Peptidoglycan ormurein is a unique large macromolecule, apolysaccharide, consisting of sugars andamino acids that forms a mesh-like layer (sacculus) that surrounds thebacterial cytoplasmic membrane.[1] The sugar component consists of alternating residues of β-(1,4) linkedN-acetylglucosamine (NAG) andN-acetylmuramic acid (NAM). Attached to theN-acetylmuramic acid is anoligopeptide chain made of three to five amino acids. The peptide chain can be cross-linked to the peptide chain of another strand forming the 3D mesh-like layer.[1][2] Peptidoglycan serves a structural role in the bacterial cell wall, giving structural strength, as well as counteracting theosmotic pressure of thecytoplasm. This repetitive linking results in a dense peptidoglycan layer which is critical for maintaining cell form and withstanding high osmotic pressures, and it is regularly replaced by peptidoglycan production. Peptidoglycan hydrolysis and synthesis are two processes that must occur in order for cells to grow and multiply, a technique carried out in three stages: clipping of current material, insertion of new material, and re-crosslinking of existing material to new material.[3]
The peptidoglycan layer is substantially thicker ingram-positive bacteria (20 to 80 nanometers) than ingram-negative bacteria (7 to 8 nanometers).[4] Depending on pH growth conditions, the peptidoglycan forms around 40 to 90% of thecell wall'sdry weight of gram-positive bacteria but only around 10% of gram-negative strains. Thus, presence of high levels of peptidoglycan is the primary determinant of the characterisation of bacteria as gram-positive.[5] In gram-positive strains, it is important in attachment roles andserotyping purposes.[6] For both gram-positive and gram-negative bacteria, particles of approximately 2 nm can pass through the peptidoglycan.[7]
It is difficult to tell whether an organism is gram-positive or gram-negative using a microscope;Gram staining, created byHans Christian Gram in 1884, is required. The bacteria are stained with the dyes crystal violet andsafranin. Gram positive cells are purple after staining, while Gram negative cells stain pink.[8]
The peptidoglycan layer within the bacterial cell wall is acrystal lattice structure formed from linear chains of two alternating aminosugars, namelyN-acetylglucosamine (GlcNAc or NAG) andN-acetylmuramic acid (MurNAc or NAM). The alternating sugars are connected by a β-(1,4)-glycosidic bond. Each MurNAc is attached to a short (4- to 5-residue)amino acid chain, containingL-alanine,D-glutamic acid,meso-diaminopimelic acid, andD-alanine in the case ofEscherichia coli (a gram-negative bacterium); orL-alanine,D-glutamine,L-lysine, andD-alanine with a 5-glycine interbridge between tetrapeptides in the case ofStaphylococcus aureus (a gram-positive bacterium). Peptidoglycan is one of the most important sources ofD-amino acids in nature.[citation needed]
By enclosing the inner membrane, the peptidoglycan layer protects the cell fromlysis caused by theturgor pressure of the cell. When the cell wall grows, it retains its shape throughout its life, so a rod shape will remain a rod shape, and a spherical shape will remain a spherical shape for life. This happens because the freshly added septal material of synthesis transforms into a hemispherical wall for the offspring cells.[9]
Cross-linking betweenamino acids in different linear amino sugar chains occurs with the help of the enzymeDD-transpeptidase and results in a 3-dimensional structure that is strong and rigid. The specific amino acid sequence and molecular structure vary with the bacterialspecies.[10]
The different peptidoglycan types of bacterial cell walls and their taxonomic implications have been described.[11]Archaea (domainArchaea)[12] do not contain peptidoglycan (murein).[13] Some Archaea containpseudopeptidoglycan (pseudomurein, see below).[14]
Peptidoglycan is involved inbinary fission during bacterial cell reproduction.L-form bacteria andmycoplasmas, both lacking peptidoglycan cell walls, do not proliferate by binary fission, but by abudding mechanism.[15][16]
In the course of early evolution, the successive development of boundaries (membranes, walls) protecting first structures of life against their environment must have been essential for the formation of the first cells (cellularisation).
The invention of rigid peptidoglycan (murein) cell walls in bacteria (domainBacteria[12]) was probably the prerequisite for their survival, extensive radiation and colonisation of virtually all habitats of the geosphere and hydrosphere.[17][18]
The peptidoglycan monomers are synthesized in thecytosol and are then attached to a membrane carrierbactoprenol. Bactoprenol transports peptidoglycan monomers across the cell membrane where they are inserted into the existing peptidoglycan.[19]
Each of these reactions requires the energy source ATP.[20] This is all referred to as Stage one.
Stage two occurs in the cytoplasmic membrane. It is in the membrane where a lipid carrier calledbactoprenol carries peptidoglycan precursors through the cell membrane.
In somearchaea, i.e. members of theMethanobacteriales and in the genusMethanopyrus,pseudopeptidoglycan (pseudomurein) has been found.[14] In pseudopeptidoglycan the sugar residues are β-(1,3) linkedN-acetylglucosamine andN-acetyltalosaminuronic acid. This makes the cell walls of such archaea insensitive tolysozyme.[23] The biosynthesis of pseudopeptidoglycan has been described.[24]
Peptidoglycan recognition is an evolutionarily conserved process.[25] The overall structure is similar between bacterial species, but various modifications can increase the diversity. These include modifications of the length of sugar polymers, modifications in the sugar structures, variations in cross-linking or substitutions of amino acids (primarily at the third position).[25][26] The aim of these modifications is to alter the properties of the cell wall, which plays a vital role inpathogenesis.[25]
Peptidoglycans can be degraded by several enzymes (lysozyme, glucosaminidase,endopeptidase...[25]), producing immunostimulatory fragments (sometimes called muropeptides[27]) that are critical for mediatinghost-pathogen interactions.[26] These include MDP (muramyl dipeptide), NAG (N-acetylglucosamine) or iE-DAP (γ-d-glutamyl-meso-diaminopimelic acid).[25][27]
Peptidoglycan fromintestinal bacteria (both pathogens and commensals) crosses the intestinal barrier even under physiological conditions.[27] Mechanisms through which peptidoglycan or its fragments enter the host cells can be direct (carrier-independent) or indirect (carrier-dependent), and they are either bacteria-mediated (secretion systems,membrane vesicles) or host cell-mediated (receptor-mediated, peptide transporters).[27]Bacterial secretion systems are protein complexes used for the delivery of virulence factors across the bacterial cell envelope to the exterior environment.[28] Intracellular bacterial pathogens invade eukaryotic cells (which may lead to the formation ofphagolysosomes and/orautophagy activation), or bacteria may be engulfed byphagocytes (macrophages,monocytes,neutrophils...). The bacteria-containingphagosome may then fuse withendosomes andlysosomes, leading to degradation of bacteria and generation of polymeric peptidoglycan fragments and muropeptides.[27]
Innate immune system senses intact peptidoglycan and peptidoglycan fragments using numerous PRRs (pattern recognition receptors) that are secreted, expressed intracellularly or expressed on the cell surface.[25]
PGLYRPs are conserved frominsects tomammals.[27] Mammals produce four secreted soluble peptidoglycan recognition proteins (PGLYRP-1,PGLYRP-2,PGLYRP-3 andPGLYRP-4) that recognize muramyl pentapeptide or tetrapeptide.[25] They can also bind toLPS and other molecules by using binding sites outside of the peptidoglycan-binding groove.[28] After recognition of peptidoglycan, PGLYRPs activatepolyphenol oxidase (PPO) molecules, Toll, or immune deficiency (IMD) signalling pathways. That leads to production ofantimicrobial peptides (AMPs).[28]
Each of the mammalian PGLYRPs display unique tissue expression patterns. PGLYRP-1 is mainly expressed in the granules ofneutrophils andeosinophils.[25] PGLYRP-3 and 4 are expressed by several tissues such as skin, sweat glands, eyes or the intestinal tract.[27] PGLYRP-1, 3 and 4 form disulphide-linkedhomodimers andheterodimers essential for their bactericidal activity.[27] Their binding to bacterial cell wall peptidoglycans can induce bacterial cell death by interaction with various bacterial transcriptional regulatory proteins.[25] PGLYRPs are likely to assist in bacterial killing by cooperating with other PRRs to enhance recognition of bacteria by phagocytes.[25]
PGLYRP-2 is primarily expressed by theliver and secreted into the circulation.[25] Also, its expression can be induced in skinkeratinocytes, oral and intestinalepithelial cells.[27] In contrast with the other PGLYRPs, PGLYRP-2 has no direct bactericidal activity. It possesses peptidoglycan amidase activity, it hydrolyses the lactyl-amide bond between theMurNAc and the first amino acid of the stem peptide of peptidoglycan.[25][27] It is proposed, that the function of PGLYRP-2 is to prevent over-activation of the immune system andinflammation-induced tissue damage in response toNOD2 ligands (see below), as these muropeptides can no longer be recognized by NOD2 upon separation of the peptide component from MurNAc.[27] Growing evidence suggests that peptidoglycan recognition protein family members play a dominant role in thetolerance of intestinal epithelial cells toward the commensal microbiota.[28][29] It has been demonstrated that expression of PGLYRP-2 and 4 can influence the composition of the intestinalmicrobiota.[28]
Recently, it has been discovered, that PGLYRPs (and also NOD-like receptors and peptidoglycan transporters) are highly expressed in the developing mousebrain.[30] PGLYRP-2 and is highly expressed inneurons of several brain regions including theprefrontal cortex,hippocampus, andcerebellum, thus indicating potential direct effects of peptidoglycan on neurons. PGLYRP-2 is highly expressed also in the cerebral cortex of young children, but not in most adult cortical tissues. PGLYRP-1 is also expressed in the brain and continues to be expressed into adulthood.[30]
Probably the most well-known receptors of peptidoglycan are theNOD-like receptors (NLRs), mainlyNOD1 andNOD2. The NOD1 receptor is activated after iE-DAP (γ-d-glutamyl-meso-diaminopimelic acid) binding, while NOD2 recognizes MDP (muramyl dipeptide), by theirLRR domains.[28] Activation leads to self-oligomerization, resulting in activation of two signalling cascades. One triggers activation ofNF-κB (through RIP2,TAK1 andIKK[31]), second leads toMAPK signalling cascade. Activation of these pathways induces production of inflammatorycytokines andchemokines.[25]
NOD1 is expressed by diverse cell types, including myeloid phagocytes, epithelial cells[25] and neurons.[30] NOD2 is expressed in monocytes and macrophages, epithelial intestinal cells,Paneth cells,dendritic cells,osteoblasts, keratinocytes and other epithelial cell types.[27] Ascytosolic sensors, NOD1 and NOD2 must either detect bacteria that enter the cytosol, or peptidoglycan must be degraded to generate fragments that must be transported into the cytosol for these sensors to function.[25]
Recently, it was demonstrated thatNLRP3 is activated by peptidoglycan, through a mechanism that is independent of NOD1 and NOD2.[27] In macrophages, N-acetylglucosamine generated by peptidoglycan degradation was found to inhibit hexokinase activity and induce its release from themitochondrialmembrane. It promotes NLRP3inflammasome activation through a mechanism triggered by increased mitochondrial membrane permeability.[27]
NLRP1 is also considered as a cytoplasmic sensor of peptidoglycan. It can sense MDP and promoteIL-1 secretion through binding NOD2.[28][26]
C-type lectins are a diverse superfamily of mainly Ca2+-dependent proteins that bind a variety ofcarbohydrates (including the glycan skeleton of peptidoglycan), and function as innate immune receptors.[27] CLR proteins that bind to peptidoglycan include MBL (mannose binding lectin),ficolins,Reg3A (regeneration gene family protein 3A) and PTCLec1.[28] In mammals, they initiate thelectin-pathway of thecomplement cascade.[27]
The role ofTLRs in direct recognition of peptidoglycan is controversial.[25] In some studies, has been reported that peptidoglycan is sensed byTLR2.[32] But this TLR2-inducing activity could be due to cell walllipoproteins andlipoteichoic acids that commonly co-purify with peptidoglycan. Also variation in peptidoglycan structure in bacteria from species to species may contribute to the differing results on this topic.[25][27]
Peptidoglycan is immunologically active, which can stimulate immune cells to increase the expression of cytokines and enhance antibody-dependent specific response when combined withvaccine or asadjuvant alone.[28] MDP, which is the basic unit of peptidoglycan, was initially used as the active component ofFreund's adjuvant.[28] Peptidoglycan fromStaphylococcus aureus was used as a vaccine to protect mice, showing that after vaccine injection for 40 weeks, the mice survived fromS. aureus challenge at an increasedlethal dose.[33]
Someantibacterial drugs such aspenicillin interfere with the production of peptidoglycan by binding to bacterial enzymes known aspenicillin-binding proteins orDD-transpeptidases.[6] Penicillin-binding proteins form the bonds between oligopeptide crosslinks in peptidoglycan. For a bacterial cell to reproduce throughbinary fission, more than a million peptidoglycan subunits (NAM-NAG+oligopeptide) must be attached to existing subunits.[34] Mutations in genes coding for transpeptidases that lead to reduced interactions with an antibiotic are a significant source of emergingantibiotic resistance.[35] Since peptidoglycan is also lacking in L-form bacteria and in mycoplasmas, both are resistant against penicillin.
Other steps of peptidoglycan synthesis can also be targeted. The topical antibioticbacitracin targets the utilization ofC55-isoprenyl pyrophosphate.Lantibiotics, which includes the food preservativenisin, attack lipid II.[36]
Lysozyme, which is found in tears and constitutes part of the body'sinnate immune system exerts its antibacterial effect by breaking the β-(1,4)-glycosidic bonds in peptidoglycan (see above). Lysozyme is more effective in acting againstgram-positive bacteria, in which the peptidoglycan cell wall is exposed, than againstgram-negative bacteria, which have an outer layer ofLPS covering the peptidoglycan layer.[31] Several bacterial peptidoglycan modifications can result in resistance to degradation by lysozyme. Susceptibility of bacteria to degradation is also considerably affected by exposure toantibiotics. Exposed bacteria synthesize peptidoglycan that contains shorter sugar chains that are poorly crosslinked and this peptidoglycan is then more easily degraded by lysozyme.[28]
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