Bacterial Cell Wall & Capsule Synthesis: Peptidoglycan, Lipid A, & Polysaccharides - Prof., Study notes of Health sciences

Download Bacterial Cell Wall & Capsule Synthesis: Peptidoglycan, Lipid A, & Polysaccharides - Prof. and more Study notes Health sciences in PDF only on Docsity! Bacterial Physiology HSCI 5607 Chapter: 11 Cell Wall and Capsule Biosynthesis Introduction: The cell wall and capsule are important external cell structures which are biosynthesized in the cytoplasm and assembled at the site where they are located. The subunits of the cell wall and capsule are synthesized as a water-soluble precursors in the cytoplasm and are transported across the cell membrane to the site of their assembly. There is no source of energy available for their assembly outside the cell membrane but this energy is obtained by means of specific biochemical reactions taking place outside the membrane. The major constituent of gram-positive bacterial cell wall is peptidoglycan while in gram-negative bacterial cell wall also has an outer membrane which is made up of lipopolysaccharides. Bacterial capsules are usually made up of polysaccharides with few exceptions where they are made up of polypeptides. L-alanine D-glutamate < L-R; 4 D-alanine ; OL CH, L-alanine CH,—CH,—OH L-homoserine CH,—CH,—NH, _ _L-diaminobutyric acid . CH,—CH,—COOH L-glutamic acid (CH,)2—CH,—NH), L-ornithine (CH,)3—CH,—NH) L-lysine COOH (CH2);—CHS LL-DAP and meso-DAP  Cross links: The tetrapeptide chains are cross-linked to each other by peptide bonds between the carboxyl group of the terminal D-alanine of one tetrapeptide and amino group of R3 of another tetrapeptide. Sometime this linkage is direct peptide while in some cases they are linked with one or more amino acid bridge. For example in Staphylococcus aureus five glycine bridge and three L-alanine and one L-threonine in Micrococcus roseus link the tetrapeptides. Synthesis: There are several stages of peptidoglycan synthesis: 1. Synthesis of UDP-aminosugar derivatives in the cytoplasm; 2. The transfer of amino sugars to the lipid carrier in the membrane which carries the precursors across the membrane; 3. Polymerization of the peptidoglycan; 4. Transpeptidation reaction to cross-link the tetrapeptides of the peptidoglycan. a 3 e fe gf i mn Oe 82 g zt __s 4 a 3 e PB oO = =z 6 . ° ° a ‘3 Oo ‘s “hes LS ‘gi QW yg 6 =. Ro Oo =. s 0. 77 * Synthesis of UDP derivatives: HOH cHo CH,0®) é=0 auamae H-C—NH, 9 CoASH ©, i -Ca HO-C-H_—_alotamine 77 Ho-C—H oe < see Hog OH H—C—OH T Brie oe 2 NH H—-C—OH H-C—OH 1. I I fo CH,O(P) cH,0®) CH, fructose-6-P glucosamine-6-P N-acetylglucosamine-6-P 3 CH,OH CH,OH CH,OH °. J g Oo P, OH PP, HO ‘O-()-UMP ‘ PEP Ke Youve , UTP HO ne 5 NH “aS H,C=C—COOH | _ = = < CH, CH; CH, UDP-N-acetylglucosamine- UDP-N-acetylglucosamine N-acetylglucosamine-1-P 3-enoylpyruvylether i NADPH + Ht ‘NADP+ CH,OH o CH,OH HOY O-()-UMP CH. =o V4 s—CH—-C=0 | _ Tejlanine I CH, Tae: TBs UDP-N-acetylmuramic acid Dtenine D-alanine UDP-muramyl-pentapeptide Fig. 11.4 Synthesis of N-acetylglucosamine and N-acetylmuramyl-pentapeptide. Enzymes: 1, glutamine: fructose-6-phosphate aminotransferase; 2, glucosamine phosphate transacetylase; 3, N-acetylglucosamine phosphomutase; 4, UDP-N-acetylglucosamine pyrophosphorylase; 5, enoylpyruvate transferase; 6, UDP-N acetylenolpyruvoylglucosamine reductase. Steps 7: The UDP-N- acetylmuramic acid is converted to the pentapeptide derivative by the sequential additions of L-alanine, D-glutamate, L-R3, and D-alanine-D-alanine by separate enzymes.  Transfer of UDP derivative to the membrane carrier: The lipid carrier bactoprenol not only serve as a carrier for peptidoglycan precursors but also serve as carrier for the other cell wall components, e.g. lipopolysaccharide and teichoic acids. The UDP derivative diffuses to the membrane where the bactoprenol-P attacks the UDP-NAcMur-Pentapep and displaces UMP to form Lipid-PP-NAcMur-Pentapep. Next NAcGlc is transferred from UDP-NAcGlc to NAcMur on the lipid carrier displacing the UDP. The lipid-disaccharide derivative moves to the other side of the membrane facilitated by some unidentified proteins. On the other side of the membrane the lipid-disaccharide is transferred to the growing end of the glycan chain with the displacement of the lipid-PP of the growing end. This reaction is catalyzed by membrane bound enzyme transglycosylase. The lipid-PP released is hydrolyzed to form lipid-P which is recycled for the continuation of the growth while the energy thus liberated is utilized to drive glycosylation reaction. Extension of Glycan Chain: cytoplasm cell membrane P. 1 | P-lipid --PP-lipid L-PP-lipid - ~~ 5 lip-PP A ‘ \ \ \ lip-PP-+ Ly M-G—-M-G ++ lip-PP M-G-M-G—M-G 87 HOH,C-CHOH Fig. 11.9 The structure of KDO. The linkage between KDO residues has been reported to be a glycosidic linkage between a C2 hydroxyl in one KDO with either the C4 or C5 hydroxyl in a second KDO. The linkage to lipid A has been suggested to be from the C2 hydroxyl of KDO to the C6 hydroxyl of lipid A. * Gane gal hep PP-etn man-rha-gal iakgegeekDO | GIcN-GIcN KDO-KDO 5 O-antigen n r nl | ‘|i acids core lipid A Fig. 11.8 The LPS of Salmonella typhimurium. Abbreviations: abe, abequose; man, mannose; rha, rhamnose; gal, galactose; GlcNAc, N-acetylglucosamine; gic, glucose; hep, heptose (L-glycero-D- mannoheptose); KDO, 3-deoxy-D-mannooctulosonic acid; etn, ethanolamine; P, phosphate; GlcN, glucosamine.  Synthesis of Lipid A: The synthesis of lipid A begins with UDP-GlcNAc, which is made from fructose-6-phosphate. Lipid A is synthesized in the cytoplasmic membrane but the beginning steps are catalyzed by three cytoplasmic enzymes. 1. UDP-GlcNAc acyltransferase, transfers -OH myristic acid from an ACP derivative to C3 of UDP-GlcNAc to form monoacyl derivative. 2. Deacetylase next removes acetate from the C2 nitrogen. 3. Second molecule of -OH-myristic acid is transferred from ACP derivative to nitrogen on C2 to form the 2,3-diacyl derivative by an enzyme N-acyl transferase. 4. Some of the 2,3-Diacyl derivative loses UMP to form 2,3-diacyl glucosamine-1-P (lipid X) which condenses with UDP-2,3- diacylglucosamine to form disaccharide linked with -1,6 linkage. 5. ATP donates phosphate to the disaccharide derivative to form 1,4 diphosphate derivative. 6. Finally, this is modified by the addition of KDO from CMP derivative and esterification of fatty acids, lauryl and myristoyl from ACP derivatives to the OH of the -OH-myristic acid moieties. Synthesis of O-Antigen: O-antigen is synthesized as a separate polymer on a lipid carrier and then transferred as a unit to the core. The lipid carrier is the same as in case of peptidoglycan synthesis, the bactoprenol-P. First, the repeat unit of the O-antigen is synthesized on the lipid carrier where the sugar moiety is transferred from nucleoside diphosphate carrier to the growing non-reducing end of the repeat unit. Then the repeat unit is transferred as a block to the growing oligosaccharide chain. The lipid-PP of the growing chain is displaced and it again enters in to the cycle after its hydrolysis by enzyme to lipid-P. This enzyme is sensitive to bacitracin. Finally, the completed oligosaccharide chain is transferred to the lipid core displacing the lipid pyrophosphate. NDP —_ (CH,0),—(CH,0),—PP—lipid NDP—(CH,0), NDP—(CH,0), ‘ ; NDP (CH,O);—PP—lipid (CH,0),—(CH,0),--(CH,O);—PP—lipid NDP—(CH,0)q NMP- i NDP NDP—(CH,0), (CH,0), lipid —P 4 (CH,O),—(CH,0), —(CH,0)- PP—lipid ‘.._ subunit of oligosaccharide chain _. pid—PP+{_ growing oligosaccaride chain | ‘ ' lipid —PP—+] LH fs _-completed oligosaccharide lipid A || core O-Antigen Synthesis tipid AL core FH lpopolysaccharide  The Assembly of LPS: The O-antigen tetrasaccharide subunit is probably synthesized on the lipid carrier on the cytoplasmic side of the membrane then moves to the periplasmic side. On the periplasmic side it is added to the growing O-antigen anchored to the membrane by its lipid carrier displacing the lipid carrier. The core lipid A region also may be assembled to the cytoplasmic surface and translocated to the periplasmic surface, where the O-antigen is transferred to the core-lipid A, displacing the lipid-PP. Lipid-PP is then hydrolyzed to enter into another transfer cycle. How the LPS moves through the periplasm to the outer membrane is not known. E.coli Capsules: E.coli possesses extracellular capsule that masks the ‘O’ antigen polysaccharides in the LPS. The capsular antigens are called K antigens and there are at least 80 types that differ in their antigenicity and composition. They are acidic polysaccharides divided into two groups: Group I, having high molecular weight (>100,000) containing either glucuronic acid or galacturonic acid and Group II, having molecular weight (>50,000) and contain N-acetylneuraminic acid (NeuNAc), also called sialic acid, 2-keto-3deoxymannosamine (KDO), N-acetyl mannosamine or phosphate in addition to glucuronic acid. The repeating unit varies from two to six monosaccharides. Many have their reducing ends substituted with either lipid A (group I) or KDO- phosphatidic acid (group II) which presumably anchor them to the outer membrane. Many other bacteria produce similar or identical capsular polysaccharides, e.g. Neisseria.meningitidis and Haemophilus influenzae. Synthesis of Extracellular Polysaccharides: Most extracellular polysaccharides are synthesized from intracellular nucleoside diphosphate-sugar precursors and must be transported across the cell membrane to out side of the cell. The nucleoside diphosphate sugar derivatives are synthesized in the same way as UDP-N-acetylglucosamine. In gram-negative bacteria they must be transported across the cell membrane, periplasm, and outer membrane. Some extracellular polysaccharides are synthesized via undecaprenol intermediates in a pathway similar to the synthesis of oligosaccharides of the LPS and are also transported through the membrane in an undecaprenol-dependent manner. Some polysaccharides are not synthesized or transported in this manner but are synthesized differently and transported through specific membrane transporters. Synthesis of Polysaccharides through Undecaprenol-Diphosphate: Several bacteria synthesize certain exopolysaccharides via undecaprenol intermediates through the similar pathways for the synthesis of oligosaccharide repeat unit in lipopolysaccharide and the disaccharide repeat unit in peptidoglycan. The examples are group I K antigen of E.coli, the extracellular xanthan made by Xanthomonas campestris, and the capsular polysaccharides of Klebsiella aerogenes. K.aerogenes: The capsule in this organism is composed of repeating tetrasaccharides made up of galactose, mannose, and glucuronic acid in the molar ratio of 2:1:1. The glucuronic molecule is attached as a branch at each mannose residue. The tetrasaccharide is synthesized on undecaprenol diphosphate from nucleoside diphosphate sugar intermediates. Each repeating unit is then added as a block to the growing oligosaccharide attached to undecaprenol diphosphate. Synthesis of Polysaccharides without Undecaprenol derivatives: The synthesis of some polysaccharides do not involve undecaprenol derivatives which include, group II K antigen capsules, such as K5 in E.coli, alginate synthesized by Azotobacter vinelandii and P.aeruginosa, cellulose synthesized by Acetobacter xylinum. These polysaccharides are synthesized from nucleoside diphosphate precursors added to the growing oligosaccharide chain. For example, alginate, which is a linear copolymer of D- mannuronic and D-guluronic acid, is synthesized by brown algae from GDP-mannuronic acid and GDP-guluronic acid. Similarly cellulosic capsules are synthesized by Acetobacter xylinum from UDP-glucose by membrane bound cellulose synthetase. Export of Polysaccharides: Undecaprenol Phosphate dependent Translocation: There must be a mechanism through which the newly synthesized polysaccharides are transported across the cell membrane. One of the mechanism postulates that the undecaprenol phosphate serves as a part of a transmembrane-assembly process, which synthesizes and moves completed polysaccharides to the periplasmic surface of the cell membrane. (Fig) Translocation through ABC transporters: There are evidence that some polysaccharides are transported through the specific ABC type inner membrane transporters which are the protein products of KpsM and KpsT genes. When these genes are mutated the cells accumulate polysaccharides in the cytoplasm. These transporters are the protein channel through which the polysaccharides are transported. translocation periplasm and surface assembly ligation transfer and reaction polymerization / dephosphorylation cytoplasmic } and lipid recycling membrane iSeatag ual «Qoo synthesis, cytoplasm precursors B translocation outer and surface membrane assembly imteraction with periplasm periplasmic otra oJ export system _ ligation Sethe aad reaction oer cytoplasmic polymerization bind possibly involving ATP-binding membrane undecaprenol precursors Fig. 11.16 Models for synthesis and export of polysaccharides in gram-negative bacteria. (A) rfe-independent O-polysaccharide biosynthesis in Salmonella enterica. (B) Group II capsular polysaccharide biosynthesis in Escherichia coli. C55, undecaprenol; P, phosphate. Source: Whitfield C,, and M. A. Valvano. 1993, Biosynthesis and expression of cell-surface polysaccharides in gram- negative bacteria, Adu. In Microbial Physiology 35:135-246. Academic Press, New York.