Membranes of the stable protoplast L-form of Proteus mirabilis strain VI were highly immunogenic carriers of lipopolysaccharide when compared with the.
INFECTION AND IMMUNITY, Nov. 1980, p. 349-352
Vol. 30, No. 2
0019-9567/80/11-0349/04$02.00/0
Comparison of Quantitative and Qualitative AntibodyProducing Cell Responses to Lipopolysaccharide in Cell Walls of the Bacterial Form and in Membranes of the Protoplast LForm of Proteus mirabilis HELGE KARCH AND KATHRYN NIXDORFF* Institute fur Mikrobiologie, Technische Hochschule Darmstadt, D-6100 Darmstadt, West Germany
Membranes of the stable protoplast L-form of Proteus mirabilis strain VI were highly immunogenic carriers of lipopolysaccharide when compared with the immune responses to lipopolysaccharide contained in cell walls of the bacterial form of this organism.
L-forms of bacteria may be classified into different types according to whether or not they are able to revert to the bacterial form (unstable or stable) and whether or not they can be shown to possess a cell wall by cytological or chemical analyses (spheroplast or protoplast) (9, 14). Lforms are produced frequently as a result of treatment of bacteria with antibiotics that inhibit cell wall biosynthesis (4, 9). These organisms are able to survive and even grow in the presence of such antibiotics and therefore represent a potential medical problem as far as the control of pathogenic bacterial infections is concerned. The establishment of L-forms as pathogenic agents is extremely difficult, and the difficulty is compounded by the fact that the status of Lform cells (whether stable protoplast or revertible spheroplast form) in disease situations is often not well defined. Some reports in the literature indicate that although L-forms do not generally produce an acute phase of disease characteristic of pathogenic bacterial forms (21), they can cause low-level chronic infections that persist for long periods of time (8). Surface antigens in cell walls of intact bacteria are highly immunogenic and surely play a decisive role in protecting the host through specific humoral and cell-mediated reactions. The persistence of L-forms in the tissues of the infected host suggests a certain refractiveness to host defense mechanisms and raises the question of the ability of L-forms to elicit strong immune responses. In general, the question of the potential immunogenicity of L-forms is unclear. Good immune responses to Vibrio cholerae L-forms (2) or L-form lysates (3) have been reported. On the other hand, a comparison of the immune responses in lymphoid organs of mice to either the bacterial form of a group A Streptococcus or its
stable L-form induced by penicillin treatment has indicated that the bacterial form induced higher responses than the L-form (16). We have chosen to investigate this problem using the model system of Proteus mirabilis strain VI and its stable protoplast L-form. This L-form has lost the cell wall components peptidoglycan and the major proteins of the outer membrane, but still contains lipopolysaccharide (LPS) as a component of the protoplast membrane (7). Since LPS represents a common surface antigen in both types of cells, it was of interest to determine the potential immunogenicity of this antigen when incorporated into cell walls or with protoplast membranes as carriers. In one type of experiment, mice were immunized intraperitoneally with membranes of the protoplast L-form containing a defined amount of LPS, or with cell walls of the bacterial form containing the same amount of LPS. The immunoglobulin M (IgM) and the IgG antibody-producing cell responses were measured daily in both groups of mice after a primary (on day 0) and secondary (day 14) stimulation. In another type of experiment, mice received the same dry weight amount of either protoplast membranes or cell walls. The results indicate that protoplast membranes are highly immunogenic carriers for LPS.
MATERIALS AND METHODS P. mirabilis VI and its stable protoplast L-form were from the collection of this laboratory. The stable L-form was originally obtained from U. Taubeneck (Jena, Germany) and was described previously (9, 14). The bacterial form was cultivated according to Martin et al. (15), and the protoplast L-form was cultivated according to Gmeiner and Martin (7). P. mirabilis VI cell walls, free of cytoplasmic membranes, were obtained by shaking aqueous suspensions of bacteria with glass beads (0.17-mm diameter) in a cooled cell 349
350
KARCH AND NIXDORFF
INFECT. IMMUN.
mill (E. Buhiler, Tubingen, Germany) in the presence of 0.4% sodium dodecyl sulfate as previously described (18). Membranes of the stable protoplast L-form were obtained by osmotic lysis according to Kroll et al. (H.P. Kroll, J. Gmeiner, and H. H. Martin, Arch. Microbiol., in press). LPS I of P. mirabilis VI was extracted from the bacterial form with phenol-water (22) and purified according to Gmeiner (6). All samples were electrodialyzed (5). Protein content of cell walls and protoplast membranes was determined by a modification of the Lowry technique (13). Fatty acids of phospholipids and LPS were analyzed by gas-liquid chromatography as previously described (7). Amounts of LPS were calculated from the 3-hydroxy myristic acid (C14-OH) content, and amounts of phospholipid were calculated from the total content of palmitic acid (C16) minus the C16 content of LPS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins in cell walls or protoplast membranes was carried out in a slab gel apparatus (GE-4; Deutsche Pharmacia, Freiburg, Germany) according to Lugtenberg et al. (12) as previously described (18). White, female, specific pathogen-free NMRI mice weighing 22 to 25 g (Wiga, Sulzfeld, Germany) were injected intraperitoneally with 0.2 ml of a suspension of either cell walls or protoplast membranes in 0.9% NaCl. A primary injection was given on day 0, and a secondary injection containing the same amount of material was given on day 14. Details of dosage are presented later in the text. The hemolytic plaque assay (11) employed was a modification of the microscope slide assay of Mishell and Dutton (17) carried out for our system with sheep erythrocytes (Deutsche bioMerieux, Niirtingen, Germany) coated with alkali-treated LPS (20) as previously described (19). For the measurement of indirect plaques, a rabbit anti-mouse immunoglobulin-serum was prepared (10). This serum was diluted in the system to obtain an optimal number of indirect plaques with the least amount of inhibition of IgM plaques (approximately 7 to 10%). The number of IgG antibody-producing cells was obtained by subtracting the number of direct plaques (IgM) from the number of indirect plaques (IgM + IgG).
TABLE 1. Comparison ofprotein, phospholipid, and LPS contents of P. mirabilis VI cell walls and membranes of its protoplast L-forma Phospholipid Preparation (nnol of Leo Pro- (nmol of CI6 / LPS Prepaation C14-OH'/Mg tein~ mg [(fry [dry weight]) weight]) 47 52.66 Cell walls 70.37 44 179.09 Protoplast 24.01 membranes a Values in the table represent the mean of two to three separate determinations. b C16, Palmitic acid. eC14-OH, 3-Hydroxy myristic acid.
RESULTS
FIG. 1. Sodium dodecyl sulfate-polyacrylamidegel electrophoresis of proteins extracted from cell walls of P. mirabilis VI or from membranes of its stable protoplast L-form. (A) Standard proteins: bovine serum albumin (molecular weight, 67,000), ovalbumin (molecular weight, 45,000), chymotrypsinogen (molecular weight, 25,00X) and cytochrome c (molecular weight, 12,500); (B) protoplast membrane proteins (40 g); and (C) cell wall proteins (30 pg).
Cell walls and protoplast membranes were analyzed for content of protein, LPS, and phospholipids. Results are shown in Table 1. On a dry weight basis, protoplast membranes contained approximately the same amount of protein as did the cell walls of the bacterial form, but they contained 3.4 times more phospholipid and 2.9 times less LPS than the latter. For the same amount of LPS, protoplast membranes contained approximately 3 times as much protein and 10 times as much phospholipid as did cell walls. Protein composition of protoplast membranes and cell walls was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 1). The protein patterns of the protoplast membranes were typical of membranes of stable protoplast L-forms of P. mirabilis (Kroll et al., in
ABC
wlow
press) and did not appear to contain significant amounts of the three major proteins characteristic of the outer membrane of the cell wall complex of the P. mirabilis bacterial form (18). Mice were given a primary injection on day 0 of 50 ,ug of LPS contained in cell walls or protoplast membranes and a secondary injection of the same amount of material on day 14. The amounts of cell walls injected per mouse contained 50 ,ig of LPS, 119 ,ug of protein, and 10
VOL. 30, 1980
IMMUNOGENICITY OF PROTOPLAST L-FORM MEMBRANES
Mg of phospholipids. Protoplast membranes injected per mouse contained 50 ,g of LPS, 278 Mg of protein, and 110 Mig of phospholipids. Figure 2 shows a comparison of the IgM and the IgG antibody-producing cell responses to both preparations. The IgM/IgG antibody-producing cell ratio was similar in both groups of mice, but the group receiving protoplast membranes gave consistently higher responses, especially after the second injection (Fig. 2B). After the first injection (Fig. 2A), IgM antibody-producing cells reached a peak titer on day 4. IgG antibodyproducing cells appeared first on day 5 and increased slowly up to day 14, the time of secondary stimulation. After the second injection (Fig.
100°
0 A
80-
60~~~~~~ 40-
0
20
-J 0-
Z
12
w w
351
2B), IgM-producing cells in the group that received protoplast membranes increased approximately twofold over the number measured after primary injection, whereas the number of IgMproducing cells in the group that received cell walls increased only slightly. Peak titers were once again reached on day 4 after secondary stimulation. In contrast, IgG antibody-producing cells reached a peak titer on day 5 after secondary injection, and the numbers of this cell type were approximately fivefold greater than the numbers of IgM-producing cells. When mice were given 25 ,ug of LPS contained in either cell walls or protoplast membranes (data not shown), the kinetics and general features of the responses were the same as those of mice given 50 jig of LPS, although the quantitative responses were approximately twofold lower. The immunogenic potential of protoplast membranes was also tested in another way by injection of mice with the same dry weight amounts of cell walls or protoplast membranes (0.25 mg per mouse). In this case, mice injected with protoplast membranes received approximately the same amount of protein, but three times more phospholipid and only about onethird as much LPS (antigen) as did mice injected with the same dry weight amount of cell walls. Nevertheless, the mice responded as strongly to LPS contained in protoplast membranes as they did to LPS contained in cell walls (Table 2).
JB.
DISCUSSION too
These results indicate that protoplast membranes are highly immunogenic carriers for LPS,
U LL
TABLE 2. IgM and IgG antibody-producing cell responses of micea Immunization with:
Cell walls
0
2
4
DAYS
6
AFTER
8
10
12
14
INJECTION
FIG. 2. IgM and IgG antibody-producing cell reeither of its stable protoplast L-form. (A) Primary injection on day 0; (B) secondary injection on day 14. Symbols: *, IgM responses to cell walls; 0, IgM responses to protoplast membranes; A, IgG responses to cell walls; A, IgG responses to protoplast membranes. Points on the curves represent geometrical mean values for two separate experiments. PFC, Plaque-forming cells. sponses of mice to 50 pg of LPS contained in cell walls of P. mirabilis VI or membranes
Antibody type
IgM IgG
PFCb/10 spleen cells on day: 4
5
14
18
19
81 0
43 1
3 29
112 263
71 515
51 25 5 93 73 Protoplast IgM membranes 0 2 34 280 592 IgG 'Responses to a primary injection (day 0) and a secondary injection (day 14) of 0.25 mg (dry weight) each of either cell walls of the bacterial form or membranes of the stable protoplast L-form of P. mirabilis. A 0.25-mg amount of cell walls contained approximately 50 ,ug of LPS, 119 pg of protein, and 10 ,ug of phospholipids. A 0.25-mg amount of protoplast membranes contained approximately 17 ug of LPS, 117 1Ag of protein, and 37 ,g of phospholipids. b PFC, Plaque-forming cells. Values in the table represent the geometrical mean of two separate experiments.
352
KARCH AND NIXDORFF
when compared with the responses to LPS contained in cell walls. These responses were much stronger than responses of mice to the same amount of LPS administered in isolated form (19), which suggests that bacterial membrane components exerted a pronounced adjuvant effect. Since peptidoglycan, which has known adjuvant activity (1), was lacking in protoplast membranes, phospholipids and proteins could be likely candidates for natural adjuvants in this model system. We have recently shown that membrane phospholipids extracted from P. mirabilis can change both the strength and type of immune response in mice to LPS when that antigen is incorporated into phospholipid vesicles in an artificial model membrane system (19). In that investigation, LPS in isolated form induced predominantly IgM-type immune responses, whereas the same amount of LPS incorporated into phospholipid vesicles induced stronger responses, primarily of the IgG type. Thus, not only did phospholipids act as adjuvant to increase the response, but they also effected a change in the type of response elicited by LPS. In the present investigation, responses to LPS contained in natural model membrane systems, i.e., cell walls or protoplast membranes, were also predominantly of the IgG type. Studies are now under way to test the effect of proteins isolated from P. mirabilis membranes on the immune responses to LPS. ACKNOWLEDGMENTS This investigation was supported by the Deutsche For-
schungsgemeinschaft. We thank Cornelia Tauchmann for excellent technical assistance. We are particularly indebted to H. H. Martin for his invaluable advice and support throughout the course of this work.
LITERATURE CITED 1. Adam, A., R. Ciorbaru, J. F. Petit, and E. Lederer. 1972. Isolation and properties of a macromolecular, water-soluble, immunoadjuvant fraction from the cell wall of Mycobacterium smegmatis. Proc. Natl. Acad.
Sci. U.S.A. 69:851-854. 2. Agarwal, S. C., and N. K. Ganguly. 1972. Experimental oral immunization with L-forms of Vibrio cholerae. Infect. Immun. 5:31-34. 3. Agarwal, S. C., and T. Sandaraaj. 1977. Cell-mediated immunity after oral immunization with ribonucleic acidprotein fractions of Vibrio cholerae L-form lysates. Infect. Immun. 16:527-530. 4. Dienes, L. 1949. The development of Proteus cultures in
the presence of penicillin. J. Bacteriol. 57:529-538.
5. Galanos, C., and 0. Luderitz. 1975. Electrodialysis of lipopolysaccharides and their conversion to uniform salt forms. Eur. J. Biochem. 54:603-610. 6. Gmeiner, J. 1975. The isolation of two different lipopoly-
INFECT. IMMUN. saccharide fractions from various Proteus mirabilis strains. Eur. J. Biochem. 58:621-626. 7. Gmeiner, J., and H. H. Martin. 1976. Phospholipid and lipopolysaccharide in Proteus mirabilis and its stable protoplast L-form. Difference in content and fatty acid composition. Eur. J. Biochem. 67:487-494. 8. Gutman, L. T., M. Turck, R. G. Petersdorf, and R. J. Wedgewood. 1965. Significance of bacterial variants in urine of patients with chronic bacteriuria. J. Clin. Invest. 44:1945-1952. 9. Hofschneider, P. H., and H. H. Martin. 1968. Diversity of surface layers in L-forms of Proteus mirabilis. J. Gen. Microbiol. 51:23-33. 10. Hudson, L., and F. C. Hay. 1976. Practical immunology, p. 2, 3, 8, and 9. Blackwell Scientific Publications, London. 11. Jerne, N. K., and A. A. Nordin. 1963. Plaque formation in agar by single antibody-producing cells. Science 140: 405. 12. Lugtenberg, B., J. Meijers, R. Peters, P. van der Hoek, and L van Alphen. 1975. Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Lett. 58:254258. 13. Markwell, M. A. K., S. M. Haar, L. L. Bieber, and N. E. Tolbert. 1978. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87:206-210. 14. Martin, H. H. 1964. Composition of the mucopolymer in cell walls of the unstable and stable L-form of Proteus mirabilis. J. Gen. Microbiol. 36:441-450. 15. Martin, H. H., C. Maskos, and R. Burger. 1975. DAlanyl-D-alanine carboxypeptidase in the bacterial form and L-form of Proteus mirabilis. Eur. J. Biochem. 55: 465-473. 16. Mauss, H., and J. Schmitt.Slomska. 1977. Changes due to streptococcal L-forms in parathymic lymph nodes and in the spleen of the mouse, p. 345-354. In J. Roux (ed.), Spheroplasts, protoplasts and L-forms of bacteria. Les Colloques de l'Institut National de la Sante et de la Recherche Medicale, 21-25 Sept. 1976. INSERM, Paris. 17. Mishell, R. I., and R. W. Dutton. 1967. Immunization of dissociated spleen cell cultures from normal mice. J. Exp. Med. 126:423-442. 18. Nixdorff, K., H. Fitzer, J. Gmeiner, and H. H. Martin. 1977. Reconstitution of model membranes from phospholipid and outer membrane proteins of Proteus mirabilis. Role of proteins in the formation of hydrophilic pores and protection of membranes against detergents. Eur. J. Biochem. 81:63-69. 19. Ruttkowski, E., and K. Nixdorff. 1980. Qualitative and quantitative changes in the antibody-producing cell response to lipopolysaccharide induced after incorporation of the antigen into bacterial membrane phospholipid vesicles. J. Immunol. 124:2548-2551. 20. Schlecht, S., and 0. Westphal. 1967. Uber die Herstellung von Antiseren gegen die somatischen (O-) Antigene von Salmonellen. II. Mitteilung: Untersuchungen uber Hamagglutinintiter. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 205:487-501. 21. Timakov, V. 1977. Bacterial L-forms in pathology, p. 357-364. In J. Roux (ed.), Spheroplasts, protoplasts and L-forms of bacteria. Les Colloques de l'Institut National de la Sante et de la Recherche Medicale, 21-25 Sept. 1976. INSERM, Paris. 22. Westphal, O., 0. Luderitz, and F. Bister. 1952. Uber die Extraktion von Bakterien mit Phenol/Wasser. Z. Naturforsch. Teil B. 7:148-155.
