BCH550/MS8114

Glycobiology

Week 6: Glycans of Eubacteria – Capsular

Polysaccharides (CPS), Exopolysaccharides (EPS), and
Bacterial Biofilms
Dr. Dustin Little
Conceptual Organization of the Cell Envelopes of Gram-Negative
and -Positive Bacteria

Chapter 21, Figure 1. Essentials of Glycobiology, Third Edition

Chapter 21, Figure 1. Essentials of Glycobiology,

Third Edition
 Cross-sectional representation of layers constituting the
bacterial cell wall of a typical Gram-negative bacteria

Cress et al, FEM Micro Rev (2014)

 CPS is distinctly different from LPS in Gram-negative bacteria

Cress et al, FEM Micro Rev (2014)

 Structures of Capsular Polysaccharides

Chapter 21, Figure 6. Essentials of Glycobiology, Third Edition

What is the function of Capsular polysaccharides (CPS)?

1. It provides protection against desiccation

- Helps retain water

- Capsules are highly hydrated and about 95% water

E. coli K5 Capsule
What is the function of Capsular polysaccharides (CPS)?

2. It can aid in the attachment and colonization of surfaces

- Can be an abiotic or biotic environment

- Capsules are highly hydrated and sticky

- They can attached to a non-biological surface through hydrogen
bonding or Van der Waals forces
- They can specifically interact with cells through surface-glycan
receptors
 What is the function of Capsular polysaccharides (CPS)?

3. Plays a role in defense against bacteriophages and toxins

- Instead of modifying LPS – can produce CPS

- Capsules are chemically different and longer than LPS

- Bacteria can make multiple types of chemicals and
produce them under the right environmental
conditions

Samson et al, 2013, Nat Rev Micro

 What is the function of Capsular polysaccharides (CPS)?

4. Plays a major role in virulence by acting as a diffusion barrier

from host innate immune defenses

- Can block antimicrobial peptides

- Can aid in the blocking of phagocytosis
- Can cloak the bacteria from immune detection
AMPs are typically cationic
and contain fewer then 50
amino acids

AMPs are a first line of

defense against pathogens
in the gastrointestinal tract
And often target the
disruption of the bacterial
membrane
Mookherjee et al., Nat Rev Drug Discovery(2020)
 CPS aids in the blocking of phagocytosis
• Opsonization of bacteria via Cb3-IgG targeting can be
blocked by capsules

Mookherjee et al., Nat Rev Drug Discovery(2020)

 How can Phage or Phagocytes overcome the CPS barrier?

Samson et al, 2013, Nat Rev Micro

Mookherjee et al., Nat Rev Drug Discovery(2020)

 Host mimicry during infection: Non-immunogenic bacterial CPSs

Cress et al, FEM Micro Rev (2014)

 Neisseria meningitidis CPS is a poly(α-2,8)-sialic acid saccharide
that mimics glycosylation of the Neural Cell Adhesion Molecule
• NCAM is found on the surface of neurons, glial
cells, and natural killer cells.

• NCAM found during embryonic

development is highly polysialylated
across the brain.

• This induces steric hindrance and

repulsion between preventing the
binding of opposing NCAM molecules
and other species on the surface of cells.

• The brain has a high degree of

plasticity during early development,
but In contrast, adult NCAM is under-
polysialylated.

Kareem and Wang, CAB Rev (2006)

 Neisseria meningitidis CPS aids in crossing the blood-brain
barrier that can lead to meningitis
1. Colonization of mucosa
2. Invasion of
bloodstream
3. Survival and
multiplication
4. Crossing of the blood–
brain barrier
5. Invasion of the
meninges and CNS
6. Increased permeability
of the blood–brain
barrier
7. Pleocytosis
8. Oedema and increased
intracranial pressure
9. Release of pro-
inflammatory.
10. Neuronal injury or
menigitis

Kwang Kim, Nat Rev Neuro (2003)

 Biosynthesis of CPS (I)
Wzx/Wzy-dependent pathway

Synthesis is initiated by WbaP - a phospho- Wzy is the oligosaccharide polymerase,

glycosyltransferase (PGT) - that transfers a Und-P is then recycled back into cell
hexose phosphate or acetamido sugar to
Wzc is important for chain length regulation
undecaprenyl phosphate (Und-P)
and export
GTs associate with WbaP to synthesize the Wza is the outer-membrane exporter
oligosaccharide repeat
Wzi is involved in CPS attachment to the
Wzx is the Und-P-oligosaccharide transporter
cell surface
Whitfield, Annu Rev Biochem (2006)
 Biosynthesis of CPS (II)
KpsM/KpsD ABC-dependent pathway

KpsS and KpsC are -Kdo-glycosyltransferases. KpsM/T is ABC transporter

KpsS adds the first Kdo to
diacylglycerophosphate. KpsC adds on the KpsD is the outer membrane exporter
other Kdo sugars in the core (n=2-8).
KpsE is an adapter protein required for
GTs associate with KpsC to synthesize the export
oligosaccharide repeat

KpsF/U synthesize CMB--Kdo

Whitfield, Annu Rev Biochem (2006)
 Visualizing CPS biosynthesis and strain variability

CPS can be separated by SDS-PAGE, and visualized using a specific

stain
- CPS antibody can be used
- Specific polysaccharide stains

Can easily compare LPS from different strains for bacteria

Drummelsmith and Whitfield, EMBO (2000)

 Visualizing CPS biosynthesis by microscopy
Bacterium Capsule

Thomassin et al, PLoS One (2013)

Background
CPS can be visualized under a light microscope using specific dyes
- Acidic dye stains the background (black)
- Positive dye strains the bacteria (grey)

The CPS (if uncharged) prevents stain diffusion and creates a zone of occlusion around the
bacteria (white)
 Quick Summary
1. CPS is chemically different from LPS

2. CPS is a much longer glycan molecule than LPS

3. CPS serves fundamental roles in bacterial protection and

homeostasis

4. Pathogenic bacteria usually have a CPS that mimics host

glycans to evade immune detection

5. CPS can be produce in a Wzx/Wzy or KpsM/KpsD dependent

mechanism

6. CPS can be visualized via gel electrophoresis or microscopy

Conceptual Organization of the Cell Envelopes of Gram-Negative
and -Positive Bacteria

Chapter 21, Figure 1. Essentials of Glycobiology, Third Edition

Chapter 21, Figure 1. Essentials of Glycobiology,

Third Edition
 What is the function of Exopolysaccharides (EPS)?

1. Is a major structural component of bacterial biofilms

- Aids in the acquisition of nutrients and removal of waste
products

2. Provides a diffusion barrier against certain types of antibiotics

or antimicrobials

3. It can act as a diffusion barrier from host innate immune

defenses
- Can block antimicrobial peptides
- Can aid in the blocking of phagocytosis

4. I can aid in the attachment and colonization of surfaces

- Can be an abiotic or biotic environment
 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
 Biofilm-related bacterial infections
CNS shunt Contact lens Middle ear Cochlear implant
infection infections infection infection

Burn-related
Sinusitis infection

Intravascular
catheter infection
Breast
implant
infection
Bacteria infection of
prosthetic heart
valves
Ventilator
associated
pneumonia Pacemaker
infection

Lung Inflammation of heart

infections in due to electro-
CF patient physiological wire

Intravascular Biliary stent

stent infection infection

Urinary stent Prosthetic joint Peritoneal dialysis

infection infection catheter infection

Canon of Proportions by Leonardo da Vinci From del Pozo & Patel Clin. Pharm. & Therap. (2007) 82, 204–209.
 Exopolysaccharides found in bacterial biofilms
O157:H7, TEM image

PNAG
Poly-β(1,6)-GlcNAc
pgaABCD
Escherichia coli
Cellulose
Poly-β(1,4)-Glc
bcsQABZC Birgit Pruess, North Dakota State University (2011)

PA14, SEM image

Alginate
Poly-β(1,4)-ManUA-GulUA
algD844KEGXLIJFA
Pseudomonas aeruginosa
PEL
Poly-α(1,4)-GalNAc
pelABCDEFG
Franklin et al., Front Microbiol (2011)
Alginate production is overproduced by P. aeruginosa found in
the lungs of Cystic Fibrosis (CF) patients
Alg - Alg +

Cystic Fibrosis patients have a mutation in the CFTR gene that causes
disregulation of Na+/Cl- (ion transport) across the epithelial membrane

P. aeruginosa is the leading cause of morbidity and mortality among

Cystic Fibrosis patients due to biofilm formation in the lungs
Ramsey and Wozniak (2005) Mol. Microbiol. Centers for Disease Control and Prevention
 Biosynthesis and export of Exopolysaccharides (EPS)
Synthase-dependent Mechanism

Adapted from Whitney & Howell, Trends Microbiol (2013)

Biosynthesis and export of Exopolysaccharides (EPS)
Synthase-dependent Mechanism
GREEN – Synthase or Glycosyltransferase (GT) of the EPS system and Inner Membrane
transport

RED – Inner membrane transport, and/or regulator of biosynthesis through c-di-GMP

binding

ORANGE – Modification of the EPS by O-acetylation or De-N-acetylation

PINK – Epimerase (unique to Alginate)

PURPLE – EPS cleaving

BLUE – Tetratricopeptide repeat (TPR) scaffold protein for transport

YELLOW – Outer membrane porin for EPS export

WHITE – Unknown function

 Biosynthesis and export of Exopolysaccharides (EPS)
Synthase-dependent Mechanism Alg44 – c-di-GMP receptor and inner
membrane transporter

Alg8 – GT that uses GDP-ManUA

AlgI – Acetyl donor transport

AlgJ – Acetyltransferase
AlgX – Acetyltransferase
AlgF – Carbohydrate binding module

AlgG – Epimerase (ManUA -> GulAc)

AlgL – Alginate Lyase

AlgK – TPR Scaffold protein

AlgE – Outer membrane Porin

Adapted from Whitney & Howell, Trends Microbiol (2013)
 Biosynthesis and export of Exopolysaccharides (EPS)
Synthase-dependent Mechanism
PelD – c-di-GMP receptor and inner
membrane transporter

PelE – Inner membrane transporter

PelF – GT that uses UDP-GalNAc

PelG - Unknown Function

PelA – N-Terminal De-N-acetylase domain

C-Terminal Glycoside Hydrolase domain

PelC - Unknown Function

UDP-Ga lNAc
PelB - N-Terminal TPR scaffold domain
C-Terminal outer membrane porin domain
Adapted from Whitney & Howell, Trends Microbiol (2013)
 Biosynthesis and export of Exopolysaccharides (EPS)
Synthase-dependent Mechanism
BcsQ – Unknown function

BcsA – c-di-GMP receptor domain

GT domain that uses UDP-Glc and inner
membrane transporter

BcsB – Inner membrane transporter and

periplasmic scaffold

BcsZ – Glycoside hydrolase (cellulase)

BcsC – N-Terminal TPR scaffold domain

C-Terminal outer membrane porin domain

Adapted from Whitney & Howell, Trends Microbiol (2013)

 Biosynthesis and export of Exopolysaccharides (EPS)
Synthase-dependent Mechanism
PelD – c-di-GMP receptor and inner
membrane transporter

PgaC – GT that uses UDP-GlcNAc and inner

membrane transporter

PgaB – Terminal De-N-acetylase domain

C-Terminal Glycoside Hydrolase domain

PgaA – N-Terminal TPR scaffold domain

C-Terminal outer membrane porin domai
Adapted from Whitney & Howell, Trends Microbiol (2013)
 Visualizing EPS biosynthesis
Deletion of PgaABCD proteins
WT
Western blot
using anti-
PNAG Cell Attached
antibody

Cell Free

PNAG (ng)

D’Gama et al, mBio (2019)

Itoh et al, J Bact (2008)

EPS can be isolated and added to immunoblot membranes for antibody analysis

Some EPS can separated by SDS-PAGE, and visualized using a specific antibody
- EPS is usually a very high molecular weight species
 Visualizing EPS by microscopy
Transmission electron microscopy (TEM)

Use an antibody
conjugated
to a gold particle

Yoong et al, mBio (2012)

Use an antibody conjugated

to a fluorescence probe

OR use a carbohydrate binding

protein fused to GFP specific for
that polysacchairde
Itoh et al, J Bact (2008)

EPS can be isolated and added to immunoblot membranes for antibody analysis

Some EPS can separated by SDS-PAGE, and visualized using a specific antibody
- EPS is usually a very high molecular weight species
 Things to think about…
1. What is the similarities and differences between CPS and EPS
biosynthesis in Gram-negative cells?

2. What are the similarities and differences between the various

EPS biosynthesis steps?

3. How can CPS and EPS be visualized and analyzed in the lab?

4. What are the properties of different EPS and how do they

contribute to biofilm formation?

5. Think about what each of the enzymes do – are some of them

CAZy family members? Do some of them serve “similar”
function in context of their respective system?