidoglycan layer that supports the bacterial cell wall but are released as bacteria grow. These fragments are detected by a variety of organisms, including human cells, other diverse bacteria, and, as we show here for the first time, UPEC. Together, these results show that for UPEC, (i) sensing of PG stem peptide and uptake of l-lysine modulate the quorum-regulated decision to proliferate and (ii) quiescence can be prevented by both intra- and interspecies PG peptide signaling. Uropathogenic (UPEC) is the leading cause of urinary tract infections (UTIs). During pathogenesis, UPEC cells adhere to and infiltrate bladder epithelial cells, where they may form intracellular bacterial communities (IBCs) or enter a nongrowing or slowly growing quiescent state. Here, we show that UPEC strains at low population density enter a reversible, quiescent state by halting division. Quiescent cells resume proliferation in response to sensing a quorum and detecting external signals, or cues, including peptidoglycan tetra- and pentapeptides.
Topics: Anti-Bacterial Agents; Cell Division; Epithelial Cells; Escherichia coli Infections; Humans; Peptidoglycan; Quorum Sensing; Urinary Tract Infections; Uropathogenic Escherichia coli
PubMed: 32778561
DOI: 10.1128/JB.00157-20 -
Nature Microbiology Oct 2022The bacterial division apparatus catalyses the synthesis and remodelling of septal peptidoglycan (sPG) to build the cell wall layer that fortifies the daughter cell...
The bacterial division apparatus catalyses the synthesis and remodelling of septal peptidoglycan (sPG) to build the cell wall layer that fortifies the daughter cell poles. Understanding of this essential process has been limited by the lack of native three-dimensional views of developing septa. Here, we apply state-of-the-art cryogenic electron tomography (cryo-ET) and fluorescence microscopy to visualize the division site architecture and sPG biogenesis dynamics of the Gram-negative bacterium Escherichia coli. We identify a wedge-like sPG structure that fortifies the ingrowing septum. Experiments with strains defective in sPG biogenesis revealed that the septal architecture and mode of division can be modified to more closely resemble that of other Gram-negative (Caulobacter crescentus) or Gram-positive (Staphylococcus aureus) bacteria, suggesting that a conserved mechanism underlies the formation of different septal morphologies. Finally, analysis of mutants impaired in amidase activation (ΔenvC ΔnlpD) showed that cell wall remodelling affects the placement and stability of the cytokinetic ring. Taken together, our results support a model in which competition between the cell elongation and division machineries determines the shape of cell constrictions and the poles they form. They also highlight how the activity of the division system can be modulated to help generate the diverse array of shapes observed in the bacterial domain.
Topics: Amidohydrolases; Cell Division; Cell Shape; Cell Wall; Escherichia coli; Peptidoglycan
PubMed: 36097171
DOI: 10.1038/s41564-022-01210-z -
Microbiology Spectrum Oct 2022Diaminopimelic acid (DAP) is a unique component of the cell wall of Gram-negative bacteria. It is also an important component of organic matter and is widely utilized by...
Diaminopimelic acid (DAP) is a unique component of the cell wall of Gram-negative bacteria. It is also an important component of organic matter and is widely utilized by microbes in the world's oceans. However, neither DAP concentrations nor marine DAP-utilizing microbes have been investigated. Here, DAP concentrations in seawater were measured and the diversity of marine DAP-utilizing bacteria and the mechanisms for their DAP metabolism were investigated. Free DAP concentrations in seawater, from surface to a 5,000 m depth, were found to be between 0.61 μM and 0.96 μM in the western Pacific Ocean. DAP-utilizing bacteria from 20 families in 4 phyla were recovered from the western Pacific seawater and 14 strains were further isolated, in which bacteria were dominant. Based on genomic and transcriptomic analyses combined with gene deletion and activity detection, DAP decarboxylase (LysA), which catalyzes the decarboxylation of DAP to form lysine, was found to be a key and specific enzyme involved in DAP metabolism in the isolated strains. Interrogation of the Oceans database found that most LysA-like sequences (92%) are from , which are widely distributed in multiple habitats. This study provides an insight into DAP metabolism by marine bacteria in the ocean and contributes to our understanding of the mineralization and recycling of DAP by marine bacteria. DAP is a unique component of peptidoglycan in Gram-negative bacterial cell walls. Due to the large number of marine Gram-negative bacteria, DAP is an important component of marine organic matter. However, it remains unclear how DAP is metabolized by marine microbes. This study investigated marine DAP-utilizing bacteria by cultivation and bioinformational analysis and examined the mechanism of DAP metabolism used by marine bacteria. The results demonstrate that bacteria are likely to be an important DAP-utilizing group in the ocean and that DAP decarboxylase is a key enzyme involved in DAP metabolism. This study also sheds light on the mineralization and recycling of DAP driven by bacteria.
Topics: Bacteria; Carboxy-Lyases; Diaminopimelic Acid; Gram-Negative Bacteria; Lysine; Peptidoglycan
PubMed: 36040174
DOI: 10.1128/spectrum.00691-22 -
Molecular Microbiology Mar 2020The peptidoglycan (PG), as the exoskeleton of most prokaryotes, maintains a defined shape and ensures cell integrity against the high internal turgor pressure. These... (Review)
Review
The peptidoglycan (PG), as the exoskeleton of most prokaryotes, maintains a defined shape and ensures cell integrity against the high internal turgor pressure. These important roles have attracted researchers to target PG metabolism in order to control bacterial infections. Most studies, however, have been performed in bacteria grown under laboratory conditions, leading to only a partial view on how the PG is synthetized in natural environments. As a case in point, PG metabolism and its regulation remain poorly understood in symbiotic and pathogenic bacteria living inside eukaryotic cells. This review focuses on the PG metabolism of intracellular bacteria, emphasizing the necessity of more in vivo studies involving the analysis of enzymes produced in the intracellular niche and the isolation of PG from bacteria residing within eukaryotic cells. The review also points to persistent infections caused by some intracellular bacterial pathogens and the extent at which the PG could contribute to establish such physiological state. Based on recent evidences, I speculate on the idea that certain structural features of the PG may facilitate attenuation of intracellular growth. Lastly, I discuss recent findings in endosymbionts supporting a cooperation between host and bacterial enzymes to assemble a mature PG.
Topics: Bacteria; Bacterial Infections; Bacterial Proteins; Cell Wall; Eukaryotic Cells; Host-Pathogen Interactions; Humans; Peptidoglycan; Symbiosis; Virulence
PubMed: 32185832
DOI: 10.1111/mmi.14452 -
Journal of Extracellular Vesicles Apr 2021Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and...
Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each strain. MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host.
Topics: A549 Cells; Autophagy; Cell Wall; Cytokines; DNA; Extracellular Vesicles; HEK293 Cells; Humans; Immunity, Innate; Peptidoglycan; RNA; Receptors, Immunologic; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 2
PubMed: 33815695
DOI: 10.1002/jev2.12080 -
MBio Aug 2022Bacteria in general serve two main tasks: cell growth and division. Both processes include peptidoglycan extension to allow cell expansion and to form the poles of the...
Bacteria in general serve two main tasks: cell growth and division. Both processes include peptidoglycan extension to allow cell expansion and to form the poles of the daughter cells, respectively. The cyanobacterium forms filaments of communicated cells in which the outer membrane and the peptidoglycan sacculus, which is engrossed in the intercellular regions between contiguous cells, are continuous along the filament. During the growth of , peptidoglycan incorporation was weak at the cell periphery. During cell division, midcell peptidoglycan incorporation matched the localization of the divisome, and incorporation persisted in the intercellular septa, even after the division was completed. MreB, MreC, and MreD were located throughout the cell periphery and, in contrast to other bacteria, also to the divisome all along midcell peptidoglycan growth. In mutants bearing inactivated , or genes, which showed conspicuous alterations in the filament morphology, consecutive septal bands of peptidoglycan growth were frequently not parallel to each other and were irregularly spaced along the filament, reproducing the disposition of the Z-ring. Both lateral and septal growth was impaired in strains down-expressing Z-ring components, and MreB and MreD appeared to directly interact with some divisome components. We propose that, in , association with the divisome is a way for localization of MreB, MreC, and MreD at the cell poles, where they regulate lateral, midcell, and septal peptidoglycan growth with the latter being involved in localization and maintenance of the intercellular septal-junction protein structures that mediate cell-cell communication along the filament. Peptidoglycan surrounds the bacterial cell, being essential for the determination of the bacterium-specific morphology and survival. Peptidoglycan growth has been thoroughly investigated in some model rod-shaped bacteria, and more recently some representatives with disparate morphologies became into focus, revealing that patterns of peptidoglycan growth are much more diverse than previously anticipated. forms filaments of communicated cells exhibiting features of multicellular organisms, such as the production of morphogens and coupled circadian oscillations. Here, we showed that presented a distinct pattern of peptidoglycan growth characterized by continuous incorporation of material at the polar intercellular regions, contributing to assembling and maintaining the protein complexes that expand the septal peptidoglycan mediating intercellular molecular exchange in the filament.
Topics: Anabaena; Bacterial Proteins; Cell Division; Cell Wall; Peptidoglycan
PubMed: 35876506
DOI: 10.1128/mbio.01165-22 -
MBio Jul 2019species are obligate intracellular bacteria lacking a classical peptidoglycan sacculus but relying on peptidoglycan synthesis for cytokinesis. While septal...
species are obligate intracellular bacteria lacking a classical peptidoglycan sacculus but relying on peptidoglycan synthesis for cytokinesis. While septal peptidoglycan biosynthesis seems to be regulated by MreB actin and its membrane anchor RodZ rather than FtsZ tubulin in , the mechanism of peptidoglycan remodeling is poorly understood. An amidase conserved in is able to cleave peptide stems in peptidoglycan, but it is not clear how peptidoglycan glycan strands are cleaved since no classical lytic transglycosylase is encoded in chlamydial genomes. However, a protein containing a SpoIID domain, known to possess transglycosylase activity in , is conserved in We show here that the SpoIID homologue of the -related pathogen is a septal peptidoglycan-binding protein. Moreover, we demonstrate that SpoIID acts as a lytic transglycosylase on peptidoglycan and as a muramidase on denuded glycan strands As SpoIID-like proteins are widespread in nonsporulating bacteria, SpoIID might commonly be a septal peptidoglycan remodeling protein in bacteria, including obligate intracellular pathogens, and thus might represent a promising drug target. species are obligate intracellular bacteria and important human pathogens that have a minimal division machinery lacking the proteins that are essential for bacterial division in other species, such as FtsZ. Chlamydial division requires synthesis of peptidoglycan, which forms a ring at the division septum and is rapidly turned over. However, little is known of peptidoglycan degradation, because many peptidoglycan-degrading enzymes are not encoded by chlamydial genomes. Here we show that an homologue of SpoIID, a peptidoglycan-degrading enzyme involved in sporulation of bacteria such as , is expressed in , localizes at the division septum, and degrades peptidoglycan , indicating that SpoIID is not only involved in sporulation but also likely implicated in division of some bacteria.
Topics: Amino Acid Sequence; Bacterial Proteins; Cell Division; Chlamydia; Chlamydia Infections; Chromatography, High Pressure Liquid; Gene Expression; Humans; Peptidoglycan; Protein Binding; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spores, Bacterial
PubMed: 31311880
DOI: 10.1128/mBio.01128-19 -
The Journal of Biological Chemistry Jun 2023Microbial recognition is a key step in regulating the immune signaling pathways of multicellular organisms. Peptidoglycan, a component of the bacterial cell wall,...
Microbial recognition is a key step in regulating the immune signaling pathways of multicellular organisms. Peptidoglycan, a component of the bacterial cell wall, exhibits immune stimulating activity in both plants and animals. Lysin motif domain (LysMD) family proteins are ancient peptidoglycan receptors that function in bacteriophage and plants. This report focuses on defining the role of LysMD-containing proteins in animals. Here, we characterize a novel transmembrane LysMD family protein. Loss-of-function mutations at the lysMD3/4 locus in Drosophila are associated with systemic innate immune activation following challenge, so we refer to this gene as immune active (ima). We show that Ima selectively binds peptidoglycan, is enriched in cell membranes, and is necessary to regulate terminal innate immune effectors through an NF-kB-dependent pathway. Hence, Ima fulfills the key criteria of a peptidoglycan pattern recognition receptor. The human Ima ortholog, hLysMD3, exhibits similar biochemical properties. Together, these findings establish LysMD3/4 as the founding member of a novel family of animal peptidoglycan recognition proteins.
Topics: Animals; Humans; Cell Wall; Drosophila melanogaster; Drosophila Proteins; Immunity, Innate; Peptidoglycan; Membrane Proteins
PubMed: 37116706
DOI: 10.1016/j.jbc.2023.104758 -
Acta Biochimica Polonica Sep 2020The insect immune system is responsible for maintaining the homeostasis of organisms. If the pathogen is able to breach the defensive barriers of the host, cellular and... (Review)
Review
The insect immune system is responsible for maintaining the homeostasis of organisms. If the pathogen is able to breach the defensive barriers of the host, cellular and humoral mechanisms are triggered. Initiation of effective defence response is possible thanks to pathogen-associated molecular patterns, among which peptidoglycan recognition proteins play a prominent role. They recognize pathogen-associated molecular patterns and some of them also have enzymatic activity. The main aim of peptidoglycan recognition proteins is to activate pathways regulating the synthesis of immune peptides. Some of the peptidoglycan recognition proteins are involved in the phagocytosis process, activation of the prophenoloxidase cascade, and regulation of the xenophagy process. The structural diversity and high specificity of peptidoglycan recognition proteins suggests that they can serve many previously unknown functions in insect's systemic response.
Topics: Animals; Carrier Proteins; Catechol Oxidase; Enzyme Precursors; Host-Pathogen Interactions; Immunity, Innate; Insect Proteins; Insecta; Pathogen-Associated Molecular Pattern Molecules; Peptidoglycan; Receptors, Pattern Recognition
PubMed: 32940448
DOI: 10.18388/abp.2020_5345