Intermolecular forces and enthalpies
in bacterial adhesion
Henk J. Busscher, Henny C. van der Mei and Willem Norde
University Medical Center Groningen and University of Groningen
Department of BioMedical Engineering
Groningen
The Netherlands
F
Twenty years ago, a typical paper on microbial adhesion to surfaces would start:
“microbial adhesion results from highly specific interaction forces
between stereo-chemical component on the interacting surfaces”
OR
“”microbial adhesion is a result of colloidal interactions
involving macroscopic properties of the interacting surfaces,
such as charge and hydrophobicity ”
Set-up of this talk
Molecular interaction forces and enthalpies in
specific and non-specific microbial adhesion approaches
The phenomena
(adhesion and aggregation, co-adhesion and co-aggregation
Interaction forces and their measurement
Interaction enthalpies and their measurement
The magnitude of specific and non-specific interaction forces, and
the number of receptors involved in microbial adhesion to surfaces
There are only few fundamental physico-chemical forces:
Lifshitz-van der Waals forces
Lewis acid-base interactions (“basis for hydrophobicity”)
Electrostatic forces
Three types of LW forces
Van der Waals
1837-1923
LW forces between molecules are
“weak” and “short-ranged”
distance r
E = A/r12 – B/r6
Electrostatic interactions between ions
q1
q2
E = 1/4πεrε0 q1 x q2/r
Lewis acid-base interactions
A Lewis acid is a substance, such as the H+ ion, that can accept electrons (γ++).
A Lewis base is a substance, such as the OH- ion, that can donate electrons (γ--).
e
Lewis, 1875-1946
Van Oss, 1923The distance dependence of hydrogen interaction depends
on the substratum hydrophobicity and folows an exponential decay
E (:) exp (l0 - l)/λ
λ equals 0.2 nm for water,
and is suggested to be 0.6 nm up to 1 nm (in the repulsive mode)
l00 = 0.157 nm
From intermolecular to macroscopic interaction forces
Additivity concept by Hamaker in 1937
E=Volume 1
dv1 dv2 n1 x n2 x F12(r12)
Volume 2
where n1 and n2 are the molecular densities in volumes 1 and 2, respectively
Distance dependence of the interaction forces
for macrocopic configurations
(sphere (radius R)-plate)*
LW interactions
E = - A132 x R/6 x l
where A132 follows from ΔG132LW
EL interactions
E = ε x R x Ψ2 ln(1+exp(- κ x l))
AB interactions
E = 2 x π x R x λ x ΔG132AB x exp((l0 – l)/ λ)
* Distance dependence, but not coefficients, are the same for sphere-sphere configuration
The thermodynamic approach
Classical DLVO theory
Electrostatic repulsion
Left panel:
Low ionic strength
Κ-1 = 0.96 nm
Right panel:
High ionic strength
Κ-1 = 0.3 nm
Electrostatic attraction
Extended DLVO theory
Strong electron-donating (mono-polar) repulsion
between microorganism and substratum
Microbial adhesion can be (sometimes) qualitatively explained
by the (extended) DLVO theory,
but there are at least as many exceptions to as
confirmations of the rule.
This is because we do not know the nature and distribution
of local high affinity sites on the microbial cell surfaces,
although also at that level
the same fundamental interaction forces operate.
Busscher and Weerkamp,
Specific and non-specific interactions in bacterial adhesion to solid substrata
FEMS Microbiology Reviews 46(1987)165-173
Busscher, Cowan and Van der Mei,
On the relative importance of specific and non-aspecific approaches to (oral) microbial adhesion
FEMS Microbiology Reviews 88(1992)199-210
All the same,
physico-chemical forces
Lewis Acid-base interactions
Forces strive to yield a thermodynamic equilibrium
At constant temperature T and pressure p, all physico-chemical interactions
contribute to changes in the Gibbs energy (G) of a system.
For a spontaneous process, the change in Gibbs energy (ΔG) is negative.
ΔG is composed of a change in enthalpy (H) and in entropy (S), according to
ΔG = ΔH – T ΔS
where T is the temperature in Kelvin.
The enthalpy tends to reach a minimum value reflecting the energetically most stable state,
whereas the entropy strives for a maximum corresponding to the highest degree of randomness.
Enthalpy-Entropy compensation
Ligand-receptor binding can occur in three ways:
The receptor becomes
(i) more ordered
(beneficial enthalpy, entropically expensive)
(ii) less ordered
(enthalpic costs, entropic benefits)
(iii) remain unchanged
(ΔS = 0).
Williams et al., J Mol Biol 340(2004)373-383
Aim of this talk is to answer three questions:
1. Is the interaction force influential
on microbial adhesion phenomena?
2. Can we separately measure the values
of specific and non-specific forces
and enthalpies in microbial adhesion?
3. How many receptors-specific bonds are
actually involved in specific adhesion?
The phenomena
Bacterial adhesion in the absence and presence of specific receptor sites
Co-aggregating and non co-aggregating oral bacterial pairs
Surface aggregation of enterococci with and without Agg
The phenomena
Bacterial adhesion in the absence and presence of specific receptor sites
Co-aggregating and non co-aggregating oral bacterial pairs
Surface aggregation of enterococci with and without Agg
Adhesion kinetics of S. mutans LT11 (■) and IB03987 (▲)
to laminin films in a parallel plate flow chamber at pH 6.8
30
6
-2
n (x10 .cm )
25
20
15
10
5
0
0
5000
10000
time (sec)
15000
Adhesion of S. mutans LT11 and isogenic mutant without antigen I/II, IB03987
to laminin and salivary conditioning films
in a parallel plate flow chamber
(shear rate 10 s-1) from adhesion buffer at pH 5.8 and 6.8.
Suspension pH
Initial deposition rate
[cm-2 s-1]
Number after 4 h
[106 cm-2]
LT11
IB03987
LT11
IB03987
5.8
1433 ± 178
137 ± 72
21.8 ± 1.7
0.8 ± 0.4
6.8
1957 ± 399
363 ± 250
26.1 ± 0.9
1.1 ± 0.7
5.8
1315 ± 28
1258 ± 169
12.7 ± 1.1
10.5 ± 2.1
6.8
1679 ± 165
1441 ± 119
9.6 ± 2.3
2.5 ± 0.7
The phenomena
Bacterial adhesion in the absence and presence of specific receptor sites
Co-aggregating and non co-aggregating oral bacterial pairs
Surface aggregation of enterococci with and without Agg
“Mixed suspensions of co-aggregating pairs
form visibly discernable aggregates
consisting of both cell types”
The phenomena
Bacterial adhesion in the absence and presence of specific receptor sites
Co-aggregating and non co-aggregating oral bacterial pairs
Surface aggregation of enterococci with and without Agg
Background:
Often, the bile is drained after an operation
E. faecalis most frequent microorganism in bile
Biofilm formation may yield clogging of the drain
References:
- Waar et al., Enterococcus surface proteins determine
its adhesion mechanisms to bile drain materials
Microbiology 148(2002)3855-3858.
- Waar et al., AFM on specificity and non-specificity
of E. faecalis with and without aggregation substance
Microbiology (2005) 151(2005)2459-2464.
Adhesion of Enterococcus faecalis
to hydrophobic biomaterials surfaces
Agg-
Agg+
Analysis by Radial distribution functions
dr
r
Radial distribution function
g(r)
gmax
3
2
1
0
0
10
20
r
30
40
50
Radial distribution function gmax
Strain
FEP
PE
SR
Agg-
1.4
1.8
2.3
Agg1+
3.2
2.7
2.8
Agg373+
2.0
2.7
2.6
Non specific interaction
between bacteria
Agg-
P
P
Agg+
Receptor
Aggregation substance
P
Sex pheromone plasmid
P
Specific interaction
between bacteria
Interaction forces and their measurement:
Atomic force microscopy
Binnig, 1947(Nobel price, 1986)
Hinterdorfer and Dufrene, Nature Methods 3(2006)347-355
Atomic force microscopy:
single contact strategies I
Protein physisorption
Dupres et al., Biomaterials 2006
Thiols on gold
Silanes on silicon
Atomic force microscopy:
single contact strategies II
Single lectin (concavalin A)-carbohydrate adhesion is accompanied
by an adhesion force of around 100 pN
Molecular bond
Interaction force
[nN per single bond]
Reference
Avidin-biotin
Avidin-iminobiotin
Streptavidin-biotin
Avidin-desthiobiotin
Streptavidin-iminobiotin
0.160
0.085
0.257
0.094
0.135
Moy et al., Science 1994
Florin et al., Science 1994
VSM cell receptor-fibronectin
0.039
Sun et al., 2005
S. carlsbergensis-carbohydrate
S. carlsbergensis-mannose spec. lectin
0.121
0.117
Touhami et al., 2003
Fv fragment of antilysozyme-lysozyme
0.050
Berquand et al., 2005
“In summary”
0.117
“Blocked single bonds” ≤ 0.005 nN
Single
Single biotin-streptavidin
biotin-streptavidin bonds
bonds demonstrate
demonstrate
aa shift
shift in
in peak
peak position
position and
and width
width
with
with an
an increase
increase in
in loading
loading rate.
rate.
Merkel
Merkel et
et al.,
al., Nature
Nature 397(1999)50-53
397(1999)50-53
Real life adhesion: Multiple contacts over an unknown surface area
Immobilization
For (co-)aggregation
For adhesion to protein films
A method for anchoring round shaped cells for atomic force
microscopy. Kasas and Ikai, Biophysics 68 (1995) 1678-1680.
Bacteria on poly-L-lysine coated
tipless cantilever
Bacteria on poly-L-lysine coated glass
Proteins on 20 nm radius AFM tips
Experimental procedure and
analysis for AFM
Force (nN)
12
10
approach
8
retraction
6
4
2
0
-2
-4
0
50
100
150
200
250
Separation distance (nm )
0
2μm
300
350
Distribution of the adhesion force Fadh
pH 5.8
250
S. mutans LT11 (black bars) and
Frequency
IB03987 (grey bars)
200
150
100
50
in the retracting mode
of a laminin coated AFM tip
toward the cell surfaces.
0
0
-0.5
S. mutans LT11
-1
-1.5
-2
-2.5
-3
Fmax (nN)
S. mutans IB03987
-3.5
-4
-4.5
-5
pH 6.8
250
Frequency
200
150
100
50
0
0
-0.5
S. mutans LT11
-1
-1.5
-2
Fmax (nN)
-2.5
-3
S. mutans IB03987
-3.5
-4
-4.5
-5
Each histogram involves 200-300 force-distance curves, over5 different bacteria.
Bacterial adhesion
Streptococci to salivary films
pH 5.8
pH 6.8
Streptococci to laminin films
pH 5.8
pH 6.8
Co-aggregation between
actinomyces and streptococci
Aggregation between
enterococci
“In summary”
pH dependence
Force value
Interaction force
in presence of specific
phenomenon
[nN]
Median 0.0 Range -1.2
Median -0.4 Range -2.9
Median 0.0 Range -5.0
Median -0.1 Range -4.9
Mean -3.0 to -4.0
Interaction force
in absence of specific
phenomenon
[nN]
Median 0.0 Range -0.1
Median 0.1 Range -0.4
Median 0.0 Range -1.5
Median 0.1 Range -2.1
Mean -1.0
Mean -2.3 to -2.6
Mean -1.2 to -1.5
Increases with pH
-3 to -5
Increases with pH
0 to -2
Reference
Xu et al.,
2006
Busscher et al.,
2006
Postollec et al.,
2006
Waar et al.,
2005
Interaction enthalpies and their measurement:
Isothermal Titration Calorimetry
The enthalpy of a system is directly related to its heat content
and at constant pressure,
and if no work other than
that related to volume change is involved,
changes in the enthalpy can be determined
as the heat exchange
between a system and its environment.
Isothermal Reaction Calorimetry
1
2
3
4
4x 60 μl protein
solution
Streptococcal
suspension
1003μg/ml,
saliva
at 1.4 mg/ml)
-1
3(laminin
times 80 at
μml,
x 109 ml
2
3
1
Actinomycessuspension
Streptococcal suspension
1.5
1.5ml,
ml,35xx10
1099ml
ml-1-1
4
reaction ampoule
reference ampoule
Peltier element
heat sink
80
coaggregating pair
Power (µW)
75
70
65
60
55
50
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
Time (s)
75
non-coaggregating pair
Power (µW)
70
65
60
55
50
0
1000
2000
3000
4000
5000
Time (s)
6000
7000
8000
9000
Molecular bond
Avidin-biotin
Avidin-iminobiotin
Streptavidin-biotin
Avidin-desthiobiotin
Streptavidin-iminobiotin
Oligosaccharides with
Pseudomonas lectin PA-IIL
“In summary”
Interaction enthalpy
[10-16 mJ/molecule]
-1.4
-0.8
-2.2
-0.9
NA
-0.3 to -0.6
-1.3
Reference
Moy et al., Science
266(1994)257-259
Perret et al., Biochem J
389(2005)325-332
Bacterial adhesion
Salivary proteins to
streptococci
pH 5.8
pH 6.8
Laminin to
streptococci
pH 5.8
pH 6.8
Co-aggregation
between actinomyces
and streptococci
“In summary”
Interaction enthalpy
Interaction enthalpy
in presence of specific
phenomena
[10-9 μJ per bacterium]
in absence of specific
phenomena
[10-9 μJ per bacterium]
Reference
Xu et al.,
2006
-614
-2073
-60
-165
Busscher et al.,
2006
-61
-63
+115
-1
-18000
-3000
Always negative to very
negative
Little negative,
Sometimes positive
Postollec et al.,
2006
How many specific receptors per bacterium??
Specific forces measured in phenomena -4 nN
Interaction force per molecule
Yields
0.117 nN
30 molecules per bond
Contact radius is about 1/50 of the bacterial cell radius
Yields
7-8 x 104 binding sites per bacterium
How many specific receptors per bacterium??
Interaction enthalpies measured
in protein adsorption phenomena -500 x 10-12 mJ/bacterium
Interaction enthalpy
Yields
-1.3 x 10-16 mJ/molecule
5 x 106 binding sites per bacterium
BUT, …
conformations will differ
Summary of conclusions ans synthesis:
We can separate specific and non-specific microbial interaction phenomena
in AFM and ITC.
A factor of 2-3 in interaction force, has major impact on microbial adhesion
to protein films under flow and microbial (co-)aggregation.
Considering 100 nm22 per specific sites (IgG),
we can maximally accomodate 4 x 1044 (7-8 x 1044 from AFM!)
sites per bacterium, hence
-sites must be arranged along surface structures.
-2 sites per laminin molecule
Immuno-gold labeled streptococci with and without antigen I/II
F
Thank you for your attention!
(this presentation and references herein can be downloaded from www.bme-umcg.nl)