Bacterial Adhesion on

Host
Why do bacteria adhere
 The host has a number of innate
defenses against bacteria
 Skin and mucus – physical barriers
 Peristalsis of the gut and the
esophagus
 Ciliated epithelium in the respiratory
tract
 Flushing of bodily fluids
Bacterial adherence

 Bacteria are more resistant to normal clearing

mechanisms, antibiotics, bacteriolytic
enzymes and immune killing when they are
adhered to surfaces such as the host cell
 Therefore, adhesion is essential for bacterial
colonization
 Maintaining the normal flora in and on the host
 But, it also the crucial first step in many
infectious disease processes
 Evidences for requirement of attachment:

 Susceptibility for certain bacteria to infect

specific tissue is directly proportional to its
ability to adhere to that tissue.
 Bacterial variants that are found to have a
reduced capacity to adhere in vitro have
decreased infectivity in vivo.
 The bacterial binding capacity of epithelial
cells from individuals prone to certain
bacterial infections is sometimes higher than
those tissues from uninfected individuals.
 Both bacterial & tissue cell surfaces are
negatively charged, it is overcome by
electrostatic ( cations bridging eg. Ca++) and
hydrophobic forces (Lipoteichoic acid).
 Adherence prevents microorganism from
flushing activity of saliva, mucous etc. , enzymes
and antibodies.
 It enable bacteria to deliver its product close to
the cell & colonization.
 Crude mechanical device &/or surface receptor
molecules on the surface of protozoan and
worms.
 All the organism uses multiple binding sites for
survival.
How do bacteria adhere to
the host?
 Surface-expressed bacterial proteins
 Microbial Surface Components Recognizing Adhesive
Matrix Molecules - ECM (extracellular matrix )
 Proteins with other receptors
 Fimbriae (or pili)
 Protein fibers extending from the bacterial cell
 Enzymes
 Bacterial enzymes can expose cryptic host cell receptors
(neuraminidase, α-enolase)
 Biofilm formation
 Communities of bacteria adhering to a solid surface which
can be host tissue
Why adhere to the host ECM
 The ECM is ubiquitous in the body, especially at
mucosal surfaces
 It is abundant (enough receptors for everybody)
 It is present in all vertebrates
 Individual components share structural
characteristics (not completely host-specific)
 It is differentially present in normal and diseased
tissue
 May signal a pathogenic opportunity
Location of Adhesions
molecules
 Fimbrillae of gram-positive bacteria
 Fimbriae of gram-negative bacteria
 Filamentous heamagglutinin (FHA) of
Bardetella pertussis
 Membrane protein of Mycoplasma of foot.
 Protein II of N. gonorrhoeae
 Capsid / envelope protein of viruses ( eg.
Heamagglutinin of influenza A virus).
 Prokaryotic and Eukaryotic Interactions
Eukaryotic Cell Pili or adhesins Prokaryotic Cell

Intracellular
Control of virulence factors: Virulent Bacteria
(Pilin, capsule, invasins, toxins etc)

Adherence blockers
Receptor
 Prokaryotic and Eukaryotic Interactions
Eukaryotic Cell Pili or adhesins Prokaryotic Cell

Intracellular
Control of virulence factors: Virulent Bacteria
(Pilin, capsule, invasins, toxins etc)

Adherence blockers
Receptor

COLONIZATION
 Prokaryotic and Eukaryotic Interactions
Eukaryotic Cell Pili or adhesins Prokaryotic Cell

Intracellular
Control of virulence factors: Virulent Bacteria
(Pilin, capsule, invasins, toxins etc)

Adherence blockers
Receptor

COLONIZATION INVASION
 TERMS USED TO DESCRIBE ADHERENCE FACTORS IN HOST-PARASITE INTERACTIONS

ADHERENCE
DESCRIPTION
FACTOR
Adhesin A surface structure or macromolecule that binds a bacterium to a specific surface
A complementary macromolecular binding site on a (eukaryotic) surface that binds specific adhesins
Receptor
or ligands
Lectin Any protein that binds to a carbohydrate
Ligand A surface molecule that exhibits specific binding to a receptor molecule on another surface
Mucous The mucopolysaccharide layer of glucosaminoglycans covering animal cell mucosal surfaces
Filamentous proteins on the surface of bacterial cells that may behave as adhesins for specific
Fimbriae
adherence
Common pili Same as fimbriae
Sex pilus A specialized pilus that binds mating procaryotes together for the purpose of DNA transfer
Fimbriae in Enterobacteriaceae which bind specifically to mannose terminated glycoproteins on
Type 1 fimbriae
eukaryotic cell surfaces
A layer of exopolysaccharide fibers on the surface of bacterial cells which may be involved in
Glycocalyx
adherence to a surface
A detectable layer of polysaccharide (rarely polypeptide) on the surface of a bacterial cell which may
Capsule
mediate specific or nonspecific attachment
A distinct cell wall component of the outer membrane of Gram-negative bacteria with the potential
Lipopolysaccharide (LPS)
structural diversity to mediate specific adherence. Probably functions as an adhesin
Teichoic acids and Cell wall components of Gram-positive bacteria that may be involved in nonspecific or specific
lipoteichoic acids (LTA) adherence
specificity of adherence of
bacteria to host cells or tissues
 Tissue tropism: particular bacteria are known to have
an apparent preference for certain tissues over others,
 S. mutans - dental plaque
 S. salivarius -epithelial cells of the tongue
 Species specificity: certain pathogenic bacteria infect
only certain species of animals,
 N. gonorrhoeae - humans;
 Enteropathogenic E. coli K-88 - pigs;
 E. coli CFA (colonizationfactor antigens) I and II – humans.
 Genetic specificity within a species: certain strains or
races within a species are genetically immune to a
pathogen ,
 Susceptibility to Plasmodium vivax infection (malaria) is
dependent on the presence of the Duffy antigens on the host's
redblood cells.
Mechanisms of
Adherence to Cell
or Tissue Surfaces
Nonspecific adherence:

 Reversible attachment of the bacterium to the eukaryotic surface

(sometimes called "docking") Possible interactions and forces
involved are:
 hydrophobic interactions
 electrostatic attractions
 atomic and molecular vibrations resulting from fluctuating
dipoles of similar frequencies
 Brownian movement
 Recruitment (Quorum Sensing) and trapping by biofilm
polymers interacting with the bacterial glycocalyx (capsule)
Specific adherence:
 Reversible permanent attachment of the microorganism to the
surface (sometimes called "anchoring"). Direct evidence that
receptor and/or adhesin molecules mediate specificity of adherence
of bacteria to host cells or tissues. These include:
 The bacteria will bind isolated receptors or receptor analogs.

 The isolated adhesins or adhesin analogs will bind to the

eukaryotic cell surface.

 Adhesion (of the bacterium to the eukaryotic cell surface) is

inhibited by:
 isolated adhesin or receptor molecules

 adhesin or receptor analogs

 enzymes and chemicals that specifically destroy adhesins or

receptors
 antibodies specific to surface components (i.e., adhesins or

receptors)
SPECIFIC ATTACHMENTS OF BACTERIA TO HOST CELL OR TISSUE SURFACES

Bacterium Adhesin Receptor Attachment site Disease

Streptococcus Amino terminus of Pharyngeal

Protein F Sore throat
pyogenes fibronectin epithelium

Streptococcus Glycosyl
Salivary glycoprotein Pellicle of tooth Dental caries
mutans transferase

Streptococcus Lipoteichoic Buccal epithelium of

Unknown None
salivarius acid tongue

Streptococcus Cell-bound N-acetylhexosamine-

Mucosal epithelium pneumonia
pneumoniae protein galactose disaccharide

Staphylococcus Cell-bound Amino terminus of

Mucosal epithelium Various
aureus protein fibronectin

Neisseria N-methylphenyl- Glucosamine-galactose Urethral/cervical

Gonorrhea
gonorrhoeae alanine pili carbohydrate epithelium
Adhesion Mechanism

gram negative
bacteria
Types of bacterial secretion used by  gram
negative bacteria
Type III Secretion System
 A cylindrical base, similar to the flagellar
hook-basal body complex, spans the
periplasm and is associated with the two
bacterial membranes where ring-like
structures are detected, ensuring
stabilization of the whole structure upon the
bacterial cell envelope. An elongated hollow
extracellular structure called the needle
extends around 50 nm outside the bacterial
cell wall (it varies according to the different
bacterial species) and can be inserted into
eukaryotic membranes. Energy derived from
ATP hydrolysis drives translocation of
bacterial proteins (known as TTSS effectors)
from the bacterial cytoplasm to the
eukaryotic cell cytoplasm, where they can
hijack host signaling pathways.
 Tir (translocated intimin receptor),/Intimin

Interaction
EPEC, the bacteria provides both
the ligand and the receptor, via its
type III secretion system (TTSS),
injects into the cytosol of target cells
the protein Tir, which integrates into
the host-cell plasma membrane,
dimerizes, and functions as a receptor
for the bacterial outer membrane
intimin. Tir/intimin interaction
promotes Tir phosphorylation by Fyn
and Abl ( Host Kinase), inducing the
recruitment of the protein adaptor Nck,
which in turn recruits N-WASP
(Wiskott-Aldrich syndrome
protein) and the Arp2/3 complex
(actin-related protein 2/3), leading
to actin polymerization and the
formation of structures known as
pedestals. Actin binding proteins such
as talin are recruited to the pedestal,
stabilizing the structure.
Adhesion Mechanism

gram positive
bacteria
 MSCRAMMS
 Microbial Surface Components Recognizing Adhesive
Matrix Molecules
 S. aureus adhesion to fibronectin and collagen binding
was described in the early-mid 1980s
 The first MSCRAMM was cloned and characterized in
1992 – Cna (collagen adhesin)
 Most (all) Gram positive pathogens and commensals
have ECM-binding MSCRAMMs
 Many non-pathogenic Gram positives do not have
MSCRAMMS
 E.g., truly environmental bacteria
 MSCRAMM domain structure

 Gram positive MSCRAMMs have a number of

unique features
 MSCRAMMs are anchored into the cell wall
 Surface exposed
 The proteins must have –
 Signal sequence for secretion by the generalized Sec
pathway
 Cell wall anchor sequence for insertion into the cell wall by
sortase
 ECM binding domains, which will depend on the type of
ECM bound (may have one or more)
Collagen binding MSCRAMMs

 Cna – S. aureus
 CbpA – A. pyogenes
 CpCna – C. perfringens
 Ace – E. faecalis
 Acm – E. faecium
 Cne – S. equi
 Cpa – S. pyogenes
 Other Gram positive bacteria bind collagen, but the
genes responsible have not been identified
 Collagen binding MSCRAMM
domain structure
 N-terminal signal sequence and C-terminal
cell wall anchor
 A domain
 Contains the collagen binding domains
 B domains
 1-4 repeated domains
 ~80-200aa long (60-100% aa identity)

Signal A domain B domains Cell wall

sequence anchor
 Cna – A domain
 The structure of the
collagen‑binding A domain
(green) contains a trench
similar to that seen in the
collagen binding domain of
β1 family integrins
 The orange lines represent
the collagen triple helix
 Cna – B domains

 The number of B domains in a collagen

binding protein varies from strain to
strain
 This is the case for collagen binding proteins
from different bacteria
 Modeling studies show that B domains
pack in a zig-zag fashion
 They may expand and contract from the
bacterial cell wall and so aid in the
projection of the A domain away from
the cell surface
 Specificity

 Although all collagen types are unique, there is some

structural conservation between some of the types
 Most collagen adhesins bind preferentially to
one/two types
 Higher affinity binding
 However, many collagen adhesins will bind to more
than one type of collagen
 Most collagen adhesins require the collagen
structure to be intact for binding to occur
 CpCna

 A collagen binding protein of C. perfringens

 Binds type I collagen (only type tested so far)
 Only plasmid-encoded MSCRAMM known
 Immediately adjacent to the cna gene is a
gene encoding sortase
 Only shares 15.4% amino acid identity and
35.2% similarity to Cna from S. aureus
 Role of collagen binding in
disease
 Cna – osteomyelitis, endocarditis
 In mice were infected IV with wildtype S. aureus or a cna
knockout (model of hematogenous osteomyelitis)
 70% of mice infected with wildtype S. aureus had osteomyelitis
in the hind leg vs. 5% of mice infected with the cna knockout
 Rats with surgically traumatized heart valve (model of
endocarditis) were infected with wildtype S. aureus or a cna
knockout
 The wildtype adhered better than the cna knockout
 When both wildtype and cna knockout were co-administered,
the wildtype out-competes the mutant
 Role of collagen binding in
disease
 CbpA – osteomyelitis (?)
 100% of A. pyogenes osteomyelitis isolates (n=5)
carry cbpA
 In contrast, only 48% of all A. pyogenes isolates
(n=75) carry this gene
 Ace – periodontal disease (?)
 Wildtype E. faecalis adheres to exposed tooth roots
(dentin – collagen type I) better than an ace knockout
Fibronectin binding MSCRAMMs

 SfbI (PrtF1), F2 (PFBP), M1 and M3 proteins,

Fbp54, Fba, FbaB - S. pyogenes
 ScpB - S. agalactiae
 FnBB – S. dysgalactiae
 FNE, FNZ, SFS – S. equi
 FnbpA, FnbpB - S. aureus
 Similarly, a number of other bacteria bind
fibronectin, but the genes involved have not
been characterized
 SbfI binding to fibronectin

 Best studied fibronectin adhesin of S. pyogenes

 Binds to fibronectin through two domains
 C-terminal repeat region – fibrin binding domain
 Non-repetitive domain UR – collagen binding domain

 Binding through these two domains is important for

subsequent invasion
 S. pyogenes SfBI
protein
 SfBI can also “recruit”
collagen type I or IV
via pre-bound
fibronectin
 This enables the
bacteria to form
collagen-coated
aggregates and allows
the bacteria to adhere
to the collagen matrix
(without having a collagen
adhesin)
 S. aureus FnbpA and FnbpB
 Most isolates express these related proteins encoded
by linked genes
 These proteins bind the N-terminus of fibronectin by
their C-terminal repeats, in a similar manner to that of
SfbI
 These proteins also bind to fibrinogen and elastin
 Both proteins can adhere to platelets, but only FnbpA
can aggregate them
Fibrinogen binding
MSCRAMMs
 Clumping factor A and B (ClfA, ClfB) – S.
aureus
 FnbpA and B – S. aureus also binds
fibrinogen
 Fbe – S. epidermidis
 FbsA and FbsB – S. agalactiae
 Fibrinogen binding MSCRAMM
domain structure

Signal A domain R domain Cell wall

sequence anchor

 A: Fibrinogen binding domain

 R: Serine-aspartate repeat region; forms a stalk
 ClfA and ClfB have an identical domain structure
 Fibrinogen binding proteins appear to have a less complex
domain structure compared with fibronectin and collagen
binding proteins
 S. aureus ClfA and ClfB

 Enables S. aureus to clump in the presence of

fibrinogen – hence the name
 Allows S. aureus to adhere to fibrinogen-containing
substrates such as plaques in blood vessels
 For ClfA, fibrinogen binding occurs through binding of
the A domain to the γ chain of fibrinogen
 For ClfB, binding is through the α and β chains
 ClfB also binds cytokeratin
 Thought to be important in nasal colonization
Fibrinogen binding in disease
 S. aureus is an important cause of infective
endocarditis in patients without a history of prior
heart valve damage
 S. aureus uses ClfA to coat itself with fibrinogen
 The fibrinogen-coated bacteria engage resting platelet
glycoprotein GPIIb/IIIa and anti-ClfA antibodies
 Subsequent signal transduction leads to activation of
GPIIb/IIIa and aggregation of platelets
 A clfA- mutant has reduced virulence in a rat model of
endocarditis
 Also, L. lactis strains expressing ClfA can adhere to heart
tissue
Downstream regulation

 Once bacteria are successfully attached to the host

they have limited options
 They can remained attached, but will eventually
become displaced when host cells turn over
 Gut - 1-2 days
 Most other mucous membranes - 5-7 days
 Bacteria can reattach to a new host cell, but they are
still at the mercy of host specific and innate
defenses
 This is not a problem for commensal bacteria
 Bacteria can invade into the host cell
 Requires both interaction of bacteria with host cell
molecule(s) AND reorganization of host cytoskeletan
 Bacteria can invade into healthy cells
 However, tissue damage through trauma,
inflammation and/or other microbial infections can
expose “different” tissue for the bacteria to adhere to
and subsequently invade
 For this to occur, bacteria may need additional
adhesins
 Linking adhesion to invasion

 Binding of the SfbI repeat region to

the N-terminal fibrin-binding domain
of fibronectin co-operatively activates
the adjacent SfbI UR domain to bind
the fibronectin collagen binding
domain
 The repeat region of SfbI mediates
adherence and constitutes a
prerequisite for subsequent invasion
 The SfbI UR domain efficiently
triggers invasion into host cells
Prophylactic potential of
MSCRAMMs
 Due to increasing antibiotic resistance, we need
novel methods for disease prophylaxis and/or
treatment
 Anti-adhesion therapy and immunity can:
 Preventing adhesion of the bacteria with a vaccine
 Reversing adhesion of the bacteria with an agonist
 The major drawback is that most bacteria have
multiple mechanisms for host cell adhesion
 It may be necessary to use multiple agents or a
broadly-effective agent
 Receptor analogs - adhesion
agonists

Bacteria that lack adhesins are In the presence of adhesion agonists,

swept away bacteria are no longer able to bind
 Adhesion agonists

 This approach has been successful with pathogens

that bind via carbohydrate-specific adhesins
 The agonist is structurally similar to the
glycoprotein or glycolipid receptor
 There have been few clinical trials that
have shown efficacy of adhesion agonist therapy
 However, drinking cranberry juice (which contains
mannose) can -
 Displace uropathogenic E. coli from the urinary tract
epithelium preventing bladder infections
 Reduce colonization by S. mutans, a cause of dental caries
 Passive protection

 Aurexis®
 Humanized monoclonal antibody against ClfA
 For treatment of S. aureus bacteremia in adults
 Completed Phase II trials in 2005
 Is proceeding into Phase III testing
 No more information is available
 Vaccines

 Antibody binding to the MSCRAMM should block

binding to the ECM receptor
 With multi-factorial adhesion, it may not be possible
to prevent all infections
 However, by targeting specific MSCRAMMs, such
as collagen binding proteins, it may be possible to
prevent specific diseases
 Osteomyelitis, septic arthritis
 Periodontal disease
Experimental MSCRAMM
vaccines
 FnbpA and ClfA vaccination to prevent mastitis
 DNA vaccine was administered to dairy cattle with S. aureus
challenge
 Vaccinates had a 50% reduction in number of mastitis infections
compared with non-vaccinated controls
 IN vaccination with Sfb1 of S. pyogenes protects against IN
challenge, but not SC challenge
 SC challenge by-passes the need to adhere
 May not be a good model for skin infections
 Immunization with a fibronectin binding protein of S. equi
prevents a strangles in a mouse model
 Vaccinated horses have good antibody responses
 How do MSCRAMM antibodies
work?
 Opsonization of bacteria for PMN-
macrophage ingestion and killing
 The role of complement-mediated killing may
also be involved
 However, it is still unclear to what extent
inhibition of bacterial adhesion contributes to
the in vivo prophylactic or therapeutic effect