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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…