sedimentation-equilibrium studies of the polysaccharide components of pseudomonas aeruginosa

Sedimentation-Equilibrium Studies of the Polysaccharide Components of Pseudomonas

aeruginosa

DEREK HORTON* and DAVID A. RILEY, Department of Chemistry, T h e Ohio S ta te University, Columbus, Ohio 43210; and

POUL M. T. HANSEN, Department of Food Science and Nutrit ion, T h e Ohio State University, Columbus, Ohio 43210

Synopsis

Mild acid hydrolysis of lipopolysaccharide antigens from seven different serotype strains antigen immunotypes nos. 1-7 [in the classification of Fisher, M. W., Devlin, H. B. & Gnabasik, F. J. (1969) J . Bacteriol. 98, 835-8361 of Pseudomonas aeruginosa gave polysaccharide components of high molecular weight, which were isolated by gel filtration and dialysis. These components were examined by ultracentrifugation at equilibrium with the Rayleigh interferometric optical system. The partial specific volumes were calculated from densities obtained by using a mechanical oscillator. The average molecular weights (B,,, G,,,, and %fz) were calculated and compared to evaluate the polydispersity of the polysaccharides. The nonideality was investigated by varying the rotor speed, the height of the solution column, and the concentrations of the polysaccharide fractions. The molar masses were found to range from 14,000 for the polysaccharide from immunotype two to 24,000 for that from immunotype one, when extrapolated to zero rotor speed and solution column height.

INTRODUCTION

Fisher et al.' have developed a classification scheme that accommodates practically all known bacteriological serotypes of Pseudomonas aeruginosa into seven immunotypes of cross-protective homogeneity. Lipopolysac- charide antigens from these seven immunotypes have been isolated and subjected to analytical characterization.2

Lipopolysaccharides having antigenic properties are characteristic cell-wall components of gram-negative bacteria, and those from the family Enterobacteriaceae have been investigated quite extensively. Numerous review articles have described the chemical composition and immunological properties of lipopolysaccharide complexes isolated from a wide variety of b a ~ t e r i a . ~ - ~ The relationship of lipopolysaccharide structure to the bacterial virulence of Escherichia, Salmonella, and Shigella has also been reviewed r e ~ e n t l y . ~ In addition to the immunological properties of lipopolysaccharide antigens, they have also been shown to affect the necrosis of tumors,* and to possess mitogenic a ~ t i v i t y . ~

Luderitz and Westphal originally proposed a generalized model for a

* T o whom requests for reprints should be addressed.

Biopolymers, Vol. 19,1801-1814 (1980) 0 1980 John Wiley & Sons, Inc. 0006-3525/80/0019-1801$01.40

1802 HORTON, RILEY, AND HANSEN

bacterial lipopolysaccharide, based on studies on the smooth (“wild-type”) strains of S a l r n ~ n e l l a . ~ J ~ The model comprises three main components (side chain, core, and lipid A) that are covalently linked, with the polymeric side chains being responsible for the 0-antigenic specificity of the complex. In the seven Fisher immunotypes of P. aeruginosa, the 0-specific side chains are composed of repeating oligosaccharide units containing neutral carbohydrates and amino ~ u g a r s . ~ J - ~ ~ Certain strains, not included in the Fisher classification, have been shown to contain other rare sugars.15

Although the serological determinant is primarily attributed to the structure of the repeating oligosaccharide unit of the high-molecular-weight polysaccharide, the molar mass of the polysaccharide has also been shown to be of considerable importance. Studies on dextrans have shown that those polysaccharides having a mean molar mass of 90,000 are good immunogens in man, whereas those having a molar mass of 50,000 and below are not immunogenic.16J7 Further studies on the capsular polysaccharide of Pneurnococcus type I11 have led to similar results.lB A polysaccharide fraction of high molecular weight (>lOO,OOO), isolated from the slime of P. aeruginosa immunotype 1, has been shown to be effective in the immuni- zation of mice.l9

This paper describes experiments undertaken to characterize the molecular size of the high-molecular-weight polysaccharides (presumably, the antigenic 0-specific side chains) present in all seven Fisher _ _ immunotypes of P. aeruginosa. The molecular-weight distributions (M,,, Mu, and mz) have been evaluated and compared to establish the extent to which these polysaccharides are polydisperse, and possibly heterogeneous.

EXPERIMENTAL

Preparation of Polysaccharide Components of Pseudomonas Antigens

Lipopolysaccharide antigens (Fisher immunotypes 1-7) were supplied by Parke Davis & Co., Detroit, Michigan. The isolation procedure used has already been described.20 Each antigen was heated for 3 hr a t 100°C in a solution of 1% aqueous acetic acid (75 ml). The antigen from immunotype 6 required only 90 min of hydrolysis. The optimal time of hydrolysis was determined by the point at which visible precipitation of hydrophobic material ceased. The resulting biphasic mixtures were centrifuged at 6000 rpm for 1 hr at 4OC. The supernatant solutions were then extracted three times with chloroform (75 ml). The organic layers were combined with the precipitated material and stored as combined lipoidal material (lipid A plus other cell-membrane lipids13). The aqueous layer was, in each instance, lyophilized to yield the water-soluble hydrolysis products.

The water-soluble products [constituting approximately 50% (w/w) of each antigen] were dissolved in water (1 ml) and placed in a sonic bath for 10 min to ensure that all material had been dissolved. Each solution was

SEDIMENTATION-EQUILIBRIUM STUDIES 1803

applied to a column of Sephadex G-25 (1.6 X 40 cm), with monitoring of the effluent by means of a refractive-index monitor (Pharmacia, model 300L). The high-molecular-weight peak was lyophilized to yield a fraction containing the presumed 0-specific side chains and the “core” polysaccharide.

High-molecular-weight polysaccharide fractions, having a molar mass greater than 6000, from each immunotype were isolated by gel-permeation chromatography on a column (1.6 X 40 cm) of Sephadex G-75. This column was also calibrated to estimate the molar mass of the lower-molecular-weight (“core”) polysaccharide. The high-molecular-weight polysaccharide fractions (4 mg) were weighed on a microbalance and dissolved in 0.05M sodium cacodylate (MezAsOZNa) buffer (2 ml). The densities of working solutions were determined from the resonant frequency of a mechanical oscillator (Mettler/Paar density meter, model DMA 02C) with a reported21 precision of f2.0 X 10+ g/ml. Values for the partial specific volumes of the polymers were calculated from the concentration (c) of the polymer and the densities of the sample solution (dl) and the solvent (dz) by using the equationz1

Partial specific volume = l/d2[1 - (dl - dZ)/c]

Molecular-Weight Determinations

The molecular weights were obtained by using a Spinco model E analytical ultracentrifuge under meniscus-depletion conditions at equilibrium. This instrument was equipped with both Rayleigh and schlieren optics, electronic speed controls, and a resistance temperature-indicator and control (RTIC) unit. All experiments were performed with the green light of mercury, 546 nm (Tiffen photo filter 77A), as the light source, and the interference patterns for solute distribution were recorded on Kodak me- tallographic plates. The experimental conditions pertaining to the different samples are listed in Table 111. In a typical experiment, a polysaccharide fraction (10 mg) was dissolved in 0.05M MezAsOzNa buffer (2 ml) containing 0.25M NaCl (pH 7, density: 1.013 g/ml at 20.0°C) and dialyzed against the same buffer (100 ml) for 48 hr in a cleaned and preconditioned dialysis tube (Spectrapor Membrane, nominal molecular-weight cutoff 6000-8000). This procedure removed approximately 30% of the material by weight, corresponding to the “core” polysaccharide fraction. The sample solution was placed in one sector of a lz-rnm, synthetic boundary, double-sector cell, with the corresponding dialysate of the sample solution in the other sector.22 The rotor, with cell and counterbalance, was accel- erated to a speed sufficient to ensure meniscus depletion of the solute after establishment of sedimentation equilibrium (48 hr). Meniscus depletion was manifested by the distinct appearance of a horizontal fringe-plateau near the meniscus. In experiments at low speeds and short column heights, where meniscus depletion was not achieved, the concentration at the meniscus was accounted for, and the conventional “low-speed” method was


used. The photographic plates were examined on a Nikon microcompar- ator at 1OX magnification, and the positions of fringes intersecting with the horizontal axis relative to the center of rotation were measured.

The experimental plots of In C (C = fringe member) vs r2 (the squared radial position) were analyzed by a nonlinear, curve-fitting procedure in- volving multiple exponential terms.23 The number (anl), the weight (awl), and the 2 - (az1) average apparent reduced molecular weights at each experimental point were determined by the following equation^.^^.^^

d In C ( r ) d r 2 / 2 a,(r) =

where C is the concentration in terms of fringes, r the radial position, and a the first experimental fringe.

To determine @,(a), a,(a) and o,(a) were combined so that their linear combination was equal to (T, (a) to first order in c o n c e n t r a t i ~ n ~ ~ . ~ ~ :

a,(a) = p aw(a> - 4 az(a)

The reduced molecular weights were then converted into the (apparent) molecular-weight averages (Mnl, MUl, and Mzl) by the following rela- tion24.25:

(apparent) = [RT/(1 - vp)w2]a ,

where R is the gas constant, T the temperature (in Kelvin), v the partial specific volume of the macromolecular species, p the solvent density, and w the angular velocity.

The weight-average molecular weight (MWJ was also calculated from the experimental data by use of a linear, least-squares analysis.

By varying the height of the solution column and the rotor speed, the applied pressure was determined from the following relation (assuming the pressure at the meniscus was zero):

P = pw2/2(rg - r;)

where r i is the radial squared position at the cell bottom and r$ is the radial squared position at the meniscus.

The apparent molecular-weight averages were then plotted against these values to evaluate the nonideality with respect to rotor speed and column height.


RESULTS

The lipopolysaccharide antigen from each immunotype of P. aeruginosa was readily hydrolyzed by dilute acetic acid at 100°C. This lability is characteristic of all lipopolysaccharides containing an acid-labile 3- deoxy-D-manno-octulosonic acid residue, which is considered to constitute the link between the polysaccharides and the lipid A component.26 A 3-hr hydrolysis time is somewhat longer than that generally reported for this type of molecular complex.15J6 When shorter hydrolysis times were at- tempted in this study, the lipid and other hydrophobic material did not precipitate fully from the acidic aqueous mixture. Attempts to sediment the partially precipitated material at 6000 rpm failed, and extractions with chloroform gave rise to emulsions. With the longer hydrolysis times uti- lized, processing of the water-soluble, hydrolysis products was greatly fa- cilitated. The lipopolysaccharide antigen from P. aeruginosa immunotype 6 did not require acid hydrolysis for 3 hr; complete separation of the hydrophobic and hydrophilic material was effected after 90 min.

Gel-permeation chromatography of the water-soluble products on Sephadex G-25 with water as the eluent gave two peaks, as indicated by the refractive index measured as a function of effluent volume. The first peak was presumed to contain the high-molecular-weight, 0-specific side chains and the “core” polysaccharide; the second peak was mainly inorganic salts and small amounts of monosaccharides liberated during partial hydrolysis of the polysaccharides. The “core” polysaccharide was separated by dialysis prior to the preparation of each sample for equilibrium ultracentrifugation. In other studies performed in this laboratory,2 this separation has been effected by gel-permeation chromatography on Sephadex G-75. The “core” polysaccharide from each immunotype appeared to have a molar mass of -3000, as estimated on a column of Sephadex G-75, calibrated with polysaccharides and oligosaccharides of known molecular weight. The hydrolysis of the antigen of immunotype 6 revealed that this lipopolysaccharide contained an additional component not found in the other six immunotypes. It appeared to have a molecular weight ranging between 3000 and 2000, and is now under investigation to determine its chemical composition. The percentages of polysaccharide (side chain and “core”) present in each immunotype are given in Table I.

The term for partial specific volume in the calculation of the apparent molecular-weight averages is a very important value that must be known with good accuracy. It serves to account for the “buoyancy” force, and any error in this value is magnified in the final calculation of molecular weight. To limit the error to 1% in the calculation of the molecular weight, the partial specific volume should be knownz1 to f l pl/g. The measurement of the partial specific volume of each polysaccharide was determined from density measurements taken on a commercial, digital, oscillator densimeter. When the temperature is controlled to fO.Ol°C, the accuracy of these readings is21 f 2 X g/ml. The value of the partial specific volume varies substantially between polysaccharides having different structures (see Table 11).


TABLE I Degradation of Pseudomonas aeruginosa Lipopolysaccharide Antigens by 1% Acetic Acid

a t 100°C

Percent Percent High-Molecular- Hydrolysis Time Polysaccharide Weight Polysaccharide

Immunotypea (hr) (w/w) (W/W)b

3.0 32.0 3.0 37.0 3.0 42.0 3.0 44.0 3.0 35.0 1.5 25.0 3.0 37.0

22.0 19.0 25.0 23.0 18.0

-17.0 -20.0

a From Ref. 1. Data include unpublished results by Dr. H. Saeki of this laboratory.

The sedimentation profile of each polysaccharide fraction was monitored by using both Rayleigh interferometric and schlieren optical systems. Although no sedimentation-velocity experiments were performed to yield quantitative results, the schlieren image of each polysaccharide in the synthetic-boundary cell appeared as a single, Gaussian-shaped band that is typical for a monodisperse, homogeneous, macromolecular solute. Al- though this observation does not preclude the possibility that these bacterial polysaccharides might be somewhat polydisperse or heterogeneous, an essential monodispersity does appear to be characteristic of them. The results from the Rayleigh interferometric optical system were quantitated to determine the fringe displacement a t equilibrium. Figure 1 is an example of a typical plot of the experimental data. The apparent, average molecular weights calculated for each polysaccharide fraction, under meniscus-depletion conditions at equilibrium, are listed in Table 111.

To ensure that 48 hr was sufficient time to achieve equilibrium, the polysaccharide fraction isolated from immunotype 1 was centrifuged for time intervals ranging from 4 to 60 hr. Although 4 hr appeared insufficient to attain equilibrium, a period of 24 hr did appear to be sufficient. The calculated, apparent, weight-average molecular weight was determined to be 18,000 f 1000 by using the linear least-square analysis. By using a polynomial expression to obtain a distribution of apparent, weight-average molecular weights, the mean (apparent) weight-average molecular weight was determined to be 20,000 f 1000. The number-average and 2-average molecular weights were found to be 18,000 f 1000 and 23,000 f 1000, respectively. Table IV gives a summary of variations in the calculated molecular weights for the polysaccharide fraction from immunotype 1 at different time intervals.

To investigate the possible roles which the rotor speed and solution column height may have had in the calculation of the molecular weights, the polysaccharide fraction isolated from immunotype 2 was centrifuged at a variety of rotor speeds and solution column heights. By converting


40.00 49 .00 50.00 51.00

r 2 (squared radio1 posit ion)

Fig. 1. Sedimentation-equilibrium study of lipid-free polysaccharides from P. aeruginosa lipopolysaccharide immunotype 3.

these experimental values into applied pressure, it was found that the apparent molecular-weight averages decreased as the applied pressure increased (Fig. 2). A linear least-squares analysis indicated that the weight-average molecular weight was 13,800, with a linear correlation coefficient of -0.972, at zero speed and solution column height. The plot of molecular weight vs pressure was also analyzed in terms of a polynomial expansion. Extrapolation of this expansion to zero pressure revealed the weight-average molecular weight to be 14,400. Table V gives the molecular-weight average for all seven immunotypes extrapolated to zero pressure.

The nonideality was also investigated by changing the concentration of the polysaccharide fraction at a particular rotor speed. Over a limited, dilute concentration range (1-7 mg/ml), the molecular weight values decreased as the concentration was increased. Using a linear, least-squares analysis, it was found that the weight-average molecular weight of the polysaccharide fraction from immunotype 2 was 13,800 at zero concentration (20,000 rpm, 2O.OOC). The linear correlation coefficient for the relation was -0.983, At a concentration of 1.5 mg/ml, the weight-average molecular weight was determined to be 13,300.

DISCUSSION

The lipopolysaccharide antigens used in these experiments were obtained from seven serotypes of P. aeruginosa belonging to immunotypes 1-7 in the classification of Fisher et a1.l These seven antigens are components


of a vaccine formulation (Pseudogen); this heptavalent vaccine has been shown useful as an immunogen for protecting humans against infection by potentially dangerous, opportunistic strains of P. aeruginosa .27,28 The high-molecular-weight polysaccharides, considered to be the antigenic determinants of these macromolecular complexes, may also be capable of serving as immunogens. Furthermore, they may not elicit some of the undesirable side reactions associated with the use of the whole lipopolysaccharide antigens in u ~ u o . ~ ~

In the course of structural determinations on these antigenic 0-specific side chains, the questions of possible polydispersity and heterogeneity of polysaccharide fractions have remained consistent problems, often leading to anomalous analytical results. The heterogeneity of polysaccharide fractions from the lipopolysaccharide complexes of some strains of P. aeruginosa has been demonstrated by observing variations in the mono- saccharide composition of fractions of different molecular weight that have been partially separated by gel-permeation chr~matography.~~ However, other investigators have isolated high-molecular-weight fractions that appear to be paucidisperse and homogeneous by gel-permeation chromatography.15J6 Attempts to characterize the molecular size of these polysaccharides, as isolated from lipopolysaccharides by gel-permeation chromatography, have led to qualitative results. Pier et al.31 reported that a high-molecular-weight polysaccharide fraction isolated from the slime of P. aeruginosa immunotype 1 may have a molecular weight ranging between 350,000 and 100,000. The classical problem of polydispersity is not limited to bacterial polysaccharides. Starch amylopectin was shown to have a weight-average molecular weight of 80,000,000 by light ~ c a t t e r i n g ~ ~ , ~ ~ and a number-average molecular weight of 300,000 by ~ s m o m e t r y . ~ ~ , ~ ~

16,000 L

I I I I I I I 1 1 I I 0 10 20 30 40 5 0 60 70 80 90 100

Pressure (atm)

Fig. 2. Apparent weight-average molecular weight of the polysaccharide component of immunotype 2 vs applied pressure.

TA

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TABLE V Molecular Weights Extrapolated to Zero Rotor Speed and Solution Column Height

Immunotypea Z l l m w , Z l mW*

1 22,000 24,000 28,000 22,000 2 14,000 15,000 17,000 14,000 3 20,000 20,000 20,000 20,000 4 14,000 17,000 20,000 15,000 5 20,000 20,000 22,000 20,000 6 15,000 16,000 17,000 15,000 7 19,000 19,000 21,000 19,000

a From Ref. 1

Experimental and theoretical treatments of sedimentation-equilibrium data for polysaccharides (mainly dextrans) have been conducted in considerable detai1.34-36 This present paper does not offer exhaustive con- sideration of the extent to which these bacterial polysaccharides may exhibit nonideal properties in solution.

Within experimental uncertainty (* lOOO), the molecular-weight averages at 1.5 mg/ml agreed with those extrapolated to zero concentration at any specific rotor speed. However, there was a significant dependence of the calculated molecular weights on the rotor speed. A similar rotor-speed dependence has already been reported for ~ a r r a g e e n a n . ~ ~ The molecular-weight averages reported in Table V are subject to error resulting from concentration effects. A study on polystyrene has shown that extrapolation of the molecular weight to zero rotor speed and solution column height gives the molecular weight, plus a contribution of the product of the polymer concentration and the light-scattering, second virial ~oef f ic ien t .~~ At the concentrations used in this study, the contribution of these terms does not seem to be greater than the experimental uncertainty.

The partial specific volumes of the seven polysaccharides did differ to a considerable extent, as shown in Table 11. This is most likely a direct reflection of their being composed of different oligosaccharide repeating units. Comparing the values for the partial specific volumes to preliminary structural assignments made in this laboratory, the antigenic 0-specific side chains having more hydrophilic structures have lower partial specific volumes. For example, the oligosaccharide repeating unit present in immunotype 2 is composed of D-glucose and 2-acetamido-2,6-dideoxy-D-and L-galactose,12 and the polysaccharide fraction from this immunotype has a partial specific volume of 0.643 ml/g. In contrast, the repeating unit occurring in immunotype 5 is composed entirely of 6-deoxy sugars (namely L-rhamnose and 2-acetamido-2,6-dideoxy-~-glucose),~~ and the partial specific volume of this polysaccharide fraction is higher, namely, 0.658 ml/g.

The sedimentation-equilibrium data for the polysaccharide fractions from P. aeruginosa immunotypes 1-7 give insight into the extent to which


- _ they may be polydisperse or heterogeneous. By using the ratio of M J M , as an index of the possible heterogeneity or polydispersity, the polysaccharide fractions from immunotypes 1 and 4 appear to be the most polydisperse or heterogeneous. These fractions were determined to have M,/M, ratios of 1.17 and 1.18, whereas the polysaccharide fractions from immunotypes 2,3,5,6, and 7 had M2m, ratios of 1.13,1.20,1.00,1.10,1.06, and 1.10, respectively. The values for M,,M,, and %, were in better agreement than might have been suspected. The polydispersity or heterogeneity observed did not indicate any significant nonspecific hydrolysis of the polysaccharides during the initial removal of the covalently attached lipid A.

The polysaccharide fractions from immunotypes 1, 5, and 7 had the largest mean molecular weights, all being in excess of 18,000. By simple least-square analysis, each afforded a linear correlation coefficient of 0.999 or 0.998. The number- and weight-average molecular weights determined for these polysaccharide fractions (from the mean of the distribution gen- erated from a polynomial expression) agreed with each other, and with the weight-average molecular weights calculated from the least-square analysis, within the limits of experimental uncertainty. The polysaccharide fraction from immunotype 3 yielded the most concordant results, with all calculated molecular weights agreeing within experimental uncertainty. The fraction appeared to have a molar mass of 20,000 f 1000.

Immunotypes 2 and 6 had polysaccharide components that had the lowest molecular-weight values. Both were determined to have mean molecular weights of less than 18,000. The experiments-on these polysaccharides also gave good correlation coefficients (0.996 and 0.998), and the number-, weight-, and 2-average molecular weights were similar.

The high-molecular-weight polysaccharide fraction from the slime of immunotype 1 has already been shown to be an immunogen.1° The ability of these bacterial polysaccharides to serve as immunogens may be controlled by factors similar to those that control the immunological properties of dextrans17J8 and Pneumococcus poly~accharides.~~ If this becomes evi- dent, the polysaccharide fractions from these bacterial immunotypes will probably not be able to elicit a substantial immunological response in uiuo because their molecular weight is too low.

This study shows that the average molecular weight of polysaccharides, isolated from antigenic lipopolysaccharides of P. aeruginosa, lies between 15,000 and 20,000. These polysaccharide fractions also exhibit nonideal, paucidisperse behavior in an ultracentrifuge.

_ _

_ -

Supported in part by Grant GM-20181 from the National Institute of General Medical Sciences, National Institutes of Health, Bethesda, Md. 20014 (The Ohio State University Research Foundation Project 3943). The authors thank Dr. T. H: Haskell, Dr. H. Machamer, and Warner-LambertParke Davis & Co. for the immunotype antigens used in this work. The authors also thank Mr. Ferenc Relle, Ross Laboratories, Columbus, Ohio, for assistance with the density measurement.


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Pap. Am. Chem. SOC. Meet. 172, Carb-97.

Am. Chem. Soc./Chem. SOC. Jpn . Chem. Congr., Carb-49.

535-546.

209-210.

908-918.

2833. Received August 22,1979 Accepted April 3,1980

sedimentation-equilibrium studies of the polysaccharide components of pseudomonas aeruginosa

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