Langmuir 1994,10, 3461-3465
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Interactions between Cationic Vesicles and Escherichia coli G . N. Tapias,? S. M. Sicchierolli,t E. M. Mamizuka,* and A. M. Carmona-Ribeiro*>t Departamento de Bioquimica, Instituto de Quimica, Universidade de Sdo Paulo, CP 20780, Sdo Paulo, Brazil, and Departamento de Ancilises Clinicas e Toxicolbgicas, Faculdade de C i h c i a s FarmacCuticas, Universidade de Sdo Paulo, CP 66083, Sdo Paulo, Brazil Received January 17, 1994. I n Final Form: July 20, 1994@ The interaction between cationic vesicles composed of dioctadecyldimethylammonium bromide (DODAB) or chloride (DODAC) and Escherichia coli is described. Adsorption isotherms point to a n interaction of high affinity (nonreversible adsorption) with limiting adsorption values for the amphiphiles which are compatible with vesicle adhesion to bacteria without vesicle rupture. Bacteria (10' bacteridml) in the mixtures attain a point of zero charge (pzc)at 4 x M DODAB as determined using microelectrophoresis. From differential turbidity measurements and optical micrographs, bacteria flocculation promoted by the vesicles is characterized and quantified. Initial flocculation rate ( U O ) for bacteria ((1.4-2.0) x lo9bacterid mL) goes through a maximum as a function of amphiphile concentration at 0.4mM DODAB or DODAC. The results may be of importance for improving water quality in contaminated reservoirs.
Introduction The interaction between bilayer-forming amphiphiles and surfaces is poorly understood. A few studies of liposome adsorption on clay, asbestos, and B i ~ b e a d s , ' - ~ incidental references to liposome adsorption by gel filtration columns and membrane filter^,^!^ some indirect evidence for adsorption of phospholipid bilayers onto mica sheet^,^ and some studies on adsorption of phospholipid vesicles onto glass are available. Recently, we reported phospholipid adsorption from vesicles onto polystyrene microsphereslO and bilayer adsorption from synthetic amphiphile vesicles onto oppositely charged latexes." Here we describe the interaction in water solution between cationic vesicles made up of dioctadecyldimethylammonium bromide (DODAB) or chloride (DODAC) and Escherichia coli, a Gram negative bacterium belonging to the family Enterobacteriaceae. Adsorption isotherms for amphiphiles onto bacteria, electrophoretic mobility of the bacteria in the interacting mixtures, and data on bacteria flocculation induced by vesicles a r e presented. A model for vesicle adhesion to bacteria is proposed that accounts for the experimental data at limiting adsorption.
* To whom all correspondence should be addressed. t
Departamento de Bioquimica.
* Departamento de Analises Clinicas e Toxicologicas.
Abstract published in Advance A C S Abstracts, September 1, 1994. (1)Murase, N.;Gonda, K. J . Biochem. (Tokyo) 1982,92,271. (2)Jaurand, M. C.; Thomassin, J. H.; Baillif, P.; Magne, L.; Touray, J. C.; Bignon, J. Br. J . Int. Med. 1980,37,169. (3)Jaurand, M. C.; Baillif, P.; Thomassin,J. H.; Magne, L.; Touray, J. C. J . Colloid Interface Sci. 1983,95,1. (4)Phillippot, J.;Mutaftschiev, S.; Liautard, J. P. Biochim. Biophys. Acta 1983,734,137. ( 5 ) Huang, C. H . Biochemistry 1969,8,344. (6)Schulleqy,S. E.; Garzaniti,J. P. Chem. Phys. Lipids 1973,12,75. (7)Horn, R. G. Biochim. Biophys. Acta 1984,778,224. (8) Jackson, S.;Reboiras, M. D.; Lyle, I. G.; Jones, M. N. Faraday Discuss. Chem. SOC.1986,85,291. (9)Jackson, S.M.;Reboiras, M. D.; Lyle, I. G.; Jones, M. N. Colloids Surf. 1987,27,325. (10)Carmona-Ribeiro, A.M.; Herrington, T. M. J . Colloid Interface Sci. 1993,156,19. (11)Carmona-Ribeiro,A.M.; Midmore, B. R.Langmuir 1992,8,801.
Material and Methods Dioctadecyldimethylammonium chloride (DODAC) was obtained from Herga Industrias Quimicas S. A. (R.J.,Brazil). DODAC was purified and analyzed by mass spectrometry.12The dialkyl chain distribution was 60% Cle:Cle,38% c1&16, and 2% c1&16. Dioctadecyldimethylammonium bromide (DODAB)was obtained from Sigma and used without further purification. DODAC and DODAB concentrations were determined by microtitration13or by solubilization of a dye-amphiphile complex All other reagents were analytical grade in nonionic mi~e1les.l~ and were used without further purification. Water was Milli-Q quality. Large DODAC vesicles (LV) with 288 nm mean z-average diameter15were prepared by injecting a chloroformic solution of DODAC into a 0.264MD-glucose water solution.16 Small DODAB vesicles (SV) with 86 nm mean z-average diameterl5 were prepared by the sonication of the amphiphile in a 0.264 M D-glucose solution. SV were centrifuged at 104gfor 1h at 15 "C to remove any multilamellar liposomes. The supernatant containing the unilamellar vesicles was used within 1 h of the preparation. An enteropathogenicstrain ofEscherichiacoli (serogroup0111: H-)isolated originally from human feces was used. Bacteria were grown from a frozen stock in appropriate media overnight at 37 "C. Followingthe growth to the logarithmic phase, bacteria were washed 3 times in phosphate buffered saline at pH 7.2 (PBS)to eliminate the culture medium. A fourth and final wash was in a PBS solution containing2%formaldehyde. The bacteria number density determined from the MacFarland scale" for the bacteria preparation in formaldehyde is 2.1 x 1 O l o bacteridml. Due t o the low stability of DODAB or DODAC vesicles in the presence of salt,18-22bacteria were centrifuged (5000 rpm, 15
@
(12)Cuccovia,I. M.; Aleixo, R. M. V.; Mortara, R. A.; Berci-Filho, P.; Bonilha, J. B. S.;Quina,F. H.; Chaimovich, H . TetrahedronLett. 1979, 33,3065. (13)Schales, 0.; Schales, S. S. J . Biol. Chem. 1941,140,879. (14)Stelmo, M.; Chaimovich, H . Cuccovia, I. M. J . Colloid Interface Sci. 1987,117,200. (15)Carmona-Ribeiro, A.M.; Midmore, B. R. J . Phys. Chem. 1992, 96,3542. (16)Carmona-Ribeiro, A.M.; Chaimovich, H.Biochim.Biophys.Acta 1983,733,172. (17)Lennette, E.H., Ed. Manual of Clinical Microbiology, 3rd ed.; American Society for Microbiology: Washington, DC, 1980. (18)Carmona-Ribeiro, A.M.;Yoshida,L. S.;Chaimovich, H. J . Phys. Chem. 1985,89,2928. (19)Carmona-Ribeiro, A.M.;Chaimovich, H . Biophys. J . 1986,50, 621. (20)Carmona-Ribeiro, A. M. J . Phys. Chem. 1989,93,2630. (21)Carmona-Ribeiro, A. M. J.Phys. Chem. 1992,96,9555.
0743-746319412410-3461$04.50/00 1994 American Chemical Society
Tapias et al.
3462 Langmuir, Vol. 10, No. 10, 1994 min) and resuspended in D-glUCOSe 0.264 M before promoting interaction with vesicles. Cells and vesicles were kept in a perfectly isotonic environment throughout. Bacteria or vesicles were always diluted using the same solution in which the vesicles were prepared, namely D-glucose 0.264 M. Interaction between vesicles and bacteria was induced by adding the vesicles to the bacteria. Mixtures were thermostated at 25 "C for 1 h unless otherwise stated. Thereafter mixtures were centrifuged at 5000 rpm for 15 min to separate bacteria from vesicles. The adsorption isotherms for DODAB or DODAC onto the bacteria were obtained from DODAB or DODAC analysis in the supernatant and in the vesicle preparation. Adsorption was expressed as the number of amphiphile molecules adsorbed per bacterium. Surface area on a bacterium (S)was estimated geometrically from the radius of a sphere whose volume is equivalent to the volume of a prolate rodlike ellipsoid (a= 0.925 pm; b = 0.478 pm). Alternately, S was estimated from 2n(b2+ 2ab)by assuming the bacterium is rod-shaped. Both calculations are expected to underestimatethe bacterium surface area as the cell surface is not perfectly smooth. Total surface area on bacteria was calculated from the bacteria number density and the surface area of a bacterium estimated as above. Electrophoretic mobilities (EM) for bacteria in mixtures bacteria/DODABvesicles were measured using a Rank Brothers microelectrophoresis apparatus with a flat cell at 25 "C. Bacteria flocculation was determined by measuring turbidity at 400 nm as a function of time after vesicle addition using a Hitachi U-2000 spectrophotometer in the double beam mode. In the cuvette used as a reference, bacteria added to 0.264 M D-glucose only was used. Thus, turbidity measured after vesicle addition is basically due to bacteria aggregation induced by the vesicles. The time lag between mixingand recordingwas usually smaller than 10s. The initial flocculation rate (UO)was calculated from turbidity kinetics.
Results and Discussion 1. The Palisade Model for Cationic Vesicle Adhesion to E . coli. In this section, we present a model to calculate the number of DODAB or DODAC molecules adsorbed per bacterium at limiting adsorption in order to interpret properly experimental data described in the next section. It is assumed that vesicles adhere without rupture to the bacteria and surround the cell forming one layer of adjacent vesicles: the palisade. Calculations strongly depend on the geometric shape of the bacterium. The E . coli cell can be considered either as a prolate ellipsoid (a = 0.925 x m; b = 0.478 x m) or as a cylinder (length a and radius b). After adhesion of a palisade of spheric vesicles with radius r, we define a second prolate ellipsoid (A = a r; B = b r ) or a second cylinder (length a r and radius b r). The area of the second ellipsoid (8,)can be calculated by the equivalent sphere method. The area of the second cylinder (S,) is 2n[(b rI2 2(a r)(b r)l. S , is 5.155 x m2 for r = 0.043 x m and 6.9 x 10-l2 m2 for r = 0.140 x m. The total number of adjacent spheric vesicles surrounding the bacterium as a palisade ( n )is
+
+
+ +
+
+
+
+
where S is either S, or S,. From S,, for the small DODAB vesicles with 43 nm mean radius, n will be 697 vesicles and for the large DODAC vesicles with 140 nm mean radius n will be 88vesicles. From S,, for the small DODAB vesicles n will be 1087 vesicles and for the large DODAC vesicles it will be 137 vesicles. These figures can be converted to the total number of DODAB or DODAC molecules adhered per bacterium. From the vesicle radius (22)Carmona-Ribeiro, A. M.J . Phys. Chem. 1993,97,4247.
e
c
0 m n r-
9
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4
t
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1
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Figure 1. Isotherm for the adsorption of DODAB from small vesicles (85 nm mean diameter)onto Escherichiacoli in a water solution of 0.264 M D-glucose at 25 "C (A). At a final bacteria number density of 2 x lo9bacteridml, DODAB SV and bacteria were left interacting for 1 h before centrifugation and determination of DODAB concentration in the supernatant (C, mM). InB, at 1x lo7bacteria/&, electrophoreticmobilityforbacteria in the mixtures is shown as a function of DODAB concentration in the supernatant. The inset shows the same mobility data using a logarithmic scale for the abcissa. EM is zero at 4 x M DODAB. and the area per monomer (M)at the airlwater interfacez3 which is 0.6 nm2,the total number of DODAB or DODAC molecules adhered per bacterium (N) is given by
N = n8nr2/M
(2)
Therefore, by considering the bacterium as a prolate or as a cylinder, the palisade contains 5.4 x lo7or 8.4 x lo7 DODAB molecules per bacterium, respectively, due to adhesion of the small DODAB vesicles. For the palisade of large DODAC vesicles, the prolate and the cylinder yield, respectively, 7.2 x lo7 and 11.3 x lo7 DODAC molecules per bacterium. 2. Adhesion of Small and Largevesiclesto E . coli. Figure 1shows a typical adsorption isotherm from small DODAB vesicles onto E. coli in 0.264 M D-glucose at 25 "C. A mean curve was drawn from the experimental points. The limiting adsorption obtained from the plateau region corresponds to 8.4 x lo7 DODAB molecules per bacterium. From the surface area per bacterium (4.46 x m2 for a prolate) and the limiting adsorption value, the area per DODAB molecule adsorbed is 0.054 nm2.The usual area per monomer in DODAB monolayers at the air-water interface is 0.6 nm2.23 Thus a DODAB monolayer or bilayer cannot be the amphiphile assembly which is adsorbed onto the bacteria. From the palisade model described in section 1, the limiting adsorption expected for a smooth prolate or cylinder is 5.4 or 8.4 x lo7DODAB molecules, respectively, in a very good agreement with experimental values. It is important to state that the (23) Claesson, P. M.; Carmona-Ribeiro, A. M.; Kurihara, K. J . Phys. Chem. 1989,93,917.
Bacteria Flocculation Induced by Cationic Vesicles
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C/mM Figure 2. Isotherm for the adsorption of DODAC from large vesicles (288nm mean diameter)onto E . coli in a water solution O f D-glucose 0.264M at 25 "C. Bacteria number density in the mixtures is 1.4x logbacteridml. Large DODAC vesicles and bacteria were left interacting for 1 h before centrifugation and determinationof DODAC concentration in the supernatant (C, mM).
a
15
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0
I
2
3
t ime/min. Figure 4. Absorbance (400 nm) as a function of time after mixing E . coli and cationic vesicles in 0.264M D-glucose at 25 "C. Bacteria flocculation was induced by small DODAB (A) or large DODAC (B) vesicles. In A, final DODAB concentration is 0.20 (a), 0.40 (b), and 0.60 mM (c). In B, final DODAC concentration is 0.06 (a), 0.39 (b), and 1.00 mM (c). Bacteria number densities are 2.1 x lo9(A) and 1.4x lo9 bacteria per mL (B).
60
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IO9 bacteria
Figure 3. Adsorption of DODAB ( 0 )or DODAC (0)from small or large vesicles, respectively, as a function of the bacteria number density in the mixtures. "he total number ofmolecules adsorbed (a)or the number of molecules adsorbed per bacterium (b) are shown as a function of the total number of bacteria in the mixture. Mixtures were left interacting for 1 h before centrifugation and determination of amphiphile in the supernatant. estimate obtained from the palisade model assumes t h a t the bacterium surface is perfectly smooth, which of course it is not. Therefore, the slight overestimation in surface area for the bacterium resulting from the cylindric shape assumption seems to compensate fairly for the underestimation due to the surface roughness. Figure 2 shows the isotherm for the adsorption of DODAC from large DODAC vesicles in a 0.264M D-glucose solution at 25 "C. The 15.4 x lo7 DODAC molecules per bacterium shield the bacterium at limiting adsorption.
Once again the validity of the palisade model can be checked. For large DODAC vesicles with 280 nm diameter and a n area per monomer of 0.6 nm2, one palisade of vesicles surrounding the bacterium cylinder corresponds to 11.3 x lo7 molecules per bacterium. Therefore, more than one palisade of large DODAC vesicles seems to be adhering to the bacterium. The electrophoretic mobilities of the bacteria in mixtures DODAB SVhacteria as a function of DODAB concentration in the supernatant is in Figure 1B. At lo7bacterid mL, there is a point where EM is zero and DODAB concentration in the supernatant is 4 x M. At limiting adsorption, the mobility curve attains a plateau region at a positive EM value. Figure 3 shows DODAB or DODAC adsorption from small or large vesicles, respectively, as a function of the total number ofbacteria in the interactingmixtures. From 1 x l o 9up to 5 x lo9bacteria in the mixture, the number of amphiphile molecules adsorbed per bacterium varies between 7 and 10 x lo7DODAB or 15 and 19 x lo7DODAC molecules per bacterium. These figures are consistent with limiting adsorption values obtained from the adsorption isotherms (Figures 1and 2). Curiously, adsorption seems to increase for total number ofbacteria smaller than lo9. This might well suggest vesicle rupture and fusion in the palisade with adhesion of more vesicles and perhaps multibilayer adsorption. Defective hydrophobic regions in the DODAC bilayer were reported to exist.24 Proximity and the hydrophobic interaction could well lead to fusion. Also, the bilayer hydrophobicity could overcome the electrostatic repulsion between vesicles and attract other vesicles to the palisade. (24) Carmona-Ribeiro, A. M.
J.Phys. Chem. 1993,97, 11843.
3464 Langmuir, Vol. 10, No. 10,1994
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\
1
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&/mM Figure 5. Amphiphile concentration effect on the initial flocculation rate (VO)of E. coli. Bacteria number densities are 2 x lo9(A)and 1.4 x lo9bacteridmL(B). C,, is the final DODAB (A) or DODAC (B) concentration in the mixtures. uo was calculated from flocculation data as those in Figure 4.
3. Bacteria Flocculation Induced by Vesicles. Figure 4 shows turbidity at 400 nm as a function of time after vesicle addition to bacteria in 0.264 M D-glucose Figure 5 shows the initial flocculation rates as a function of the final amphiphile concentration in the mixtures. Although flocculation was obtained over the entire range of amphiphile concentrations studied (0.1-1.0 mM), flocculation rates go through a maximum at 4 x lom4 M DODAB or DODAC. EM is zero at 4 x M DODAB (Figure lB,inset). I n the EM experiment the number density is lo7 b a c t e r i d m l whereas in the flocculation experiment it is 2 orders of magnitude higher. Thus, the pzc of the latter is expected to occur at a amphiphile concentration 2 orders of magnitude higher and i t does indeed. Consistently, flocculation rates are highest at 0.4 mM DODAC which is the amphiphile concentration where the pzc possibly occurs. The association of a turbidity increase with bacteria aggregation for a suspension ofE. coli upon vesicle addition is not straightforward. To be sure of bacteria flocculation, we directly observed the bacterialvesicle mixtures by optical microscopy (Figure 6). Micrographs confirm our interpretation for data in Figures 4 and 5. A turbidity increase is indeed associated with bacteria flocculation. Consistently, the frequency of aggregates is highest at 4 x M DODAC or DODAB (parts b and d of Figure 6) which is the amphiphile concentration where the initial flocculation rate goes through a maximum. E. coli scatters light in accordance with the Joebst law.% Turbidity (2'') depends on the wavelength of the incident radiation (A), on the particle number density (N), on the anhydrous mass of the particle (q), and on the volume of the scattering particle (V)
T =( c o n ~ t a n t ) q ~ ~ P ' ~ ~ ~ ~ When bacteria aggregate, there is a decrease in N,an increase in q, and a n increase in V. A decrease in N and (25) Koch, A.
L.Biochim. Bwphys. Acta 1961,51,429.
Figure 6. Bacteria aggregation in the presence of cationic vesicles as seen from optical micrographs. DODAB small vesicles a t 0.15 (a)or 0.48 mM DODAB (b) and E. coli ( 2 x lo9 bacteridml) were mixed, left interacting for 1 h, and directly examined using optical microscopy. The same was done for mixtures of DODAC larere vesicles at 0.16 (c).0.39(d). and 0.79 m M DODAC (e) and E:coZi (1.4x lo9 backridmlj.
Bacteria Flocculation Induced by Cationic Vesicles an increase in V would lead to a turbidity decrease, whereas a n increase in q would lead to a turbidity increase. Because the power of q is 2, whereas the power of the other factors is smaller than 2, q predominates and causes the increase in turbidity measured in Figure 4. Quaternary ammonium compounds are extensively used as bacteriostatic or bactericidal agents. In this work, the flocculant action of vesicles made up of quaternary ammonium compounds is described. Their germicide effect on preparations in vivo is the object of current investigation in our laboratory.
Conclusions At high bacteria number densities (lo9bacteridml), the
Langmuir, Vol. 10, No. 10, 1994 3465 interaction between Escherichia coli and small DODAB or large DODAC vesicles leads to vesicle adhesion to the bacteria without vesicle rupture. At limiting adsorption, a palisade of small vesicles surrounds the bacterium and the cell becomes positively charged. Vesicle adhesion is accompanied by bacteria flocculation. The dependence of flocculation rates on the amphiphile concentration is bellshaped going through a maximum a t the point of zero charge.
Acknowledgment. FAPESP and CNPq are acknowledged for research grants. G.N.T. and S.M.S. thank FAPESP and CNPq for undergraduate fellowships.
