INFECrION AND IMMUNITY, Apr. 1992, p. 1695-1698 0019-9567/92/041695-04$02.00/0 Copyright C) 1992, American Society for Microbiology
Vol. 60, No. 4
Biochemical Studies of Helicobacter mustelae Fatty Acid Composition and Flagella SEBASTIAN SUERBAUM,t* GABRIELE GEIS, CHRISTINE JOSENHANS, AND WOLFGANG OPFERKUCH Medical Microbiology and Immunology, Ruhr-Universitat Bochum, D-4630 Bochum 1, Germany Received 26 August 1991/Accepted 22 January 1992
The fatty acid compositions of Helicobacter mustelae whole cells, isolated phospholipids, and isolated lipopolysaccharides were analyzed by gas-liquid chromatography. Major phospholipid fatty acids were C16:0, C18:09 Cl,:jl and C19.0 cyc. In isolated lipopolysaccharides, 3-OH-C16:0O 3-OH-C4:0O C14:0, C16:0, and C18:0 were found. The lipid composition of H. mustelae thus showed pronounced differences from that of H. pylori. Flagella were purified by mechanical shearing and centrifugation steps. In all H. mustelae strains, the flagellin had an apparent molecular mass of 53 kDa and was thus the same size as H. pylori flagellin. The flagellin of strain NCTC 12032 was further purified and subjected to N-terminal amino acid sequence analysis. The first 10 amino acids were identical to those of H. pylori flagellin, but the next 5 were different. Significant homology was also found with flagellins of other bacteria.
Since the first report of Helicobacter pylon, the causative agent of human chronic type B gastritis (30), this bacterium has elicited a great deal of interest among both microbiologists and gastroenterologists (for reviews, see references 2 and 26). H. mustelae is a common pathogen in ferrets, in which, like H. pylon, it colonizes the gastric mucosa and causes chronic gastritis. The disease associated with H. mustelae infection shares several features with that associated with human H. pyloni infection. H. mustelae gastritis is histologically similar to human H. pylon gastritis, ferrets infected with H. mustelae develop both gastric and duodenal ulcers, and the infection is accompanied by a pronounced humoral immune response (7, 9, 10). H. mustelae infection of ferrets is thus considered an important animal model of human H. pylon infection, and the first studies done with this model for the evaluation of therapeutic regimens for H. pylon eradication were published recently (25). The morphological and phenotypic properties of H. mustelae are remarkably similar to those of H. pylon: both are oxidase- and catalase-producing gram-negative spiral bacteria requiring a microaerophilic atmosphere for optimal growth. Both express abundant amounts of urease and are weakly hemolytic on blood agar plates. In addition to the polar sheathed flagella characteristic of H. pylon, H. mustelae has lateral flagella (8, 14). Morgan et al. (23) screened H. mustelae strains for the expression of potential virulence factors. They could not detect cytotoxic activity but found hemagglutinins similar to those described for H. pylon. Except for the urease enzyme (3), there is as yet no detailed information concerning defined cell structures of H. mustelae. Comparative studies of H. pylon and H. mustelae are a potential tool for evaluating the relevance of structures to the pathogenic potential of both organisms. We therefore performed biochemical analyses of H. mustelae lipids, lipopolysaccharides (LPS), and flagella. The data are discussed in relation to our previously published data on H. pylon (11,
used for the experiments. Bacteria were grown for 3 to 5 days on Columbia agar supplemented with sheep blood or for 3 days in brain heart infusion broth supplemented with 8% horse serum and 0.25% yeast extract with agitation (37°C; microaerophilic conditions). For determination of the fatty acid composition of H. mustelae, the bacteria were grown on brain heart infusion agar supplemented with lysed horse blood as described by Goodwin et al. (15). LPS were isolated by hot phenol-water extraction (31). Phospholipids were extracted by the method of Folch et al. (6) and evaporated with a gentle flow of nitrogen. Protein content was measured by the method of Markwell et al. (22) with bovine serum albumin as the standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Lugtenberg et al. (21). The concentration of acrylamide in the separating gel was 11%. Sixty micrograms of protein was applied to each slot of the gel. The gels were stained with Coomassie blue R-250 (Pharmacia-LKB, Freiburg, Germany). Protein mixtures of myosin (205,000 Da), ,B-galactosidase (116,000 Da), phosphorylase b (94,000 Da), albumin (67,000 Da), ovalbumin (43,000 Da), carbonic anhydrase (30,000 Da), trypsin inhibitor (20,100 Da), and a-lactalbumin (14,400 Da) were used as standards for molecular mass determinations. Fatty acid methyl esters were prepared as previously described by method A of Gmeiner and Martin (13). Conditions for gas-liquid chromatography were as previously described (12). Flagellar filaments were isolated as previously described (11). In brief, the flagella were removed from the cells by mechanical shearing in a Sorvall omnimixer (5 min; 4'C). The sheared cell suspension was centrifuged twice (5,000 x g; 20 min), and the flagellum-containing supernatant was pelleted by ultracentrifugation (90,000 x g; 1 h). For protein sequencing, the material was subjected to preparative SDSPAGE, and the flagellin band was cut out of the gel and electroeluted with a Biotrap device (Schleicher and Schull, Dassel, Germany) into a buffer containing 0.01 M NH4HCO3 and 0.025% SDS. N-terminal amino acid analysis was performed by Edman degradation with a solid-phase sequencer under the conditions described previously (28). Cellular fatty acids as well as fatty acid substitutions in isolated phospholipids and LPS were analyzed for H. mus-
12). H. mustelae ATCC 43772, NCTC 12032, Fl, and F13 were
Corresponding author. t Present address: Unite des Enterobacteries, Institut Pasteur, 28, Rue du Dr. Roux, F-75724 Paris Cedex 15, France. *
1695
1696
INFECT. IMMUN.
NOTES TABLE 1. Fatty acid compositions of H. mustelae and H. pylon
Fatty acid source
Cellular
Strain
Amt of the following fatty acid (% of total)':
C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 3-OH-C14:0 C19o0 cyc 3-OH-C16:0 3-OH-C8:0
H. mustelae ATCC 43772 H. mustelae NCTC 12032 H. pylon NCTC 11637 H. pylori 898-1
12 9 45
43
32 29 2 3
Isolated phospholipids H. mustelae ATCC 43772 H. mustelae NCTC 12032 H. pylon NCTC 11637 H. pylon 898-1
6 4 49 41
22 24 3 7
H. mustelae ATCC 43772 H. mustelae NCTC 12032 H. pylon NCTC 11637 H. pylon 898-1
10 9 3 4
12 10 10 2
Isolated LPS
7
16 28 5 6
2 2 Tr Tr
Tr Tr
17 16 4 5
14 23 5 7
8 10 Tr 2
-
11 9 23 21
4 4
Tr Tr
-
-
-
4 3 7
3 3
20 16 24
4 4 4
7
22
6
7
26 31 27 25
22 24
13 7 30 28 11 11
-
a Tr, 0.5 to 1.9%; -, less than 0.5%. The number before the colon refers to the number of carbon atoms, and the number after the colon refers to the number of double bonds; cyc refers to a cyclopropane fatty acid; OH refers to a hydroxyl group.
telae ATCC 43772 and NCTC 12032 by gas-liquid chromatography (Table 1). Previously published values for H. pylon (12) are also given to facilitate comparison. The results for the two H. mustelae strains were very similar. However, there were pronounced differences from the profiles for H. pylon. Major cellular fatty acids were palmitic acid (C16:0), C19 cyclopropane fatty acid (C19:0 cyc), oleic acid (C18:1), and myristic acid (C14:0). Stearic acid (C18:0), 1-hydroxypalmitic acid (3-OH-C16:0), B-hydroxymyristic acid (3-OHC14:0), and linoleic acid (C18:2) were present in minor amounts. Major phospholipid fatty acids were C16:0, C18:0, C18:1, and C19:0 cyc. Small amounts of C18:2 and C14:0 were detected. Isolated LPS contained predominantly 3-OH-C16:0 and nearly equal amounts of 3-0H-C14:0 C14:0, C16:0, and
considered a suitable model for studying the virulence factors involved in the pathogenesis of gastric Helicobacter infections. In this paper, we report on comparative studies concerning lipids, LPS, and flagella of H. mustelae. Our data on the cellular fatty acid composition of H. mustelae are in good agreement with those published by Goodwin et al. (14, 15), with the exceptions that they did not detect 3-OH-C14:0 and that we did not detect C15:0. We observed an extraordinary degree of variation in the fatty acid compositions dependent on the growth conditions (data not shown), and although we used exactly the same media as those described by Goodwin et al., growth conditions may
C18:0.
Flagella were mechanically detached from the cell surface of H. mustelae. After ultracentrifugation, flagella were enriched in the sediment, as demonstrated by electron microscopy. SDS-PAGE analysis of this material revealed several protein bands. All four strains expressed a markedly predominant band with an apparent molecular mass of 53 kDa, the putative flagellin (Fig. 1). For analysis of the N-terminal amino acid sequence, the 53-kDa protein of H. mustelae NCTC 12032 was further purified by preparative SDSPAGE, excision of the band, and electroelution. Table 2 shows the 15 N-terminal amino acids of this protein in comparison with those of H. pylon, Campylobacter coli, and Salmonella typhimurium flagellins. The first 10 amino acids were identical to those of H. pylon flagellin (18, 19), but the next 5 amino acids differed. Both sequences were partly homologous to those published for C coli and S. typhimurium (17, 20). The marked sequence homologies confirmed that the purified 53-kDa protein was indeed flagellin. Research on the pathogenesis of H. pylon-associated gastroduodenal disease has been hampered by the lack of good animal models. Eaton et al. (4, 5) used gnotobiotic piglets to study the role of defined H. pylon virulence factors in colonization. However, this model is labor-intensive and very expensive. An alternative approach to conventional animal models of an infectious disease is to study the relationship between a closely related animal pathogen and its natural host. H. mustelae is a bacterium closely related to H. pylon and naturally infects ferrets, causing gastritis and ulcer disease (9). H. mustelae infection of ferrets is therefore
205 116
94U 67
.*-
-
m
43
30 20.1 14.4
_
*_ i_ X--i__ __
1
2 3
4 5
FIG. 1. SDS-PAGE analysis of H. mustelae flagellar preparations. Lanes: 1, standard proteins; 2, ATCC 43772; 3, NCTC 12032; 4, Fl; 5, F13. Numbers at left are sizes in kilodaltons.
VOL. 60, 1992
NOTES
TABLE 2. N-terminal amino acid sequences of Helicobacter flagellins and flagellins of other bacteria Flagellin source (reference) Residuesa H. mustelae NCTC 12032 ......A F Q V N T N I N A L T T X A H. pylori NCTC 11637 .......... MNAHV C. coli (22) .................... G - R I - - - V A - - N A K S. typhimurium (17) ..............- QVI- - - S L S - L - Q N -
a Amino acid residues are designated by the single-letter nomenclature. -, residues homologous to H. mustelae NCTC 12032 residues; X, not identified.
be one explanation for the differences. Medium-dependent variations in lipid composition were not observed in our H. pylori study (12), and it needs to be stressed that the stability of fatty acid patterns under different growth conditions must be determined for a given species before these patterns are used for identification or chemotaxonomic purposes. The analysis of isolated phospholipids and LPS revealed remarkable differences between H. mustelae and H. pylori. Isolated phospholipids of H. mustelae contained the same fatty acids as those of H. pyloni. However, in H. mustelae we detected large amounts of C16:0, C18:0, C18:1, and C18:2, in contrast to the marked predominance of C14:0 and C19:0 cyc in H. pylori. Likewise, the LPS fatty acid substitutions in H. mustelae differed from those in H. pyloni. The unusual 3-OH-C18:0 characteristic of H. pylon LPS was not detected in H. mustelae LPS and was replaced by the more common 3-OH-C14:0. The other unusual fatty acid in H. pylori LPS, C18:0, was also present in H. mustelae LPS, but in smaller amounts.
Fatty acid substitutions are known to affect the biological effects of LPS (for a review, see reference 24). It was recently shown that LPS isolated from H. pylori were less effective than Campylobacterjejuni or S. abortusequi LPS in liberating tumor necrosis factor from human peripheral blood lymphocytes and both tumor necrosis factor and interleukin-6 from mouse peritoneal macrophages (1). Unusual fatty acid substitutions may be responsible for the low biological activity of Helicobacter LPS. The molecular masses of H. pylon and H. mustelae flagellins were virtually identical and determined to be 53 kDa. We had originally reported 51 kDa as the molecular mass of H. pylon flagellin (11), and Kostrzynska et al. (18) reported 56 kDa for the same protein (as ascertained by the identical N-terminal amino acid sequences of the two proteins). The gene encoding H. pylon flagellin was recently cloned and sequenced (19), and the molecular mass of the polypeptide deduced from the flaA sequence is 53.2 kDa, consistent with the value obtained in this study. Further comparisons between H. mustelae and H. pylon flagellins also revealed a high degree of similarity. The first 10 N-terminal amino acids were identical. H. mustelae chromosomal DNA strongly hybridized with a probe derived from the cloned H. pylon flaA gene (29). On the other hand, polyclonal antibodies directed against H. pylon flagellin showed only weak cross-reactivity with H. mustelae flagellin (data not shown). Although the antisera only detected one protein band, our data do not rule out the possibility of a second flagellin species in H. mustelae, as has been reported for H. pyloni (18) and also for Vibio parahaemolyticus which, like H. mustelae, has lateral flagella (27). Recently, the construction of aflagellated mutants of H. pylori by allelic exchange was described (16). Cloning and mutagenesis of H. mustelae flagellin genes are under way in our laboratory. These mutants will permit us to prove the
1697
importance of flagella in the virulence of the bacteria. Similar approaches will allow elucidation of the roles of other putative virulence factors in both the gnotobiotic piglet model of H. pylon infection and the H. mustelae-ferret system. We are very grateful to Hein-Peter Kroll and Werner Schroder for performing the amino acid sequencing, to D. S. Tompkins for H. mustelae Fl and F13, and to Ulrike Deiss for excellent technical assistance. REFERENCES 1. Birkholz, S., U. Knipp, C. Nietski, U. Schade, and W. Opferkuch. 1991. Mitogenicity and cytokine releasing capacity of Helicobacter pylori lipopolysaccharide in comparison with lipopolysaccharides of other intestinal bacteria, abstr. p128. Ital. J.
Gastroenterol. 23(Suppl. 2):50. 2. Dick, J. D. 1990. Helicobacter (Campylobacter) pylori: a new twist to an old disease. Annu. Rev. Microbiol. 44:249-269. 3. Dunn, B., C. Sung, N. Taylor, and J. Fox. 1991. Characterization of the urease of Helicobacter mustelae, abstr. B-77, p. 38. Abstr. 91st Gen. Meet. Am. Soc. Microbiol. 1991. 4. Eaton, K. A., C. L. Brooks, D. R. Morgan, and S. Krakowka. 1991. Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylon in gnotobiotic piglets. Infect. Immun. 59:2470-2475. 5. Eaton, K. A., D. R. Morgan, and S. Krakowka. 1989. Campylobacter pylori virulence factors in gnotobiotic piglets. Infect. Immun. 57:1119-1125. 6. Folch, J., M. Lees, and G. H. Sloane Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-505. 7. Fox, J. G., E. B. Cabot, N. S. Taylor, and R. Laraway. 1988. Gastric colonization by Campylobacter pylon subsp. mustelae in ferrets. Infect. Immun. 56:2994-2996. 8. Fox, J. G., T. Chilvers, C. S. Goodwin, N. S. Taylor, P. Edmonds, L. I. Sly, and D. J. Brenner. 1989. Campylobacter mustelae, a new species resulting from the elevation of Campylobacterpylori subsp. mustelae to species status. Int. J. Syst. Bacteriol. 39:301-303. 9. Fox, J. G., P. Correa, N. S. Taylor, A. Lee, G. Otto, J. C. Murphy, and R. Rose. 1990. Helicobacter mustelae-associated gastritis in ferrets. An animal model of Helicobacter pylori gastritis in humans. Gastroenterology 99:352-361. 10. Fox, J. G., G. Otto, N. S. Taylor, W. Rosenblad, and J. C. Murphy. 1991. Helicobacter mustelae-induced gastritis and elevated gastric pH in the ferret (Mustela putonius furo). Infect. Immun. 59:1875-1880. 11. Geis, G., H. Leying, S. Suerbaum, U. Mai, and W. Opferkuch. 1989. Ultrastructure and chemical analysis of Campylobacter pylon flagella. J. Clin. Microbiol. 27:436-441. 12. Geis, G., H. Leying, S. Suerbaum, and W. Opferkuch. 1990. Unusual fatty acid substitution in lipids and lipopolysaccharides of Helicobacterpyloni. J. Clin. Microbiol. 28:930-932. 13. Gmeiner, J., and H. H. Martin. 1976. Phospholipid and lipopolysaccharide in Proteus mirabilis and its stable protoplast L-form. Eur. J. Biochem. 67:487-494. 14. Goodwin, C. S., J. A. Armstrong, T. Chilvers, M. Peters, M. D. Collins, L. Sly, W. McConnell, and W. E. S. Harper. 1989. Transfer of Campylobacter pylon and Campylobacter mustelae to Helicobacter gen. nov. as Helicobacter pylon comb. nov. and Helicobacter mustelae comb. nov., respectively. Int. J. Syst. Bacteriol. 39:397-405. 15. Goodwin, C. S., W. McConnell, R. K. McCulloch, C. McCullough, R. Hill, M. A. Bronsdon, and G. Kasper. 1989. Cellular fatty acid composition of Campylobacter pylon from primates and ferrets compared with those of other campylobacters. J. Clin. Microbiol. 27:938-943. 16. Haas, R., H. Leying, T. F. Meyer, G. Geis, S. Suerbaum, and W. Opferkuch. 1991. Cloning of a H. pylori flagellin gene and construction of a non-flagellated mutant by transformationmediated allelic exchange, abstr. p66. Ital. J. Gastroenterol.
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NOTES
23(Suppl. 2):31. 17. Joys, T. M. 1985. The covalent structure of the phase-1 flagellar filament protein of Salmonella typhimurium and its comparison with other flagellins. J. Biol. Chem. 260:15758-15761. 18. Kostrzynska, M., J. D. Betts, J. W. Austin, and T. J. Trust. 1991. Identification, characterization, and spatial localization of two flagellin species in Helicobacterpylon flagella. J. Bacteriol. 173:937-946. 19. Leying, H., S. Suerbaum, G. Geis, and R. Haas. Submitted for publication. 20. Logan, S. M., L. A. Harris, and T. J. Trust. 1987. Isolation and characterization of Campylobacter flagellins. J. Bacteriol. 169: 5072-5077. 21. Lugtenberg, B., J. Meiers, 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. 58:254-258. 22. Markwell, M. A., S. M. Haas, 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. 23. Morgan, D. R., J. G. Fox, and R. D. Leunk. 1991. Comparison of isolates of Helicobacterpylon and Helicobacter mustelae. J. Clin. Microbiol. 29:395-397.
INFECT. IMMUN. 24. Morrison, D. C. 1983. Bacterial endotoxins and pathogenesis. Rev. Infect. Dis. 5(Suppl. 4):S733-S747. 25. Otto, G., J. G. Fox, P. Y. Wu, and N. S. Taylor. 1990. Eradication of Helicobacter mustelae from the ferret stomach: an animal model of Helicobacter (Campylobacter) pylon chemotherapy. Antimicrob. Agents Chemother. 34:1232-1236. 26. Peterson, W. L. 1991. Helicobacter pylori and peptic ulcer disease. N. Engl. J. Med. 324:1043-1048. 27. Shinoda, S., T. Miwatani, and T. Fujino. 1970. Antigens of Vibrio parahaemolyticus. II. Existence of two different subunits in the flagella of Vibrio parahaemolyticus and their characterization. Biken J. 13:241-247. 28. Siekmann, J., H. R. Wenzel, W. Schroder, and H. Tschesche. 1988. Characterization and sequence determination of six aprotinin homologues from bovine lungs. Biol. Chem. Hoppe-Seyler 369:157-163. 29. Suerbaum, S., and C. Josenhans. Unpublished data. 30. Warren, J. R., and B. Marshall. 1983. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet i:1273-1275. 31. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharides. Extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem. 5:83-91.
Vol. 60, No. 4
Biochemical Studies of Helicobacter mustelae Fatty Acid Composition and Flagella SEBASTIAN SUERBAUM,t* GABRIELE GEIS, CHRISTINE JOSENHANS, AND WOLFGANG OPFERKUCH Medical Microbiology and Immunology, Ruhr-Universitat Bochum, D-4630 Bochum 1, Germany Received 26 August 1991/Accepted 22 January 1992
The fatty acid compositions of Helicobacter mustelae whole cells, isolated phospholipids, and isolated lipopolysaccharides were analyzed by gas-liquid chromatography. Major phospholipid fatty acids were C16:0, C18:09 Cl,:jl and C19.0 cyc. In isolated lipopolysaccharides, 3-OH-C16:0O 3-OH-C4:0O C14:0, C16:0, and C18:0 were found. The lipid composition of H. mustelae thus showed pronounced differences from that of H. pylori. Flagella were purified by mechanical shearing and centrifugation steps. In all H. mustelae strains, the flagellin had an apparent molecular mass of 53 kDa and was thus the same size as H. pylori flagellin. The flagellin of strain NCTC 12032 was further purified and subjected to N-terminal amino acid sequence analysis. The first 10 amino acids were identical to those of H. pylori flagellin, but the next 5 were different. Significant homology was also found with flagellins of other bacteria.
Since the first report of Helicobacter pylon, the causative agent of human chronic type B gastritis (30), this bacterium has elicited a great deal of interest among both microbiologists and gastroenterologists (for reviews, see references 2 and 26). H. mustelae is a common pathogen in ferrets, in which, like H. pylon, it colonizes the gastric mucosa and causes chronic gastritis. The disease associated with H. mustelae infection shares several features with that associated with human H. pyloni infection. H. mustelae gastritis is histologically similar to human H. pylon gastritis, ferrets infected with H. mustelae develop both gastric and duodenal ulcers, and the infection is accompanied by a pronounced humoral immune response (7, 9, 10). H. mustelae infection of ferrets is thus considered an important animal model of human H. pylon infection, and the first studies done with this model for the evaluation of therapeutic regimens for H. pylon eradication were published recently (25). The morphological and phenotypic properties of H. mustelae are remarkably similar to those of H. pylon: both are oxidase- and catalase-producing gram-negative spiral bacteria requiring a microaerophilic atmosphere for optimal growth. Both express abundant amounts of urease and are weakly hemolytic on blood agar plates. In addition to the polar sheathed flagella characteristic of H. pylon, H. mustelae has lateral flagella (8, 14). Morgan et al. (23) screened H. mustelae strains for the expression of potential virulence factors. They could not detect cytotoxic activity but found hemagglutinins similar to those described for H. pylon. Except for the urease enzyme (3), there is as yet no detailed information concerning defined cell structures of H. mustelae. Comparative studies of H. pylon and H. mustelae are a potential tool for evaluating the relevance of structures to the pathogenic potential of both organisms. We therefore performed biochemical analyses of H. mustelae lipids, lipopolysaccharides (LPS), and flagella. The data are discussed in relation to our previously published data on H. pylon (11,
used for the experiments. Bacteria were grown for 3 to 5 days on Columbia agar supplemented with sheep blood or for 3 days in brain heart infusion broth supplemented with 8% horse serum and 0.25% yeast extract with agitation (37°C; microaerophilic conditions). For determination of the fatty acid composition of H. mustelae, the bacteria were grown on brain heart infusion agar supplemented with lysed horse blood as described by Goodwin et al. (15). LPS were isolated by hot phenol-water extraction (31). Phospholipids were extracted by the method of Folch et al. (6) and evaporated with a gentle flow of nitrogen. Protein content was measured by the method of Markwell et al. (22) with bovine serum albumin as the standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Lugtenberg et al. (21). The concentration of acrylamide in the separating gel was 11%. Sixty micrograms of protein was applied to each slot of the gel. The gels were stained with Coomassie blue R-250 (Pharmacia-LKB, Freiburg, Germany). Protein mixtures of myosin (205,000 Da), ,B-galactosidase (116,000 Da), phosphorylase b (94,000 Da), albumin (67,000 Da), ovalbumin (43,000 Da), carbonic anhydrase (30,000 Da), trypsin inhibitor (20,100 Da), and a-lactalbumin (14,400 Da) were used as standards for molecular mass determinations. Fatty acid methyl esters were prepared as previously described by method A of Gmeiner and Martin (13). Conditions for gas-liquid chromatography were as previously described (12). Flagellar filaments were isolated as previously described (11). In brief, the flagella were removed from the cells by mechanical shearing in a Sorvall omnimixer (5 min; 4'C). The sheared cell suspension was centrifuged twice (5,000 x g; 20 min), and the flagellum-containing supernatant was pelleted by ultracentrifugation (90,000 x g; 1 h). For protein sequencing, the material was subjected to preparative SDSPAGE, and the flagellin band was cut out of the gel and electroeluted with a Biotrap device (Schleicher and Schull, Dassel, Germany) into a buffer containing 0.01 M NH4HCO3 and 0.025% SDS. N-terminal amino acid analysis was performed by Edman degradation with a solid-phase sequencer under the conditions described previously (28). Cellular fatty acids as well as fatty acid substitutions in isolated phospholipids and LPS were analyzed for H. mus-
12). H. mustelae ATCC 43772, NCTC 12032, Fl, and F13 were
Corresponding author. t Present address: Unite des Enterobacteries, Institut Pasteur, 28, Rue du Dr. Roux, F-75724 Paris Cedex 15, France. *
1695
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INFECT. IMMUN.
NOTES TABLE 1. Fatty acid compositions of H. mustelae and H. pylon
Fatty acid source
Cellular
Strain
Amt of the following fatty acid (% of total)':
C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 3-OH-C14:0 C19o0 cyc 3-OH-C16:0 3-OH-C8:0
H. mustelae ATCC 43772 H. mustelae NCTC 12032 H. pylon NCTC 11637 H. pylori 898-1
12 9 45
43
32 29 2 3
Isolated phospholipids H. mustelae ATCC 43772 H. mustelae NCTC 12032 H. pylon NCTC 11637 H. pylon 898-1
6 4 49 41
22 24 3 7
H. mustelae ATCC 43772 H. mustelae NCTC 12032 H. pylon NCTC 11637 H. pylon 898-1
10 9 3 4
12 10 10 2
Isolated LPS
7
16 28 5 6
2 2 Tr Tr
Tr Tr
17 16 4 5
14 23 5 7
8 10 Tr 2
-
11 9 23 21
4 4
Tr Tr
-
-
-
4 3 7
3 3
20 16 24
4 4 4
7
22
6
7
26 31 27 25
22 24
13 7 30 28 11 11
-
a Tr, 0.5 to 1.9%; -, less than 0.5%. The number before the colon refers to the number of carbon atoms, and the number after the colon refers to the number of double bonds; cyc refers to a cyclopropane fatty acid; OH refers to a hydroxyl group.
telae ATCC 43772 and NCTC 12032 by gas-liquid chromatography (Table 1). Previously published values for H. pylon (12) are also given to facilitate comparison. The results for the two H. mustelae strains were very similar. However, there were pronounced differences from the profiles for H. pylon. Major cellular fatty acids were palmitic acid (C16:0), C19 cyclopropane fatty acid (C19:0 cyc), oleic acid (C18:1), and myristic acid (C14:0). Stearic acid (C18:0), 1-hydroxypalmitic acid (3-OH-C16:0), B-hydroxymyristic acid (3-OHC14:0), and linoleic acid (C18:2) were present in minor amounts. Major phospholipid fatty acids were C16:0, C18:0, C18:1, and C19:0 cyc. Small amounts of C18:2 and C14:0 were detected. Isolated LPS contained predominantly 3-OH-C16:0 and nearly equal amounts of 3-0H-C14:0 C14:0, C16:0, and
considered a suitable model for studying the virulence factors involved in the pathogenesis of gastric Helicobacter infections. In this paper, we report on comparative studies concerning lipids, LPS, and flagella of H. mustelae. Our data on the cellular fatty acid composition of H. mustelae are in good agreement with those published by Goodwin et al. (14, 15), with the exceptions that they did not detect 3-OH-C14:0 and that we did not detect C15:0. We observed an extraordinary degree of variation in the fatty acid compositions dependent on the growth conditions (data not shown), and although we used exactly the same media as those described by Goodwin et al., growth conditions may
C18:0.
Flagella were mechanically detached from the cell surface of H. mustelae. After ultracentrifugation, flagella were enriched in the sediment, as demonstrated by electron microscopy. SDS-PAGE analysis of this material revealed several protein bands. All four strains expressed a markedly predominant band with an apparent molecular mass of 53 kDa, the putative flagellin (Fig. 1). For analysis of the N-terminal amino acid sequence, the 53-kDa protein of H. mustelae NCTC 12032 was further purified by preparative SDSPAGE, excision of the band, and electroelution. Table 2 shows the 15 N-terminal amino acids of this protein in comparison with those of H. pylon, Campylobacter coli, and Salmonella typhimurium flagellins. The first 10 amino acids were identical to those of H. pylon flagellin (18, 19), but the next 5 amino acids differed. Both sequences were partly homologous to those published for C coli and S. typhimurium (17, 20). The marked sequence homologies confirmed that the purified 53-kDa protein was indeed flagellin. Research on the pathogenesis of H. pylon-associated gastroduodenal disease has been hampered by the lack of good animal models. Eaton et al. (4, 5) used gnotobiotic piglets to study the role of defined H. pylon virulence factors in colonization. However, this model is labor-intensive and very expensive. An alternative approach to conventional animal models of an infectious disease is to study the relationship between a closely related animal pathogen and its natural host. H. mustelae is a bacterium closely related to H. pylon and naturally infects ferrets, causing gastritis and ulcer disease (9). H. mustelae infection of ferrets is therefore
205 116
94U 67
.*-
-
m
43
30 20.1 14.4
_
*_ i_ X--i__ __
1
2 3
4 5
FIG. 1. SDS-PAGE analysis of H. mustelae flagellar preparations. Lanes: 1, standard proteins; 2, ATCC 43772; 3, NCTC 12032; 4, Fl; 5, F13. Numbers at left are sizes in kilodaltons.
VOL. 60, 1992
NOTES
TABLE 2. N-terminal amino acid sequences of Helicobacter flagellins and flagellins of other bacteria Flagellin source (reference) Residuesa H. mustelae NCTC 12032 ......A F Q V N T N I N A L T T X A H. pylori NCTC 11637 .......... MNAHV C. coli (22) .................... G - R I - - - V A - - N A K S. typhimurium (17) ..............- QVI- - - S L S - L - Q N -
a Amino acid residues are designated by the single-letter nomenclature. -, residues homologous to H. mustelae NCTC 12032 residues; X, not identified.
be one explanation for the differences. Medium-dependent variations in lipid composition were not observed in our H. pylori study (12), and it needs to be stressed that the stability of fatty acid patterns under different growth conditions must be determined for a given species before these patterns are used for identification or chemotaxonomic purposes. The analysis of isolated phospholipids and LPS revealed remarkable differences between H. mustelae and H. pylori. Isolated phospholipids of H. mustelae contained the same fatty acids as those of H. pyloni. However, in H. mustelae we detected large amounts of C16:0, C18:0, C18:1, and C18:2, in contrast to the marked predominance of C14:0 and C19:0 cyc in H. pylori. Likewise, the LPS fatty acid substitutions in H. mustelae differed from those in H. pyloni. The unusual 3-OH-C18:0 characteristic of H. pylon LPS was not detected in H. mustelae LPS and was replaced by the more common 3-OH-C14:0. The other unusual fatty acid in H. pylori LPS, C18:0, was also present in H. mustelae LPS, but in smaller amounts.
Fatty acid substitutions are known to affect the biological effects of LPS (for a review, see reference 24). It was recently shown that LPS isolated from H. pylori were less effective than Campylobacterjejuni or S. abortusequi LPS in liberating tumor necrosis factor from human peripheral blood lymphocytes and both tumor necrosis factor and interleukin-6 from mouse peritoneal macrophages (1). Unusual fatty acid substitutions may be responsible for the low biological activity of Helicobacter LPS. The molecular masses of H. pylon and H. mustelae flagellins were virtually identical and determined to be 53 kDa. We had originally reported 51 kDa as the molecular mass of H. pylon flagellin (11), and Kostrzynska et al. (18) reported 56 kDa for the same protein (as ascertained by the identical N-terminal amino acid sequences of the two proteins). The gene encoding H. pylon flagellin was recently cloned and sequenced (19), and the molecular mass of the polypeptide deduced from the flaA sequence is 53.2 kDa, consistent with the value obtained in this study. Further comparisons between H. mustelae and H. pylon flagellins also revealed a high degree of similarity. The first 10 N-terminal amino acids were identical. H. mustelae chromosomal DNA strongly hybridized with a probe derived from the cloned H. pylon flaA gene (29). On the other hand, polyclonal antibodies directed against H. pylon flagellin showed only weak cross-reactivity with H. mustelae flagellin (data not shown). Although the antisera only detected one protein band, our data do not rule out the possibility of a second flagellin species in H. mustelae, as has been reported for H. pyloni (18) and also for Vibio parahaemolyticus which, like H. mustelae, has lateral flagella (27). Recently, the construction of aflagellated mutants of H. pylori by allelic exchange was described (16). Cloning and mutagenesis of H. mustelae flagellin genes are under way in our laboratory. These mutants will permit us to prove the
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importance of flagella in the virulence of the bacteria. Similar approaches will allow elucidation of the roles of other putative virulence factors in both the gnotobiotic piglet model of H. pylon infection and the H. mustelae-ferret system. We are very grateful to Hein-Peter Kroll and Werner Schroder for performing the amino acid sequencing, to D. S. Tompkins for H. mustelae Fl and F13, and to Ulrike Deiss for excellent technical assistance. REFERENCES 1. Birkholz, S., U. Knipp, C. Nietski, U. Schade, and W. Opferkuch. 1991. Mitogenicity and cytokine releasing capacity of Helicobacter pylori lipopolysaccharide in comparison with lipopolysaccharides of other intestinal bacteria, abstr. p128. Ital. J.
Gastroenterol. 23(Suppl. 2):50. 2. Dick, J. D. 1990. Helicobacter (Campylobacter) pylori: a new twist to an old disease. Annu. Rev. Microbiol. 44:249-269. 3. Dunn, B., C. Sung, N. Taylor, and J. Fox. 1991. Characterization of the urease of Helicobacter mustelae, abstr. B-77, p. 38. Abstr. 91st Gen. Meet. Am. Soc. Microbiol. 1991. 4. Eaton, K. A., C. L. Brooks, D. R. Morgan, and S. Krakowka. 1991. Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylon in gnotobiotic piglets. Infect. Immun. 59:2470-2475. 5. Eaton, K. A., D. R. Morgan, and S. Krakowka. 1989. Campylobacter pylori virulence factors in gnotobiotic piglets. Infect. Immun. 57:1119-1125. 6. Folch, J., M. Lees, and G. H. Sloane Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-505. 7. Fox, J. G., E. B. Cabot, N. S. Taylor, and R. Laraway. 1988. Gastric colonization by Campylobacter pylon subsp. mustelae in ferrets. Infect. Immun. 56:2994-2996. 8. Fox, J. G., T. Chilvers, C. S. Goodwin, N. S. Taylor, P. Edmonds, L. I. Sly, and D. J. Brenner. 1989. Campylobacter mustelae, a new species resulting from the elevation of Campylobacterpylori subsp. mustelae to species status. Int. J. Syst. Bacteriol. 39:301-303. 9. Fox, J. G., P. Correa, N. S. Taylor, A. Lee, G. Otto, J. C. Murphy, and R. Rose. 1990. Helicobacter mustelae-associated gastritis in ferrets. An animal model of Helicobacter pylori gastritis in humans. Gastroenterology 99:352-361. 10. Fox, J. G., G. Otto, N. S. Taylor, W. Rosenblad, and J. C. Murphy. 1991. Helicobacter mustelae-induced gastritis and elevated gastric pH in the ferret (Mustela putonius furo). Infect. Immun. 59:1875-1880. 11. Geis, G., H. Leying, S. Suerbaum, U. Mai, and W. Opferkuch. 1989. Ultrastructure and chemical analysis of Campylobacter pylon flagella. J. Clin. Microbiol. 27:436-441. 12. Geis, G., H. Leying, S. Suerbaum, and W. Opferkuch. 1990. Unusual fatty acid substitution in lipids and lipopolysaccharides of Helicobacterpyloni. J. Clin. Microbiol. 28:930-932. 13. Gmeiner, J., and H. H. Martin. 1976. Phospholipid and lipopolysaccharide in Proteus mirabilis and its stable protoplast L-form. Eur. J. Biochem. 67:487-494. 14. Goodwin, C. S., J. A. Armstrong, T. Chilvers, M. Peters, M. D. Collins, L. Sly, W. McConnell, and W. E. S. Harper. 1989. Transfer of Campylobacter pylon and Campylobacter mustelae to Helicobacter gen. nov. as Helicobacter pylon comb. nov. and Helicobacter mustelae comb. nov., respectively. Int. J. Syst. Bacteriol. 39:397-405. 15. Goodwin, C. S., W. McConnell, R. K. McCulloch, C. McCullough, R. Hill, M. A. Bronsdon, and G. Kasper. 1989. Cellular fatty acid composition of Campylobacter pylon from primates and ferrets compared with those of other campylobacters. J. Clin. Microbiol. 27:938-943. 16. Haas, R., H. Leying, T. F. Meyer, G. Geis, S. Suerbaum, and W. Opferkuch. 1991. Cloning of a H. pylori flagellin gene and construction of a non-flagellated mutant by transformationmediated allelic exchange, abstr. p66. Ital. J. Gastroenterol.
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23(Suppl. 2):31. 17. Joys, T. M. 1985. The covalent structure of the phase-1 flagellar filament protein of Salmonella typhimurium and its comparison with other flagellins. J. Biol. Chem. 260:15758-15761. 18. Kostrzynska, M., J. D. Betts, J. W. Austin, and T. J. Trust. 1991. Identification, characterization, and spatial localization of two flagellin species in Helicobacterpylon flagella. J. Bacteriol. 173:937-946. 19. Leying, H., S. Suerbaum, G. Geis, and R. Haas. Submitted for publication. 20. Logan, S. M., L. A. Harris, and T. J. Trust. 1987. Isolation and characterization of Campylobacter flagellins. J. Bacteriol. 169: 5072-5077. 21. Lugtenberg, B., J. Meiers, 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. 58:254-258. 22. Markwell, M. A., S. M. Haas, 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. 23. Morgan, D. R., J. G. Fox, and R. D. Leunk. 1991. Comparison of isolates of Helicobacterpylon and Helicobacter mustelae. J. Clin. Microbiol. 29:395-397.
INFECT. IMMUN. 24. Morrison, D. C. 1983. Bacterial endotoxins and pathogenesis. Rev. Infect. Dis. 5(Suppl. 4):S733-S747. 25. Otto, G., J. G. Fox, P. Y. Wu, and N. S. Taylor. 1990. Eradication of Helicobacter mustelae from the ferret stomach: an animal model of Helicobacter (Campylobacter) pylon chemotherapy. Antimicrob. Agents Chemother. 34:1232-1236. 26. Peterson, W. L. 1991. Helicobacter pylori and peptic ulcer disease. N. Engl. J. Med. 324:1043-1048. 27. Shinoda, S., T. Miwatani, and T. Fujino. 1970. Antigens of Vibrio parahaemolyticus. II. Existence of two different subunits in the flagella of Vibrio parahaemolyticus and their characterization. Biken J. 13:241-247. 28. Siekmann, J., H. R. Wenzel, W. Schroder, and H. Tschesche. 1988. Characterization and sequence determination of six aprotinin homologues from bovine lungs. Biol. Chem. Hoppe-Seyler 369:157-163. 29. Suerbaum, S., and C. Josenhans. Unpublished data. 30. Warren, J. R., and B. Marshall. 1983. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet i:1273-1275. 31. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharides. Extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem. 5:83-91.
