Essentials of Glycobiology Lecture 33 May 28, 2002 Jeff Esko Bacterial Polysaccharides: Structure, Biosynthesis, Biological Functions and Significance …or I never met a glycan I didn’t like

Overview General structure of bacterial cell walls Structure, function and assembly of peptidoglycan (murein) Periplasmic  -glucans (MDO) Lipopolysaccharide (LPS) - endotoxin Capsular polysaccharides - mimicry Bacterial glycoproteins - a new area

LPS MDO PG Capsule

Peptidoglycan A G D A A G D A A G D A A G D A A G D A A G D A A G D A A G D A AA A G D A A G D A G D A A G D A A G D A G D A A G D A A G D A A G D A A G D A A G D A A G D A A G D A A G D A A G D A [GlcNAc  1,4MurNAc  1,4] n Heijenoort (2001) Glycobiology 11:25R

Murein Glycopeptide O NHAc O CH 2 OH HCCH 3 C O NHAc O CH 2 OH O O O L-Ala D-Glu DAP D-Ala D-Ala O Notice D-amino acids DAP = diaminopimelic acid

Peptidoglycan A G D A A G D A A G D A A G D A A G D A A G D A A G D A A G D A AA A G D A A G D A G D A A G D A A G D A G D A A G D A A G D A A G D A A G D A A G D A A G D A A G D A A G D A A G D A The final step involves cleavage between the D-Ala-D-Ala unit and transpeptidation to the amino group of DAP of another unit AAAA

Vancomycin

Monenomycins

Peptidoglycan Biosynthesis P Undecaprenyl phopsphate

MDO

Membrane Derived Oligosaccharides (MDO) Branched  -glucans Represent about 1-5% of dry weight Charged substituents act as an osmolyte and protects the inner membrane against the large difference in osmolarity inside the cell compared to outside the cell

LPS

Lipopolysaccharide (LPS) LPS consists of three domains –Lipid A, otherwise known as endotoxin –Core region composed of KDO (K), heptoses (H), and hexoses (open hexagons –Highly variable outer O-antigen region

Initiates by acylation of UDP- GlcNAc at C3, followed by N- deacetylation, and N-acylation HO O O NH OH CO HO O O P O O OH HO P O O OH Uridine HO O HO AcN HO O P O O OH P O O OH Uridine Wyckoff et al. (1998) Trends Microbiol. 6:154

Lipid A Assembly HO O O NH OH CO HO O O P O O OH HO P O O OH Uridine HO O O NH OH CO HO O O P O O OH HO HO O O NH OH CO HO O O HO HO O O NH CH 2 CO HO O O P O O OH HO + Diacylglucosamine-1-P condenses with another molecule of UDP-diacylglucosamine to form the tetraacyl disaccharide core

Lipid A Assembly KDO transferases initiate the formation of the core Additional C12 fatty acids added to  -hydroxy groups (wax) Lipid A translocates to the outer leaflet of the outer membrane by msbA (ABC transporter) Doerrler et al. (2001) J Biol Chem. 276:11461

Lipid A Biology Lipid A, the heat stable endotoxin of gram negatives Resistant strains of mice defined a locus, lps, which as positionally cloned. This turned out to be homologous to toll receptors in Drosophila, which were known to be involved in innate immunity to fungal infection (anitmicrobial peptides) lps turns out to beTlr4, a member of a family of signaling receptors (10 members known) that induce. Tlr4 binds to Lipid A. Tlr2 apparently binds and responds to muramyl- dipeptide

Takeda & Akira(2001)Genes to Cells 6:733

LPS Structural and Functional Domains

Core region contains unusual sugars The inner core contains 1-4 KDO residues, which look like an analog of sialic acid. The core also contains heptopyranoses, which can vary stereochemically The rest of the core consists of various combinations of Glc and Gal OH O COOH OH OH HOHC CH 2 OH 3-deoxy-D-manno- octulosonic acid (KDO) L-glycero-D-manno- heptulose O OH OH CHOH CH 2 OH OH OH

LPS Structural and Functional Domains

O-antigens O-antigens consist of 2- 8 sugars, repeated ≤50 times O-antigens gives rise to different serotypes and some are correlated with disease

Capsule

Mucoid strains contain a polysaccharide capsule >80 different capsules types are known just in E. coli Extraordinary diversity of structure

Mycobacteria Crick et al. (2001) Glycobiology 11:107R

Bacterial Glycoproteins Surface-layer (S-layer) glycoproteins Prevalent in Bacteria and Archaea, but structures differ, e.g., N-linked glycosylation only in Archaea Structural analyses have revealed unusual carbohydrate-linkage regions…. ….and unusual nucleoside diphosphate- linked oligosaccharides

Schaffer et al (2001) Proteomics 1:248

Notice diversity of linkages Similarity in sequence of N- linked attachment sites and mechanism of assembly of glycan (dolichol pathway) Burda &Aebi (1999) Biochim Biophys Acta 1426:239