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, -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

Intracellular

Eukaryotic Cell

Receptor

Virulent Bacteria

Prokaryotic Cell

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

Adherence blockers

Pili or adhesins

Prokaryotic and Eukaryotic Interactions

Intracellular

Eukaryotic Cell

Receptor

Virulent Bacteria

Prokaryotic Cell

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

COLONIZATION

Adherence blockers

Pili or adhesins

Prokaryotic and Eukaryotic Interactions

Intracellular

Eukaryotic Cell

Receptor

Virulent Bacteria

Prokaryotic Cell

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

COLONIZATION INVASION

Adherence blockers

Pili or adhesins

TERMS USED TO DESCRIBE ADHERENCE FACTORS IN HOST-PARASITE INTERACTIONS

ADHERENCE FACTOR

DESCRIPTION

Adhesin A surface structure or macromolecule that binds a bacterium to a specific surface

ReceptorA complementary macromolecular binding site on a (eukaryotic) surface that binds specific adhesins 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

FimbriaeFilamentous proteins on the surface of bacterial cells that may behave as adhesins for specific adherence

Common pili Same as fimbriae

Sex pilus A specialized pilus that binds mating procaryotes together for the purpose of DNA transfer

Type 1 fimbriaeFimbriae in Enterobacteriaceae which bind specifically to mannose terminated glycoproteins on eukaryotic cell surfaces

GlycocalyxA layer of exopolysaccharide fibers on the surface of bacterial cells which may be involved in adherence to a surface

CapsuleA detectable layer of polysaccharide (rarely polypeptide) on the surface of a bacterial cell which may mediate specific or nonspecific attachment

Lipopolysaccharide (LPS)A distinct cell wall component of the outer membrane of Gram-negative bacteria with the potential structural diversity to mediate specific adherence. Probably functions as an adhesin

Teichoic acids and lipoteichoic acids (LTA)

Cell wall components of Gram-positive bacteria that may be involved in nonspecific or specific 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 Mechanisms of Adherence to Cell Adherence to Cell or Tissue Surfacesor Tissue Surfaces

Nonspecific adherence:

Reversible attachment of the bacterium to the eukaryotic surface (sometimes called "docking") Possible interactions and forces involved are:

1. hydrophobic interactions

2. electrostatic attractions

3. atomic and molecular vibrations resulting from fluctuating dipoles of similar frequencies

4. Brownian movement

5. 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:

1. The bacteria will bind isolated receptors or receptor analogs.

2. The isolated adhesins or adhesin analogs will bind to the eukaryotic cell surface.

3. 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 pyogenes

Protein FAmino terminus of fibronectin

Pharyngeal epithelium

Sore throat

Streptococcus mutans

Glycosyl transferase

Salivary glycoprotein Pellicle of tooth Dental caries

Streptococcus salivarius

Lipoteichoic acid

Unknown Buccal epithelium of tongue 

None

Streptococcus pneumoniae

Cell-bound protein

N-acetylhexosamine-galactose disaccharide

Mucosal epithelium pneumonia

Staphylococcus aureus

Cell-bound protein

Amino terminus of fibronectin

Mucosal epithelium Various

Neisseria gonorrhoeae

N-methylphenyl- alanine pili

Glucosamine-galactose carbohydrate

Urethral/cervical epithelium

Gonorrhea

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 Signal sequencesequence

A domainA domain B domainsB domains Cell wall Cell wall anchoranchor

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 1 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

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

Signal Signal sequencesequence

A domainA domain R domainR domain Cell wall Cell wall anchoranchor

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 chain of fibrinogen

For ClfB, binding is through the and 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

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

Downstream regulation

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 swept away

In the presence of adhesion agonists, 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