1. Unicellular Organisms: Eubacteria, Archea, Yeast Lecture 27, Chapter 21 May 13, 2004 Jeff 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. LPS MDO PG Capsule Gram-negative bacteria cell wall

4. 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

5. 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

6. 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

7. Vancomycin

8. Moenomycins

9. Peptidoglycan Biosynthesis P Undecaprenyl phosphate

10. MDO Gram-negative bacteria cell wall

11. 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 2 2 2 2 2 2 2

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. 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

15. 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

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. 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

21. LPS Structural and Functional Domains

22. 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 O-antigenStructure O9 O6 O124 GlcLA GlcLA = glucolactillic acid

23. Capsule Gram-negative bacteria cell wall

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

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

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

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

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