Effect of Hypertonic Sucrose Upon the Immune Bactericidal Reaction

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1 INFECrION AND IMMUNITY, Jan. 1970, p Copyright 1970 American Society for Microbiology Vol. 1, No. 1 Printed in U.S.A. Effect of Hypertonic Sucrose Upon the Immune Bactericidal Reaction LOUIS H. MUSCHEL AND LINDA J. LARSEN Department of Microbiology, University of Minnesota, Minneapolis, Minnesota Received for publication 3 September 1969 This study was performed to determine the mechanism whereby hypertonic sucrose inhibits the immune bactericidal reaction. Other investigators had postulated that the initial attack of complement (C) on the cell wall was followed with lysozyme-containing whole serum by an enzymatic reaction upon the peptidoglycan substrate resulting in cell death. In the absence of serum lysozyme, secondary lethal changes might occur from damage to the cell's inner membrane as a result of osmotic forces in the presence of a defective cell wall. Hypertonic sucrose giving rise to plasmolysis and protection of the inner membrane was presumed to differentially inhibit the immune response mediated by lysozyme-free serum. The experimental results observed in this investigation have indicated, however, that the inhibitory effect of sucrose upon the bactericidal reaction may be explained simply by its anticomplementary effect and not by any effect on the bacterial cell. This view was supported by the following observations: (i) the comparability of the inhibitory effect of sucrose upon the immune hemolytic and bactericidal reactions, (ii) the comparable percentage loss in bactericidal activity of whole serum and lysozymefree serum resulting from hypertonic sucrose, (iii) bactericidal antibody titrations were relatively unaffected and C titrations markedly inhibited by sucrose, (iv) the inhibitory effect of sucrose on the bactericidal reaction was unaffected by prior growth of the organism in the presence of sucrose, (v) the kinetics of the bactericidal reactivity of lysozyme-free serum in hypertonic sucrose, compared with whole serum, did not reveal a prolonged lag phase with lysozyme-free serum, but simply diminished reactivity at all times. These observations are compatible with the view that the C attack upon the outer surface of gram-negative bacteria, which plays a part in the cell's permeability control, may account for cell death. In this regard, the immune bactericidal reaction is quite comparable to the lysis of red cells or nucleated cells by C despite the lack of overt lysis in bacteria, probably because of their underlying supporting structures. We have previously postulated that the insusceptibility of gram-positive bacteria to the complement (C) system was related to the thickness of the cell wall of these organisms (15 to 80 nm) compared to the relatively thin cell walls of gramnegative species which range from 7.5 to 10 nm (12). The surface antigens of the cell wall, which constitute the locus of the antigen-antibody complexes that activate C, were believed to be too far removed from the susceptible inner plasma membrane of gram-positive bacteria. In accord with this view, protoplasts of gram-positive cells were found to be susceptible to the C system (8). On the other hand, because of the relatively narrow cell wall of the gram-negative bacteria, the locus of the antigen-antibody complex on the surface of the organism that activated C was believed to be sufficiently close to the inner cell membrane for its destruction. The complexity of the cell surface of gramnegative bacteria, however, probably precludes this simple explanation. Treatment of Escherichia coli with ethylenediaminetetraacetic acid, which did not itself alter the growth rate of the organism or affect its viability, was recently found to result in increased permeability of the organism to actinomycin D and to other substances. Together with other evidence, this finding has led to the suggestion that gram-negative bacteria may possess two membranes separated by the rigid peptidoglycan layer, and that both of these membranes may function in permeability control (13). Because of the close proximity of the outer membrane to the surface antigens of the cell and because of the tremendous loss in the effectiveness of C when it is separated from its target by a distance equal only to that of a bovine serum albumin molecule (11), we suggested that the 51

2 52 MUSCHEL AND LARSEN INFEC. IMMUN. second outer membrane containing the antigenic lipopolysaccharide of the cell is the ultimate substrate for the C enzymes (7). This scheme is nicely in accord with the observation that the killing of bacteria by the C system results in their sensitivity to lysozyme (5) reflecting a loss of permeability control by the external barrier. A recent interesting series of observations by Feingold and his colleagues (2) has suggested that wall damage resulting from C action is not necessarily lethal unless lysozyme degrades the peptidoglycan of the wall or unless secondary damage to the inner cell membrane occurs. This concept was based upon different experimental approaches, one of which related to the effect of hypertonic sucrose upon the bactericidal reaction. They reported that when serum was tested with and without sucrose in the reaction mixture, a marked difference was observed between whole serum and lysozyme-free serum. Whole serum exerted its bactericidal effect regardless of the presence of hypertonic sucrose, but the lethal effect of lysozyme-free serum was inhibited by the sucrose. Presumably the hypertonicity provided by the sucrose protects the inner membrane by its contraction (plasmolysis) so that protrusion of the cell membrane through the wall and consequent loss of viability with lysozymefree serum does not occur. The anticomplementary action of sucrose was considered not to be involved, since the protective effect of sucrose against the lethal activity of lysozymefree serum was abolished by growth of the bacteria for several generations in the sugar. These observations seemed of significance in elucidating the mechanism of bacterial death mediated by C and worthy, therefore, of critical reexamination. MATERIALS AND METHODS Bacterial strains. Salmonella typhi 0901, E. coli W1485, and Vibrio cholerae 20-A-10 were all obtained from the Walter Reed Army Institute of Research and maintained on meat extract agar. Serum. Lyophilized guinea pig serum was purchased from the Texas Biological Laboratories and reconstituted with water prior to use. Assay methods. The immune bactericidal reaction was performed by a quantitative photometric assay (10). In certain experiments, C was assayed in the presence of an optimal amount of antiserum (4). Other experiments simply measured the bactericidal activity of whole serum (6). Hemolytic C was assayed by a standardized procedure (9). When the effects of sucrose were being determined, the sugar was dissolved in the saline diluent used for either the bactericidal or hemolytic procedures. In addition, sucrose was added to the broth containing the test organism for the bactericidal reaction so that the final concentration of the sugar was essentially equal to its concentration in the diluent and broth. It was not added, however, to test sera. The bactericidal activity of polymyxin was determined by an adaptation of the photometric assay used for the immune bactericidal reaction (15). Serum deficient in lysozyme. A suspension containing 4 mg of Bentonite (Volclay SPV, American Colloid Co.) per ml of water was prepared. This suspension was centrifuged and 1 ml of serum was added to 4 mg of sediment. The serum-bentonite mixture was gently mixed for 4 min at room temperature, and the absorbed serum then separated by centrifugation at 3,500 X g for 15 min at 2 C. Lysozyme activity was no longer detectable in the serum as determined by the method of Smolelis and Hartsell (14). RESULTS Effect of sucrose upon the hemolytic and bactericidal titer of C. Because of the marked influence of salt concentration upon the immune hemolytic activity of C (1), the effect of different concentrations of sucrose on C activity was similarly determined. Hemolytic titrations of both whole guinea pig serum and lysozyme-free serum for C activity resulted in a loss of 83% with sucrose and a loss of 37% in the presence of a concentration as low as M (Table 1). As might be expected, because of the absence of the peptidoglycan substrate of lysozyme in red cells, the activity of whole serum and lysozyme-free serum was quite comparable. There was, however, a marked difference in the bactericidal activity of C with these two serum samples. A titer of 111 was obtained with the untreated whole serum and 33 with lysozyme-free serum (Table 2). The percentage loss in activity resulting from the addition of sucrose, however, was no greater, even slightly less, with the lysozyme-free serum. In these experiments an optimal amount of antiserum was used so that only C was limiting. Bactericidal activity of whole guinea pig serum without added antiserum. The differential effect of sucrose upon the bactericidal activity of whole guinea pig and lysozyme-free samples without added antiserum was determined. Sucrose exerted a marked inhibitory effect (79%) upon the bactericidal action of lysozyme-free normal guinea pig serum, but an equally potent inhibition was noted with untreated lysozyme-containing serum (Table 3). Sensitivity of cells to serum bactericidal action after their growth in sucrose. Other studies (2) had indicated that E. coli cells grown overnight in sucrose were fully sensitive to whole serum or to lysozyme-free serum when the bactericidal reaction was conducted in the presence of sucrose. Cells put into sucrose shortly before contact with serum, however, were reported to be resistant to lysozyme-free serum. The protective effect of sucrose upon cells treated

3 VOL. 1, 1970 HYPERTONICITY AND THE BACTERICIDAL REACTION 53 TABLE 1. Effect of sucrose upon the hemolytic activity of guinea pig C Titer Sucrose molarity C titer, whole serum (CH 50/ml) titer without sucrose C titer, lysozyme-free serum titer without sucrose TABLE 2. Effect of sucrose upon the bactericidal activity of guinea pig C against S. typhi 0901 Titer Sucrose molarity C titer, whole serum (CB 50/ml)a titer without sucrose C titer, lysozyme-free serum NDb 13 3 titer without sucrose a Fifty per cent unit ricidally. bnd, not done. of C measured bacte- TABLE 3. Effect of sucrose on the bactericidal titers of whole guinea pig (GP) serum and lysozyme-free GP serum against S. typhi No 06mPer cent loss sucrose sucrose from sucrose Titer N 0. i resulting Whole GP serum a Lysozyme-free GP serum Whole GP serum titer/ lysozyme-free GP serum titer a Represents the titer or the reciprocal of the serum amount required for 50% killing of the test organism (see reference 6). with lysozyme-free serum was apparently abolished by prior growth in sucrose. The inhibitory effect of sucrose upon the bactericidal reaction could not be attributed, therefore, to its anticomplementary action. TABLE 4. Effect of prior growth of S. typhi and E. coli in sucrose on the bactericidal reaction of normal guinea pig serum Growth of: S. typhi in sucrose for 1 hr S. typhi in sucrose for 16 hr E. coli in sucrose for 16 hr a ND, not done. Sucrose in growth medium Sucrose in test medium Whole serum NDa ND ITiter Lysozyme-free serum Based on these reported results, experiments were performed in which S. typhi cells were first grown for 1 or 16 hr in Brain Heart Infusion broth containing sucrose. The results of these experiments indicated that such growth had little effect upon the subsequent bactericidal action of whole serum or lysozyme-free serum when the tests were performed in sucrose-free media (Table 4). For example, after growth for 1 hr in sucrose the lysozyme-free serum titer was 9.5 compared with 7.7 against the cells grown in broth lacking added sucrose. Similarly, prior growth in sucrose caused relatively little change in the sensitivity of the test cells when the tests were subsequently performed in sucrose. A trend may be noted toward decreased sensitivity in certain instances. For example, after 16 hr growth in sucrose, the whole serum titer was 2.2, but 5.0 after growth in sucrose-free media. Another organism, E. coli W1485, was similarly tested with comparable results. In summary, performance of the bactericidal reaction in the presence of sucrose caused a marked decline in serum bactericidal activity despite prior exposure of the test organism to sucrose. These results do not, therefore, support the view that the effect of sucrose upon the bactericidal reaction is independent of its anticomplementary activity. Effect of sucrose on the titration of antibody. In the immune hemolytic reaction, it was found that sucrose in the reaction mixture caused a marked reduction in C titer from 182 without sucrose to 31 in its presence. In the titration of hemolysin, however, sucrose caused only a two-

4 54 MUSCHEL AND LARSEN INFEC. IMMUN _ 13 _ 12_ 1L 8L w 7 6_ 4_ 320 l O _ r-- A A A-A -_ REACTION TIME3, MIN - NO SUCROSE ---- WITH 0.6M SUCROSE FIG. 1. Bactericidal titers ofwhole (A) and lysozymefree (0) guinea pig serum against S. typhi 0901 related to the reaction time. fold decline from a titer of 12,800 to 6,400. Similarly in the bactericidal reaction against V. cholerae there was a ninefold loss in C titer resulting from the presence of sucrose but only a twofold loss in antibody titer in the presence of a relatively large amount of whole guinea pig serum as a C source. V. cholerae was used rather than S. typhi in these experiments because of the relatively high resistance of the former to normal guinea pig serum. A large volume of guinea pig serum (0.45 ml) could be added, therefore, as a C source without the need for extensive absorptions to remove normally occurring antibody with consequent loss of activity. In both the hemolytic and bactericidal titrations of antibody, it is quite likely that C was not present in excess, despite the large amounts used, because of the marked anticomplementary action of hypertonic sucrose. It is reasonable, therefore, to attribute the relatively slight loss of antibody activity in the presence of sucrose to a lack of excess C. Kinetics of the immune bactericidal reaction. It had been reported that lysozyme-free serum in the absence of sucrose exerted its lethal effect within 15 min but that in the presence of sucrose there was little or no killing prior to 60 min at which time there was a reversal of plasmolysis (2). Titrations of whole and lysozyme-free guinea pig serum were performed against S. typhi in the presence and absence of sucrose. The results given in Fig. 1 indicate no essential difference between whole or lysozyme-free serum. The slope is considerably greater in the reactions performed in the absence of sucrose. Sucrose and the antibacterial activity of polymyxin. In contrast to the marked inhibitory action of sucrose upon the bactericidal reaction mediated by C, the activity of polymyxin was enhanced by the presence of sucrose in the 90 reaction mixture, with a 50% end point of 1.6 X 10-4 mg/ml in the presence of sucrose and slightly greater than 2.3 X 10-4 mg/ml in its absence. DISCUSSION Gram-negative bacteria may possess two membranes separated by the peptidoglycan structure. Recent experimental findings have suggested that both of these membranes may function in permeability control. In contrast to grampositive bacteria, the presence of a second outer membrane in gram-negative bacteria may account for their resistance to actinomycin D and relative resistance to penicillin. On the otherhand, the presence of such an outer structure with membrane functions suggests that it may serve as a target for the action of C (7). As a result of C activity, there is a loss of permeability control resulting in cell death and, in the presence of lysozyme, to lysis or to protoplast formation in the presence of a stabilizing milieu (5). A series of investigations by Feingold et al. (2) has led to the interpretation that the attack of C upon the cell wall or the outer surface of the wall is inadequate for cell death unless lysozyme degrades the peptidoglycan of the wall, or in the absence of lysozyme unless secondary damage occurs to the inner cell membrane. There is no question, as the data in this report confirm (Table 2), that lysozyme may act synergistically with C in the immune bactericidal reaction. Serum free of detectable lysozyme is nonetheless capable of exerting an easily demonstrable bactericidal effect (Tables 2-4) after a reaction period of 1 hr. In the absence of detectable serum lysozyme, Feingold et al. (2) postulated that weakening of the cell wall by C resulted in protrusion of the inner cell membrane through the wall by osmotic forces and loss of viability. They reasoned that the protective effect of sucrose, observed only with lysozyme-free serum, resulted from plasmolysis. The inner membranes in such plasmolyzed cells were thereby protected from secondary lethal changes. The experimental results obtained in this investigation, however, have not demonstrated a quantitative disproportionate effect of sucrose in the bactericidal reaction of lysozyme-free serum compared with whole serum (Table 3), whereas the experimental results previously reported were in qualitative terms (2). Moreover, lysozyme-free serum was reported to demonstrate a delayed lethal effect (15). The kinetic results obtained in this investigation (Fig. 1), on the contrary, indicated no difference in the kinetics of the response to whole serum or to lysozyme-free serum. In addition, the slope reflecting the increment in titer per unit time was greater in the tests

5 VOL. 1, 1970 HYPERTONICITY AND THE BACTERICIDAL REACTION conducted without sucrose. Consequently, our results do not support the view that there is a delay in the bactericidal reaction of lysozymefree serum in sucrose associated with plasmolysis of the cell. The previously reported lack of bactericidal activity prior to 30 min (2) may merely have resulted from the use of a single serum amount insufficiently great to give any effect. On the other hand, quantitative determination of serum titer indicated substantial killing prior to 30 min (Fig. 1). The effect of sucrose upon the immune bactericidal reaction may be explained solely in terms of its anticomplementary effect. In the first place, a comparable anticomplementary effect was exerted by sucrose in both the hemolytic and bactericidal systems (Tables 1 and 2). In the bactericidal system, the relative inhibitory effect of sucrose was comparable with lysozyme-free serum and whole serum both in the titration of C with excess antiserum (Table 2) and in the titration of whole serum alone (Table 3). In addition, the inhibitory effect of sucrose upon the titration of bactericidal antibody was relatively insignificant compared with its marked effect upon the titration of bactericidal C. The concept that sucrose-induced plasmolysis protects an organism primarily against lysozyme-free serum is simply not supported by the quantitative data in these experiments. These data suggest, on the other hand, that damage to the outer membrane with lysozyme-free serum is adequate to account for cell death. In contrast to the results obtained by others, prior growth of S. typhi or E. coli in sucrose did not reverse the inhibitory effect of sucrose on the bactericidal reaction (Table 4). There is no known reason, however, why prior growth of an organism in sucrose would annul the anticomplementary action of sucrose. Finally, polymyxin, an antibiotic that acts primarily by damaging bacterial membranes, was not inhibited by sucrose. If the inhibitory action of sucrose in the bactericidal reaction were exerted primarily upon the cell rather than upon the C system, one might have expected a similar inhibitory action with polymyxin. The locus of the antigen-antibody complex that activates C for the bactericidal reaction is obviously located on the periphery of the bacterial cell surface. The presently available evidence seems to be compatible with the simple hypothesis that cell death mediated by C results from a loss of permeability control of the outer surface layer. This is reflected by the lysozyme sensitivity of C-killed cells. The ineffectiveness of lysozyme treatment of gram-negative bacteria in promoting their lysis prior to the action of C (3, 5) is also quite compatible with this suggestion. Undoubtedly, secondary changes also occur as a result of C action, but the results of this investigation do not support the view that damage to those bacterial cell structures affected by suspension of the cells in hypertonic sucrose is of particular significance. ACKNOWLEDGMENT This work was supported by Public Health Service grant AI from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Becker, E. L., and G. H. Wirtz The salt sensitive step in immune hemolysis. Biochim. Biophys. Acta 35: Feingold, D. A., J. N. Goldman, and H. M. Kuritz Locus of the action of serum and the role of lysozyme in the serum bactericidal reaction. J. Bacteriol. 96: Glynn, A. A The complement lysozyme sequence in immune bacteriolysis. Immunology 16: Muschel, L. H Serum bactericidal actions. Ann. N. Y. Acad. Sci. 88: Muschel, L. H., W. F. Carey, and L. S. Baron Formation of bactericidal protoplasts by serum components. J. Immunol. 82: Muschel, L. H., R. Chamberlin, and E. Osawa Bactericidal activity of normal serum against bacterial cultures. I. The activity against Salmonella typhi strains. Proc. Soc. Exp. Biol. Med. 97: Muschel, L. H., and L. Gustafson Antibiotic, detergent, and complement sensitivity of Salmonella typhi after ethylenediaminetetracetic acid treatment. J. Bacteriol. 95: Muschel, L. H., and J. E. Jackson The reactivity of serum against protoplasts and spheroplasts. J. Immunol. 97: Muschel, L. H., and K. M. Lowe A new complement fixation test for syphilis. J. Lab. Clin. Med. 46: Muschel, L. H., and H. P. Treffers Quantitative studies on the bactericidal actions of serum and complement. I. A rapid photometric growth assay for bactericidal activity. J. Immunol. 76: Rowley, D., and K. J. Turmer Passive sensitization of Salmonella adelaide to the bactericidal action of antibody and complement. Nature 217: Salton, M. R. J The bacterial cell wall, p Elsevier Publishing Co., New York. 13. Salton, M. R. J In B. D. Davis and L. Warren (ed.), The specificity of cell surfaces, p. 71. Prentice-Hall, Inc., Englewood Cliffs, N. J. 14. Smolelis, A. N., and S. E. Hartsell The determination of lysozyme. J. Bacteriol. 58: Treffers, H. P., and L. H. Muschel The combined actions of chloramphenical and of bactericidal antibody plus complement on Salmonella typhosa. J. Exp. Med. 99: