BCH550/MS8114

Glycobiology

Week 6-7: Glycans of Eubacteria – Poly-N-

acetylglucosamine (PNAG)

Dr. Dustin Little

 Bacterial Biofilms

Bacteria
Extracellular
matrix

Planktonic state Biofilm state

• The extracellular matrix contains proteins, nucleic acids, and exopolysaccharides

•The extracellular matrix provides protection from environmental stresses,
antibiotics, and host defense mechanisms
Poly-(1,6)-N-acetyl-D-glucosamine (PNAG) is found in the
biofilms of numerous pathogenic bacteria

Staphylococcus epidermidis
Staphylococcus aureus

Escherichia coli
Yersinia pestis
De-N-acetylation
Bordetella pertussis
Bordetella bronchiseptica
Acinetobacter baumannii
Actinobacillus pleuropneumoniae
Pseudomonas fluorescens
Burkholderia cepacia complex
Aggregatibacter actinomycetemcomitans • Also known as PIA, PGA, hms+-ECM, and BPS
 Proposed model for PNAG biosynthesis and secretion
PgaD – Inner membrane c-di-GMP binding
protein

PgaC – Inner membrane Glycosyltransferase

that synthesizes PNAG using UDP-GlcNAc

PgaB – Two-domain periplasmic protein that

De-N-acetylates PNAG, and also hydrolases
deacetylated PNAG

PgaA – Outer membrane Porin with TPR

domain for PNAG export
Adapted from Whitney & Howell, Trends Microbiol (2013)
 PNAG is required for B. bronchiseptica biofilm formation
SEM images of B. bronchiseptica grown on glass coverslips for 120 h

WT pgaABCD

Parise et al., J Bact (2007)

PgaC and PgaD form a complex that is required for PNAG
synthesis
How can you measure PNAG biosynthesis?

Glycosyltransferase + NDP-Sugar = Exopolysaccharide + NDP

Steiner et al, EMBO (2013)

PgaC and PgaD form a complex that is required for PNAG
synthesis
How can you measure PNAG biosynthesis?

Glycosyltransferase + NDP-Sugar = Exopolysaccharide + NDP

Steiner et al, EMBO (2013)

PgaCD synthase activity is dependent on c-di-GMP

Same GT assay, but now:

v
PgaCD + UDP-GlcNAc

VS.

PgaCD + UDP-GlcNAc +
C-di-GMP

Conducting a GT assay with PgaC and PgaD in the presence of c-di-GMP shows
~40 fold decrease in Km
Steiner et al, EMBO (2013)
 Model for the allosteric activation of PNAG biosynthesis
by the PgaCD complex
v

Steiner et al, EMBO (2013)

 Proposed model for PNAG biosynthesis and secretion

Adapted from Whitney & Howell, Trends Microbiol (2013) Little et al., J Biol Chem (2012)
 PgaB is required for Bordetella biofilm architecture

SEM images of Bordetella bronchiseptica biofilms on glass coverslides,

96 hr

WT ΔpgaB

Little et al (2015) JBC

How do you test for the deacetylation of PNAG?

De-N-acetylation
Monitoring Deacetylation by Fluorescence
Monitoring Deacetylation by Fluorescence

• Modify free amines with fluorescamine

– Fluorescence increases with degree of
deacetylation
λex = 360 nm
R N O λem = 460 nm
+ R NH 2
OH
t1/2 100-500 msec OH

O O O
O Fluorophore
+ H2O
O Hydrolysis Products
t1/2 5-10 sec
(nonfluorescent)
Fluorescamine
(nonfluorescent)
 PgaB is a two-domain protein that displays length-
dependent activity on PNAG

β-1,6-(GlcNAc)4

β-1,4-(GlcNAc)4

kca t/Km for β-1,6-(GlcNAc)5 is 0.25 ± 0.01 M-1s -1

The activity of PgaB increases as the length of the oligosaccharide increase

The activity of PgaB is specific for PNAG vs. other poly-GlcNAc polymers

Little et al., J Biol Chem (2012)

 PgaB is a two-domain protein that displays metal-
dependent activity on PNAG

Asp115 Water

Metal
His189 Acetate
His184

Active site of PgaB shows a

metal coordinating triad (DHH)
AND a bound water and
acetate molecule PgaB shows increased activity with Ni and Co

PgaB shows the least amount of activity with EDTA

Little et al., J Biol Chem (2012)

Enzymatic mechanism of PgaB on PNAG
 N

What is the role of the C-terminal

Deacetylase domain domain (PgaB310-672)?
 Association of PgaB’s N- and C-terminal domains is
required for de-N-acetylation

+
Normalized de-N-acetylation (%)

500.00#
A
400.00#

#(mAu)#
(mAu)
300.00#

A280A280 200.00#

100.00#

0.00#
0.00# 5.00# 10.00# 15.00# 20.00# 25.00#

Elution Volume (mL)

Little et al., PNAS (2014)

 α4
α3
Testing if PgaB C-terminal domain binds PNAG
oligomers
C D
βHL

Fluorescence quenching (%)

L-Tryptophan

β-1,6-GlcNAc oligomer [mM]

-10 kT/e to +10 kT/e

The electronegative groove is

pinched off from 14 to 7 Å

Little et al., PNAS (2014)

 α4
α4
α3
α3
PgaB C-terminal domain binds PNAG oligomers
C D
βHL

Fluorescencequenching
Fluorescence (%)
quenching(%)
β-1,6-GlcNAc
β-1,6-GlcNAc oligomer
oligomer [mM]
[mM]
-10 kT/e to +10 kT/e

PNAG Oligomer Kd (mM)

The electronegative groove is
(β-1,6-GlcNAc)3 3.0 ± 0.4
pinched off from 14 to 7 Å
(β-1,6-GlcNAc)4 2.7 ± 0.6
(β-1,6-GlcNAc)5 1.9 ± 0.3
(β-1,6-GlcNAc)6 1.3 ± 0.2
Little et al., PNAS (2014)
 PgaB C-terminal domain in complex with β-1,6-(GlcNAc)6

12
W613

W552 11
4 5 8 9
6 7
3
10
Y432
2

W613 M572
E576 L542 β-1,6-(GlcNAc)4
Y323 L575
Y645
D320 M570 12
W552
9
W400
D322 F553 10 11
D466
D358 F540
H465
F356 L469 D476
D467 Y432
Little et al., PNAS (2014)
 GlcNAc and GlcNH3+ probability binding density contoured at an occupancy of 15%
Inter-domain linker (IDL), β-hairpin loop (βHL), Conserved residue patch on PgaB310-672
Little et al., PNAS (2014)
 Proposed mechanism for dPNAG export

(a) (b) (c)

Outer
Membrane PgaA PgaA PgaA

PgaB PgaB
PgaB

GlcNAc GlcN
 Quick Summary
1. PNAG biosynthesis in E. coli and B. bronchiseptica is through
the PGA biosynthetic system (PgaABCD)

1. PgaC and PgaD are both needed for PNAG biosynthesis

2. PgaB is a PNAG deacetylase that is required for biofilm

formation and/or biofilm architecture

3. PgaB N-terminal CE4 domain displays length and metal

dependent activity on PNAG

4. The C-terminal PgaB domain binds to PNAG and dPNAG, and

may be a dPNAG hydrolase?