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Unicellular Organisms: Eubacteria, Archea, Yeast Lecture 27, Chapter 21 May 13, 2004 Jeff Esko

Unicellular Organisms: Eubacteria, Archea, Yeast Lecture 27, Chapter 21 May 13, 2004 Jeff Esko. Overview. General structure of bacterial cell walls Structure, function and assembly of peptidoglycan (murein) Periplasmic  -glucans (MDO) Lipopolysaccharide (LPS) - endotoxin

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Unicellular Organisms: Eubacteria, Archea, Yeast Lecture 27, Chapter 21 May 13, 2004 Jeff Esko

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  1. Unicellular Organisms: Eubacteria, Archea, Yeast Lecture 27, Chapter 21May 13, 2004Jeff Esko

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

  3. Capsule LPS PG MDO Gram-negative bacteria cell wall

  4. A A A A D D D D G G G G A A A A A A A A G G G G D D D D A A A A A A A D D D G G G A A A A A A A G G G G D D D D A A A A A A A A D D D D G G G G A A A A A A A A G G G G D D D D A A A A Peptidoglycan [GlcNAcb1,4MurNAcb1,4]n Heijenoort (2001) Glycobiology 11:25R

  5. C H O H C H O H 2 2 O O O O O O O H C C H N H A c N H A c 3 O C L - A l a D - G l u D A P D - A l a D - A l a Murein Glycopeptide Notice D-amino acids DAP = diaminopimelic acid

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

  7. Vancomycin

  8. Moenomycins

  9. P Peptidoglycan Biosynthesis Undecaprenyl phosphate

  10. MDO Gram-negative bacteria cell wall

  11. b2 b2 b2 b2 b2 b2 b2 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 + Phosphoethanolamine + Phosphoglycerol + Succinate

  12. LPS Gram-negative bacteria cell wall

  13. 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

  14. H O O H H O H O H O U r i d i n e U r i d i n e N H AcN O H O H O H O H H O H O O O O O O O O P P P P O O O C O O O O O • Initiates by acylation of UDP-GlcNAc at C3, followed by N-deacetylation, and N-acylation Wyckoff et al. (1998) Trends Microbiol. 6:154

  15. O H O H O H C H 2 O O O O H O O O H O H O O O O O O H O O U r i d i n e N H P P N H N H O P O C O O O C O O C O O N H P O O H O H O O O C O O H O O O H H O H O H O H O H O H O H O H O Lipid A Assembly + Diacylglucosamine-1-P condenses with another molecule of UDP-diacylglucosamine to form the tetraacyl disaccharide core

  16. 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

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

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

  19. LPS Structural and Functional Domains

  20. C H O H 2 C H O H 2 H O H C C H O H O C O O H O H O O H O H O H O H O H O H 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 3-deoxy-D-manno-octulosonic acid (KDO) L-glycero-D-manno- heptulose

  21. LPS Structural and Functional Domains

  22. O-antigen Structure O9 a 3 a 2 a 2 a 2 a 3 O6 a 3 b 4 b 3 a 4 b 2 O124 b 3 b 6 a 3 a 4 G l c L A b 6 GlcLA = glucolactillic acid 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

  23. Capsule Gram-negative bacteria cell wall

  24. a4 b4 a4 b4 a4 a8 a8 a8 a8 a8 b4 b3 b4 b3 b4 Capsule type Structure K1, polysialic acid K5, N-acetylheparosan Group A Streptococcus (hyaluronan) • Mucoid strains contain a polysaccharide capsule • >80 different capsules types are known just in E. coli • Extraordinary diversity of structure

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

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

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

  28. 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

  29. Schaffer et al (2001) Proteomics 1:248

  30. 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

  31. Yeast make AsN-linked Glycoproteins • Yeast make membrane N-linked glycoproteins much in the same way as higher eukaryotes • Serves as a model for human genetic diseases (CDG) M Aebi (2001) Trends in Cell Biology 11:136

  32. a 6 a 6 a 2 a 3 a 2 a 6 a 2 a 3 a 2 a 6 a 2 a 3 a 2 a 3 a 6 a 2 a 3 a 2 a 3 a 3 a 2 a 6 a 6 a 3 -1-P a 2 a 4 a 3 a 6 a 6 -P- a 2 a 3 a 2 a 2 a 3 a 6 a 2 a b4 a Ser/Thr Ser/Thr b4 Vertebrate a-dystroglycan Asn Yeast Mannans Willer et al (2003) Curr Opin Struct Biol 13:621

  33. GPI proteins GPI proteins GPI proteins Pir cell wall proteins b1,6-glucans b1,6-glucans b1,6-glucans b1,3-glucans Plasma membrane Yeast Cell Walls The cell wall is made of 60% b-glucans, 40% mannoproteins, and ~1% chitin Smits et al (2001) Microbiology 147:781

  34. Summary • Bacterial cell walls are complex layered structures composed of multiple classes of glycans • Bacterial wall glycans provide an exoskeleton, define shape, protect against turgor pressure, create antigenic serotypes, and provide a way to prevent immune recognition • Glycosylated proteins are present in eubacteria and in archaea • Yeast produce many of the same glycans found in higher eukaryotes, but the mecahnism of assembly differs in subtle way • Yeast cell walls are composed of glycans (glucans and chitin) • Yeast and bacteria provide powerful genetic systems for understanding membrane and cell wall assembly

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