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04/12/23 Stenutz - Alignment of polysaccharide sequences
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Alignment of polysaccharide sequences:
Identification of the biological repeating units in LPS
Roland StenutzIsoSep AB,Dalkärrsvägen 11SE-146 36 Tullinge, Sweden
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Overview
• Biosynthesis
• Regularities in O-antigens - “Rules”
• Serotype & pathotype / Themes
• Questions
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Biosynthesis
Biosynthesis of O-antigen repeats by the polymerase-dependent pathway
LPS and some CPS (EC group 1 & 4) in Gram-negative bacteria
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Gram negative cell wall
Cytosol(inside)
inne
r m
embr
ane
pept
idog
lyca
n(m
urei
n)
Periplasmic spaceou
ter
mem
bran
e
outside
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LPS
Lipid A
Core
O-antigen, typically 10-20 repeats50-100 nm
BiologicalRU
ChemicalRU
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Initiation
Cytosol Periplasm
WecAa glycosylphosphotransferasefrom ECA biosynthesis
Undecaprenyl phosphate
UDP-GlcNAc
WbaP (Glc, colanic acid synthesis, Group 1 K)
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Elongation
transferase I
transferase II
transferase III
transferase IV
nucleotide sugars
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Translocation
Cytosol
Periplasm
Flippase (Wzx)
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Polymerisation
Polymerase (Wzy)
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Decoration
phage transferasee.g. Sf GtrI, II etc.
repeat
Und-P-Glc
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Ligation
Ligase
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Observations fromE. coli O-antigens
The structures of Escherichia coli O-polysaccharide antigens
R. Stenutz, A. Weintraub, G. Widmalm
FEMS Microbiology Reviews 30 (2006), 382–403
http://www.casper.organ.su.se/ECODAB/
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Some EC LPS
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Rule I
The first residue of the BRU isD-GlcNAc or D-GalNAc
WecA can transfer both D-GlcNAc and D-GalNActo the undecaprenyl phosphate carrier
E. coli O25
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Rule IIThe BRU is linear:
Branching occurs at the junction between two BRU:s
Added after polymerisation
except for glycosylation by by phage encoded transferases
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Rule III
The BRU is four or five residues long
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Rule IV
“Rare” residues are most often found at the end of the BRU
Common residues are D-Glc, D-GlcNAc, D-Gal, D-GalNAc, D-Man and L-Rha
Each accounts for >10% of the total number of residues
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Rule V
Violation of rules I-IV suggest that the polymer is made by the ABC-transporter dependent pathway
E. coli O20ac
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Application to EC O-antigens
How well do the rules work?
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Rule I first residue is GlcNAc or GalNAc
3)--D-GlcNAc 39%
3)--D-GlcNAc 25%
3)--D-GalNAc 15%
3)--D-GalNAc 10%
4)--D-GlcNAc 8%
exceptions 3%
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Rule IIbranches not at BRU junction
-D-Glc 33%
-D-Glc 17%
-D-GlcNAc 20%
-D-Gal 10%
-D-Gal 7%
exceptions 13%
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Rule IIIlength of BRU is 4 or 5 residues
3 residues 9%
4 residues 58%
5 residues 29%
6 residues 4%
exceptions 13%
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Rule IV“rare” residues are more frequent at
the end of a BRU
common residues
total:77% at end: 50%
rare residues
total:23% at end: 50%
“rare” residues constitute <10% of all residues
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Possible uses
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The plausibility of O-antigen structures can be tested
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Predicting the BRU providesinsights into the LPS biosynthesis
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The immunologically dominant epitope can be predicted and cross reactivities can be explained
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Relationship between serotype and pathotype
EPEC – Enteropathogenic E. coli
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BRU:s ending with -L-Rha
O18, O25, O26, O142, O158
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BRU:s ending with -Col
O55 & O111
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BRU:s ending with -L-Fuc
O86,O126, O127,O128
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EPEC serotypes
O18, O20, O25, O26, O44, O55,O86, O91, O111, O114, O119, O125,
O126, O127, O128, O142, O158
-L-Rha 5-Col 2-L-Fuc 4other 6-deoxy sugar 3other 3
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EPEC serotypes
Caveat:
Both virulence factors andLPS genes are transferred bybacteriophages / plasmids
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Structural themes shared across genera
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-D-Man-(2)--D-Man-(2)--D-Man-(3)-D-HexNAc-(
Citrobacter freundiiEnterobacter cloacaeEscherichia coliSalmonella ssp.Yersinia pseudotuberculosis
common ancestor
– Wang et al. Microbiol. 153 (2007) 2159-2167
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-Col-(2)--D-Gal-(3)--D-GlcNAc-(4)--D-Hex-(3)-D-HexNAc-(
Aeromonas trottaEscherichia coliPseudoalteromonas spp.Salmonella arizonaeVibrio cholera
common ancestor– Reeves et al. J. Bacteriol. 186 (2004) 6536-6543
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-L-Rha-(2)--L-Rha-(3)--L-Rha-(3)-D-HexNAc-(
Acinetobacter baumanniiPseudomonas solanacearumSalmonella arizonaeSerratia marcescensShigella flexneri
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Questions
Why do these regularities exist?
How does the LPS structure affect the interaction between bacteria and host, other bacteria or phages?
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Future work
Extend to other bacteria and polysaccharides
– initiating residue differs, e.g.
Glc, Gal, GlcNAc, GalNAc,
D-FucNAc, D-QuiNAc, D-Qui2N4N
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Assignment of the BRU is not always trivial:
2)--D-Man-(13)--D-Gal-(1 |-D-GlcA-(13)--D-Gal-(13)
E. coli K30 – CPS often begin with D-Gal or D-Glc
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Assignment of the BRU is not always trivial:
2)--D-Gal-(3)--D-Glc-(3)--D-Gal-(4)--D-Gal-( | | |-D-Gal-(3) -D-Gal-(4) -L-Rha-(3)
Lactobacillus delbrueckii ssp. bulgaricus LY03, strain 24
EPS often begin with D-Gal or D-Glc and contain little else…