Electron microscopic studies of the surface structure of Campylobacter fetus by the freeze-etching method showed two different types of S layer. One was in a ...
INFECTION AND IMMUNITY, Aug. 1989. p. 2563-2565 0019-9567/89/082563-03$02.00/0 Copyright © 1989. American Society for Microbiology
Vol. 57, No. 8
Hexagonal Surface Layer of Campylobacter fetus Isolated from Humans SHUJI FUJIMOTO,* AKIKO UMEDA, AKEMI TAKADE, KUNIHIKO MURATA,
Department
of Bacteriology,
AND
KAZUNOBU AMAKO
Faculty of Medicine, Kyushui University, anid Laboratory for Infectious Disease, Facdlty Medicine, Fukuoka Universitm, Fiukiuoka 812, Japan
of
Received 19 January 1989/Accepted 22 April 1989
Electron microscopic studies of the surface structure of Campylobacter fetus by the freeze-etching method showed two different types of S layer. One was in a hexagonal array, and the other was in a tetragonal array. A high-passage-number strain lost its S layer during cultivation on culture media but regained it after a single animal passage.
Bacterial cell surface structures play several important roles in pathogenesis. In Campylobacter fetius infection, McCoy et al. (5, 6) described the presence of the antiphagocytic antigens on a strain. Later, Winter et al. (9) showed that this antigen is a surface layer consisting of a single glycoprotein with a molecular size of 98 kilodaltons (kDa). Morphologically, however, complete data on this structure have not yet been obtained. Only on a portion of the cell fragments has a hexagonal pattern been recognized by negative staining (6). The very fragile nature of this layer makes it difficult to observe by electron microscopy. Recently, Dubreuil et al. suggested that the S layer of C. fcutits must be tetragonal because the freeze-etched profiles of their strain of C. feuis isolated from animals showed the array to be linear (3). The molecular size of a surface protein of this strain was reported to be 131 kDa. In our studies of the surface layer of strains of C. feuts isolated from humans, we found that a strain with a surface protein of 98 kDa had a different surface pattern. We used C. fetlus subsp. fertus TK. This is a clinically isolated strain from a patient with C. fttus infection. After isolation, the strain was kept in brucella broth (Difco Laboratories, Detroit, Mich.) in a freezer after a single passage on brucella agar plates. This strain was designated TK(LP) (LP indicates a low passage number). For serial passages on culture media, strain TK(LP) was cultured on brucella agar plates every 2 days at least 15 times at 37°C in a GasPak anaerobic system (BBL Microbiology Systems, Cockeysville, Md.) without a catalyst. This high-passage-number strain was designated TK(HP). For animal passages, 5- to 6-day-old suckling mice of strain ddY were inoculated intragastrically with 0.1 ml of a 2 x 109CFU/ml bacterial suspension of strain TK(HP) through a fine polyethylene tube attached to a small injection syringe with a 23-gauge needle. Three days after the inoculation, the mouse was killed and the bacteria were cultured from the liver by homogenizing it with a Potter-type glass homogenizer. Brucella agar plates containing antibiotics (polymyxin B, 2,500 IU/liter; vancomycin, 10 mg/liter; trimethoprim. 5 mg/liter) were used for isolation of C. fetius. This animalpassaged strain was designated strain TK(AP). Whole-cell proteins and polypeptides were analyzed b! sodium dodecyl sulfate-polyacrylamide gel electrophoresis by the Laemmli method (4) with some modifications as *
previously described (7). The analysis showed that among the many protein or polypeptide bands, two high-molecularweight bands at 98,000 and 94,000 that were always found in strain TK(LP) were missing from strain TK(HP) (Fig. 1. lane 2: arrows) and reappeared in the cells after a single passage in a mouse [strain TK(AP); Fig. 1, lane 5]. No other qualitative differences in protein profiles were observed among these three strains. The surface structures of these strains were examined by the freeze-etching technique. Freeze-etching was done with a Balzer device (type BAF301; Balzers Union, Lichtenstein). The bacterial specimen was frozen in Freon 25 in liquid nitrogen. After fracturing at -110°C at a pressure of .r;,/}.
FIG. 2. Electron micrograph showing hexagonally arranged subunits on the surface of the cell wall of C. fetus TK(AP), prepared by freeze-etching. Bar, 0.5 ,.m.
cleaned with sodium hypochloride and washed with distilled water. Cleaned replicas were picked up on copper grids and examined with an electron microscope (JEM 10OC; JEOL Co., Tokyo, Japan) at 80 kV. The external surface of C. fetus TK(AP) revealed by the freeze-etching technique was covered with a regular hexag-
:----- --:
onal array (Fig. 2). The distance from center to center of the subunits was approximately 24 nm. On the other hand, no regular structure was found on the surfaces of the cultured cells [strain TK(HP); Fig. 3]. The existence of the S layer on
------- :-A .-.. F U. *i--aj:X. -E
FIG. 4. Electron micrograph of a thin sectioned profile of C.
FIG. 3. Electron micrograph showing smooth surface of the cell
wall of C. fetuis TK(HP), prepared by freeze-etching. Bar, 0.5 ,um.
fetus TK(AP) examined by the rapid-freezing and substitutionfixation method. Bar, 0.2 p.m.
NOTES
VOL. 57, 1989
the cell surface of strain TK(AP) was confirmed on a thin, sectioned profile prepared by the freeze-substitution technique as described by Umeda et al. (8). On the surface of strain TK(AP), a thin additional layer 10 nm thick was found at the outer side of the outer membrane (Fig. 4, arrow). This surface structure was unique and differed from that of C. fetius isolated from an animal, as reported by Dubreuil et al. (3). In this report, we present two new observations on S layer of C. fetius. First, the strain which lost its S layer through in vitro passages regained its S layer after a single in vivo passage. Recently, Blaser et al. (1, 2) showed that the 100-kDa protein has a close relationship with serum resistance and that the presence of this protein inhibits the binding of C3b to the cell surface. Antiphagocytic activity of the S layer was also shown by McCoy et al. (5, 6). Our results suggest that the S layer is indispensable for the growth of the bacteria in animals. Secondly, C. fetis has at least two types of S layer: one is hexagonal and the other is tetragonal. Epidemiological studies with specific antisera against the S layer proteins could clarify whether the hexagonal pattern is prevalent in human isolates. Whether the genes encoding the surface proteins are multiple is also an interesting issue that calls for further research. This research was supported in part by grant 63770268 from the Ministry of Education and Science of Japan and a grant from the Inoue Science Foundation. We thank T. Morooka and S. Miyake for their donation of bacterial strains. M. L. Robbins and D. B. Griffiths are thankfully acknowledged for their critical reading of the manuscript.
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LITERATURE CITED 1. Blaser, M. J., P. F. Smith, J. A. Hopkins, I. Heinzer, J. H. Bryner, and W. L. Wang. 1987. Pathogenesis of Campylobacter fetius infections: serum resistance associated with high-molecular-weight surface proteins. J. Infect. Dis. 155:696-706. 2. Blaser, M. J., P. F. Smith, J. E. Repine, and K. A. Joiner. 1988. Pathogenesis of Campylobacter fetus infections: failure of encapsulated Campylohacter fetus to bind C3b explains serum and phagocytosis resistance. J. Clin. Invest. 81:1434-1444. 3. Dubreuil, J. D., S. M. Logan, S. Cubbage, D. N. Eidhin, W. D. Mccubbin, C. M. Kay, T. J. Beveridge, F. G. Ferris, and T. J. Trust. 1988. Structural and biochemical analyses of a surface array protein of Campylobacter fetis. J. Bacteriol. 170:41654173. 4. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 5. McCoy, E. C., K. D. Doyle, K. Burda, L. B. Corbeil, and A. J. Winter. 1975. Superficial antigens of Camnpylobacter (Vibrio) fetuts: characterization of an antiphagocytic component. Infect. Immun. 11:517-525. 6. McCoy, E. C., H. A. Wiltberger, and A. J. Winter. 1976. Major outer membrane protein of Canmpylobacter fetus: physical and immunological characterization. Infect. Immun. 13:1258-1265. 7. Ueki, Y., S. Fujimoto, A. Umeda, and K. Amako. 1988. Purification and antigenic analysis of flagella of Caimpylobacter jejuni. Microbiol. Immunol. 32:327-337. 8. Umeda, A., Y. Ueki, and K. Amako. 1987. Structure of the Staphylococcus aireu.s cell wall determined by the freeze-substitution method. J.Bacteriol. 169:2482-2487. 9. Winter, A. J., E. C. McCoy, C. S. Fullmer, K. Burde, and P. J. Bier. 1987. Microcapsule of Caumpylobacter fetus: chemical and physical characterization. Infect. Immun. 22:963-971.
