Intermolecular forces and enthalpies in bacterial adhesion

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

Van der Waals 1837-1923 Three types of LW forces

LW forces between molecules are weak and short-ranged distance r E = A/r 12 B/r 6

Electrostatic interactions between ions q 1 q 2 E = 1/4πε r ε 0 q 1 x q 2 /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, 1923- The distance dependence of hydrogen interaction depends on the substratum hydrophobicity and folows an exponential decay E (:) exp (l 0 -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) l 0 = 0.157 nm

From intermolecular to macroscopic interaction forces

Additivity concept by Hamaker in 1937 E = - dv 1 dv 2 n 1 x n 2 x F 12 (r 12 ) Volume 1 Volume 2 where n 1 and n 2 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 = - A 132 x R/6 x l where A 132 follows from ΔG 132 LW EL interactions E = ε x R x Ψ 2 ln(1+exp(- κ x l)) AB interactions E = 2 x π x R x λ x ΔG 132 AB x exp((l 0 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

Lewis Acid-base interactions All the same, physico-chemical forces

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

Adhesion kinetics of S. mutans LT11 ( ) and IB03987 ( ) to laminin films in a parallel plate flow chamber at ph 6.8 30 25 n (x10 6.cm -2 ) 20 15 10 5 0 0 5000 10000 15000 time (sec)

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 [10 6 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

Mixed suspensions of co-aggregating pairs form visibly discernable aggregates consisting of both cell types

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) 3 g max 2 1 0 0 10 20 30 40 50 r

Radial distribution function g max 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

Agg- Non specific interaction between bacteria Agg+ P P P Specific interaction between bacteria Receptor Aggregation substance P Sex pheromone plasmid

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 Thiols on gold Silanes on silicon Dupres et al., Biomaterials 2006

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 Avidin-biotin Avidin-iminobiotin Streptavidin-biotin Avidin-desthiobiotin Streptavidin-iminobiotin VSM cell receptor-fibronectin Interaction force [nn per single bond] 0.160 0.085 0.257 0.094 0.135 0.039 Reference Moy et al., Science 1994 Florin et al., Science 1994 Sun et al., 2005 S. carlsbergensis-carbohydrate S. carlsbergensis-mannose spec. lectin Fv fragment of antilysozyme-lysozyme 0.121 0.117 0.050 Touhami et al., 2003 Berquand et al., 2005 In summary 0.117 Blocked single bonds 0.005 nn

Single biotin-streptavidin bonds demonstrate a shift in peak position and width with an increase in loading rate. Merkel et al., Nature 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 Proteins on 20 nm radius AFM tips Bacteria on poly-l-lysine coated glass

Experimental procedure and analysis for AFM 12 10 8 approach retraction Force (nn) 6 4 2 0-2 -4 0 2μm 0 50 100 150 200 250 300 350 Separation distance (nm)

Distribution of the adhesion force F adh 250 ph 5.8 S. mutans LT11 (black bars) and IB03987 (grey bars) Frequency 200 150 100 in the retracting mode of a laminin coated AFM tip toward the cell surfaces. 50 0 0-0.5-1 -1.5-2 -2.5-3 -3.5-4 F max (nn) -4.5-5 S. mutans LT11 S. mutans IB03987 250 ph 6.8 200 Frequency 150 100 50 0 0-0.5-1 -1.5-2 -2.5-3 -3.5-4 F max (nn) -4.5-5 S. mutans LT11 S. mutans IB03987 Each histogram involves 200-300 force-distance curves, over5 different bacteria.

Interaction force Interaction force Bacterial adhesion in presence of specific phenomenon in absence of specific phenomenon Reference [nn] [nn] Streptococci to salivary films Xu et al., ph 5.8 Median 0.0 Range -1.2 Median 0.0 Range -0.1 2006 ph 6.8 Median -0.4 Range -2.9 Median 0.1 Range -0.4 Streptococci to laminin films ph 5.8 Median 0.0 Range -5.0 Median 0.0 Range -1.5 Busscher et al., 2006 ph 6.8 Median -0.1 Range -4.9 Median 0.1 Range -2.1 Co-aggregation between actinomyces and streptococci Mean -3.0 to -4.0 Mean -1.0 Postollec et al., 2006 Aggregation between enterococci Mean -2.3 to -2.6 Mean -1.2 to -1.5 Waar et al., 2005 In summary ph dependence Increases with ph Increases with ph Force value -3 to -5 0 to -2

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 Streptococcal 4x 60 μl protein suspension solution 3 (laminin times 80 at μml, 100 3 μg/ml, x 10 9 ml saliva -1 at 1.4 mg/ml) 1 reaction ampoule 2 reference ampoule 3 Peltier element 4 heat sink 2 1 3 4 Actinomycessuspension Streptococcal suspension 1.5 ml, 35 x 10 99 ml ml -1-1

80 75 coaggregating pair Power (µw) 70 65 60 55 50 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 Time (s) 75 70 non-coaggregating pair Power (µw) 65 60 55 50 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 Time (s)

Molecular bond Avidin-biotin Avidin-iminobiotin Streptavidin-biotin Avidin-desthiobiotin Streptavidin-iminobiotin Oligosaccharides with Pseudomonas lectin PA-IIL Interaction enthalpy [10-16 mj/molecule] -1.4-0.8-2.2-0.9 NA -0.3 to -0.6 Reference Moy et al., Science 266(1994)257-259 Perret et al., Biochem J 389(2005)325-332 In summary -1.3

Bacterial adhesion Interaction enthalpy Interaction enthalpy Reference in presence of specific phenomena in absence of specific phenomena [10-9 μj per bacterium] [10-9 μj per bacterium] Salivary proteins to Xu et al., streptococci 2006 ph 5.8-614 -60 ph 6.8-2073 -165 Laminin to streptococci Busscher et al., 2006 ph 5.8-61 +115 ph 6.8-63 -1 Co-aggregation between actinomyces and streptococci -18000-3000 Postollec et al., 2006 In summary Always negative to very negative Little negative, Sometimes positive

How many specific receptors per bacterium?? Specific forces measured in phenomena -4 nn Interaction force per molecule 0.117 nn Yields 30 molecules per bond Contact radius is about 1/50 of the bacterial cell radius Yields 7-8 x 10 4 binding sites per bacterium

How many specific receptors per bacterium?? Interaction enthalpies measured in protein adsorption phenomena Interaction enthalpy -500 x 10-12 mj/bacterium -1.3 x 10-16 mj/molecule Yields 5 x 10 6 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 nm 2 per specific sites (IgG), we can maximally accomodate 4 x 10 4 (7-8 x 10 4 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)