Proteus mirabilis Is an OmpA Protein and - Infection and Immunity

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ALEXANDER KORN,1 HEIN-PETER KROLL,2 HANS-PETER BERGER,1. ANDREA KAHLER,1 ... KLAUS-PETER MULLER,3 AND KATHRYN NIXDORFF1*. Department ..... Rev. 49:1-32. 18. Nixdorff, K., H. Fitzer, J. Gmeiner, and H. H. Martin.
INFECTION AND IMMUNITY, Nov. 1993, p. 4915-4918

Vol. 61, No. 11

0019-9567/93/114915-04$02.00/0 Copyright © 1993, American Society for Microbiology

The 39-Kilodalton Outer Membrane Protein of Proteus mirabilis Is an OmpA Protein and Mitogen for Murine B Lymphocytes ALEXANDER KORN,1 HEIN-PETER KROLL,2 HANS-PETER BERGER,1 ANDREA KAHLER,1 REGINA HEB3LER,1 JENS BRAUBURGER,l KLAUS-PETER MULLER,3 AND KATHRYN NIXDORFF1*

Department of Microbiology, University of Darnstadt, Schnittspahnstr. 10, D-64287 Darmstadt,' Institute for Chemotherapy, Bayer AG, D-42017 Wuppertal,2 and Institute of Immunology and Genetics, German Cancer Research Center, D-69120 Heidelberg, 3 Germany Received 4 May 1993/Returned for modification 7 June 1993/Accepted 1 September 1993

Partial amino acid sequence analysis of a ma,jor outer membrane protein of Proteus mirabiis (39-kDa protein) indicates that it is an OmpA protein. The mitogenic activities of the 39-kDa protein for murine lymphocytes were also investigated with T lymphocytes isolated by passing spleen cells over columns of nylon wool fiber and B lymphocytes obtained by treating spleen cells with monoclonal antibodies to Thyl plus complement. The 39-kDa protein showed little activity in stimulating T cells to proliferate but was strongly mitogenic for B cells.

The 39-kDa protein isolated from purified cell walls of Proteus mirabilis is a potent immunogen in mice (10). It is also a particularly effective immunomodulator of the immune responses to lipopolysaccharide (LPS) when mixed with this antigen before administration to mice (9, 11). In this regard, T-cell-dependent, immunoglobulin G responses specific for the particular LPS serotype used for immunization are predominantly enhanced by the 39-kDa protein (9, 11, 19). Furthermore, the 39-kDa protein is a strong yet very differential modulator of the actions of LPS on macrophages, although the protein alone has no significant stimulatory effects on these cells (23, 24). The location of the 39-kDa protein and its potent immunomodulating activities suggest that it may belong to the proteosome class of adjuvants. Proteosomes are multimolecular preparations of outer membrane proteins of gramnegative bacteria that act as B-cell mitogens in vitro and enhance T-cell-dependent responses to antigen in vivo (12). It has been speculated that a part of the excellent adjuvant effect of proteosomes is the promotion of enhanced uptake and processing of antigens by macrophages (12). In this regard, preliminary studies in our laboratory indicate that LPS is taken up much more efficiently in macrophages when it is complexed with the 39-kDa protein (lla). The present investigation was undertaken to definitively establish the 39-kDa protein as a characteristic outer membrane protein and B-cell mitogen, which would lend further support to its classification as a protein with the traits of proteosomes. LPS I was extracted from P. mirabilis 19 two times with phenol-water to eliminate contaminating protein (9). The 39-kDa protein was isolated from purified cell walls of P. mirabilis 19 according to methods previously described (18) and was purified further by phenol-water extraction followed by gel filtration (9). The protein purified in this manner contained no detectable LPS (less than 0.0075%) according

*

to gas chromatographic analyses of 3-hydroxymyristic acid and was not visibly contaminated with other proteins when

examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Approximately 1 mg of the highly purified 39-kDa protein was digested with trypsin after reductive carboxymethylation. Ten tryptic peptides were separated by high-performance liquid chromatography on an RP-18 column, and the isolated fragments were sequenced by automated Edman degradation according to a method previously described by Siekmann et al. (20). The amino acid sequences of the peptides were compared with those of known proteins by using the BestFit program of the University of Wisconsin Genetics Computer Group (7). Specific-pathogen-free NMRI and C57BL/6 mice were obtained from Zentralinstitut fur Versuchstierzucht, Hannover, Germany. All mice were 6 weeks old at the beginning of the experiment. The fibroblast cell line C63, used as a source of interleukin 2, was a kind gift from Peter Krammer, Institute of Immunology and Genetics, German Cancer Research Center, Heidelberg, Germany. Spleen cells of NMRI mice were enriched for T cells by being passed over columns containing nylon wool fiber (Polysciences, Ltd., St. Goar, Germany) according to the method of Julius et al. (8). Two passages were necessary for obtaining cells that no longer responded to LPS. Enriched B-cell populations were obtained by treating 4.0 ml of spleen cells at a density of 5 x 107 cells per ml in minimum essential medium (Biochrom KG, Berlin, Germany) in the absence of fetal calf serum with 4.0 ml of a 1:10 dilution of monoclonal anti-Thyl.2 (ICN Biomedicals GmbH, Eschwege, Germany) and 4.0 ml of a 1:4 dilution of guinea pig serum (Behringwerke AG, Marburg, Germany) at 37°C in a shaker water bath for 30 min. The mixtures were then cooled in an ice bath for 10 min. The Thyl.2 inbred mouse strain C57BL/6 was used for these experiments to ensure good reactivity with the antibody. Preliminary studies had shown that the mitogenic responses of spleen cells to LPS and to the 39-kDa protein were strikingly similar in NMRI and C57BL/6 mice. The enriched T cells and B cells were washed and resuspended in

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1 APSDNTWYTG ........................ 22

APKDNTWYTGAKLGWSQYHDTGFINNNGPTHENQLGAGAFGGYQVNPYVG 71

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122 VWRADTKSNVYGKNHDTGVSPVFAGGVEYAITPEIATRLEYQWTNNIGDA 171 35

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FNQETPAPVVEPAP ......... IGLRSDV 55

172 HTIGTRPDNGMLSLGVSYRFGQGEAAPVVAPAPAPAPEVQTKHFTLKSDV 221

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......... VVVIGYTNRIGSQDYNL 78

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222 LFNFNKATLKPEGQAALDQLYSQLSNLDPKDGSVVVLGYTDRIGSDAYNQ 271 79 PLSEKRAQSVVNYLVSK ..............

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322 DCLAPDR 328

FIG. 1. Comparison of the tryptic peptides designated amino acids 1 to 106 of the 39-kDa outer membrane protein of P. mirabilis 19 with the sequence of the OmpA protein (amino acids 22 to 328) of

E. coli (1, 5, 16).

complete RPMI medium (13) supplemented with 5% (vol/ vol) fetal calf serum to be used in the lymphocyte proliferation test. The proliferation test described by Mosmann and Fong (15) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide was employed. Briefly, 50 ,ul of a cell suspension at a density of 4 x 106 cells per ml was added to 50 ,ul of various concentrations of a solution of mitogen per well in a 96-well culture dish. The culture medium used was complete RPMI medium (13) supplemented with 5% (vol/ vol) fetal calf serum. For testing proliferative responses of enriched T cells, the medium was also supplemented with 5% (vol/vol) of the supernatant of C63 fibroblast cultures containing interleukin 2 in a concentration of approximately 4,300 U/ml. Reactions were measured with an enzymelinked immunosorbent assay reader (EAR 400 ATX; SLT Labinstruments Deutschland GmbH, Crailsheim, Germany) at 570 nm with a reference at 690 nm. As depicted in Fig. 1, the amino acid sequences of 10 tryptic peptides of the 39-kDa protein, which represent 32% of the intact molecule, showed a strong similarity to the OmpA protein of Escherichia coli (1, 5, 16). The calculated identity index was 74.5%, while the similarity index was 90.6%. Most convincing evidence is the composition of the 39-kDa protein peptide of amino acids designated 35 to 48. This stretch contains a sequence very similar to the protease-sensitive Ala-Pro-Val-Val-Ala-Pro-Ala-Pro-Ala-ProAla-Pro sequence characteristic of OmpA (5, 17). Furthermore, comparison of the 39-kDa protein fragments with the known amino acid sequence of OmpA of Shigella dysenteriae (4) gives a calculated identity index of 77% and a similarity index of 92%. Previous studies from our laboratory support the conclusion that the 39-kDa protein is an OmpA protein. It is heat modifiable in that its apparent molecular weight is increased by boiling in the presence of sodium dodecyl sulfate, and it is sensitive to trypsin treatment while contained in the cell walls of P. mirabilis (18). These are properties which are very characteristic of OmpA (17). It was recently shown that OmpA of E. coli can function as a porin (21). In this regard, the 39-kDa protein also has the apparent properties of a porin (18), although it must be noted that in these experiments with artificial membranes, the protein was in an

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FIG. 2. Comparison of the proliferation of spleen cells from NMRI mice after stimulation with the 39-kDa protein from P.

mirabilis 19 containing LPS (-) and purified to remove LPS (0). Proliferation was measured with the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide test. In each case, the results are reported as the geometric means ± standard errors of values taken from two separate experiments. OD, optical density.

aggregated form that surely was not representative of its native structure in the outer membrane complex as described for E. coli (14, 21). The mitogenic properties of the 39-kDa protein from P. mirabilis for murine spleen cells are shown in Fig. 2. When the protein is isolated from purified cell walls (18), it usually contains about 10% LPS. The responses of spleen cells to such a preparation increased in a dose-dependent manner to a peak at about 10 p,g of protein per ml and then decreased with additional amounts of the mitogen. Preparations of the 39-kDa protein purified by phenol-water extraction (9) contained no detectable LPS (less than 0.0075%) as determined by gas chromatographic analysis of 3-hydroxymyristic acid. The responses to the purified protein were somewhat lower than those to the impure protein in the concentration range of 1 to 20 ,ug/ml, but at higher concentrations, the activity of the purified protein was comparable to the peak activity of the impure preparation. The proliferative responses of enriched populations of T cells are presented in Fig. 3. Responses to concanavalin A (Fig. 3A) were quite strong, which shows that T lymphocytes in the treated cell population were present and viable. In contrast, responses to LPS (Fig. 3B) were not detectable to any significant degree. Only at very high doses of LPS (over 500 p,g/ml) was there a tendency toward a slight increase in proliferation. This may be an indication of the apparent ability of LPS to stimulate a small proportion of T cells in an enriched population (22). Treatment of these cells obtained after two passages over nylon wool with antimouse immunoglobulin plus complement gave no further enrichment of T lymphocytes (data not shown). Results similar to the responses to LPS were obtained with the purified 39-kDa protein (Fig. 3C). This outer membrane protein is therefore not a T-cell mitogen. In contrast, the purified 39-kDa protein was able to stimulate enriched populations of B cells to proliferation with almost the same strength of activity as LPS (Fig. 4B and C). Together, these

VOL. 61, 1993

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FIG. 3. Stimulation of T lymphocytes with concanavalin A (ConA [A]), purified LPS I from P. mirabiis (B), or purified 39-kDa protein from P. mirabiis (C). T lymphocytes were isolated by passage of spleen cells from NMRI mice over columns containing nylon wool fiber two times. Proliferation was measured by the

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test. Results are reported as geometric means standard errors of values taken from three separate experiments. OD, optical density.

FIG. 4. Stimulation of B lymphocytes with concanavalin A (ConA [A]), purified LPS I from P. mirabilis (B), or purified 39-kDa protein from P. mirabilis (C). B lymphocytes were obtained by treating spleen cells from C57BL/6 mice with monoclonal antibody to Thyl.2 plus complement. Proliferation was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test. Results are reported as geometric means standard errors of values taken from two or three separate experiments. OD, optical density.

results indicate that the 39-kDa protein is a murine B-lymphocyte mitogen, which seems to be a property shared by several outer membrane proteins of gram-negative bacteria (2, 6), including OmpA of E. coli (3).

This work was supported by the Deutsche Forschungsgemeinschaft. We especially thank Waltraud Ruiner for excellent technical assistance.

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