AND MARTIN J. BLASER1 3,4*. Division ofInfectious Diseases ...... Blaser, M. J., P. F. Smith, J. A. Hopkins, J. Bryner, I. Heinzer, and W. L. L. Wang. 1987.
Vol. 175, No. 16
JOURNAL OF BACTERIOLOGY, Aug. 1993, p. 4979-4984
0021-9193/93/164979-06$02.00/0 Copyright X 1993, American Society for Microbiology
Shift in S-Layer Protein Expression Responsible for Antigenic Variation in Campylobacterfetus ENZE WANG,' MANUEL M. GARCIA,2 MILAN S. BLAKE,3 ZHIHENG PEI,' AND MARTIN J. BLASER1 3,4* Division of Infectious Diseases, Department of Medicine, Vanderbilt University School ofMedicine, Nashville, Tennessee, 372321*; Animal Diseases Research Institute, Nepean, Ontario K2H 8P9, Canada2; Laboratory of Bacterial Pathogenesis and Immunology, Rockefeller University, New York, New York 10021-63993; and Medical Service, Department of Veterans Affairs, Nashville, Tennessee 372124 Received 18 February 1993/Accepted 3 June 1993
Campylobacter fetus strains possess regular paracrystalline surface layers (S-layers) composed of highmolecular-weight proteins and can change the size and crystalline structure of the predominant protein expressed. Polyclonal antisera demonstrate antigenic cross-reactivity among these proteins but suggest differences in epitopes. Monoclonal antibodies to the 97-kDa S-layer protein of Campylobacterfetus subsp.fetus strain 82-40LP showed three different reactivities. Monoclonal antibody iD1 recognized 97-kDa S-layer proteins from all C. fetus strains studied; reactivity of monoclonal antibody 6E4 was similar except for epitopes in S-layer proteins from reptile strains and strains with type B lipopolysaccharide. Monoclonal antibody 2Ell only recognized epitopes on S-layer proteins from strains with type A lipopolysaccharide regardless of size. In vitro shift from a 97-kDa S-layer protein to a 127-kDa S-layer protein resulted in different reactivity, indicating that size change was accompanied by antigenic variation. To examine in vivo variation, heifers were genitally challenged with Campylobacterfetus subsp. venerealis strains and the S-layer proteins from sequential isolates were characterized. Analysis with monoclonal antibodies showed that antigenic reactivities of the S-layer proteins were varied, indicating that these proteins represent a system for antigenic variation.
Campylobacter fetus is an important pathogen of both humans and animals (13, 22, 23). Campylobacter fetus subsp. venerealis lives in the penile prepuce of bulls and is venereally transmitted to susceptible cows, in whom it causes endocervicitis and infertility (23). After several weeks or months, infected cows may eliminate the organism from the upper genital tract, but carriage in the vagina may persist for months to years (20). The life cycle of Campylobacter fetus subsp. fetus in ungulates centers on the gastrointestinal tract (7, 23). After oral ingestion, bacteremia ensues rapidly, and in pregnant animals, infectious abortion may occur. The bacteremia seeds the biliary tract, and chronic carriage develops in a high proportion of hosts (7). Thus, both C. fetus subspecies cause chronic colonization of host epithelial surfaces, despite host defenses against such microbial parasitism. C. fetus, when experimentally introduced into the bovine vagina, may undergo antigenic variation, but the basis for this phenomenon has not been explored (8, 21). C fetus cells produce lipopolysaccharide (LPS) molecules with long polysaccharide side chains (18) of either serotype A or B (19). In addition, essentially all wild-type C. fetus strains contain surface layers (S-layers) which are regular paracrystalline structures external to the outer membrane (4, 10, 15). These S-layers (or surface arrays) are composed of high-molecular-mass (95- to 98-, 127-, and 149-kDa) S-layer proteins (10, 16) that are bound to the LPS side chains (11, 26). The S-layer proteins from type A strains have the same N termini and similar amino acid compositions and isoelectric points. These represent a family of closely related molecules (16) and bind only to type A and not to type B
strains (26). The S-layer proteins isolated from type B strains differ in their N termini, isoelectric points, and LPS-binding specificity (to B but not A cells) from the proteins from type A strains and represent a second family of related molecules (10, 26). Polyclonal antiserum raised to the 97-kDa type A protein recognized other type A proteins (16), but with polyclonal antisera, Dubreuil et al. reported that the S-layer proteins undergo antigenic variation (9). Fujimoto et al. showed that C. fetus strains could vary the predominant S-layer protein expressed, and with this shift, recognition by polyclonal antisera changed (12). Thus, variation in C. fetus S-layer proteins may be responsible for the antigenic variation observed in vivo. Since all of the S-layer proteins are recognized by polyclonal antisera, we produced monoclonal antibodies to define their antigenic relationships. The sapA and sapAl genes encoding type A S-layer proteins have been cloned and express products in Escherichia coli (2, 24). We also sought to localize antigenic determinants recognized by the monoclonal antibodies by using truncated proteins produced by recombinant E. coli.
MATERIALS AND METHODS Bacterial strains. The C. fetus strains used in this study were from the culture collection of the Vanderbilt University Campylobacter laboratory (Table 1). For each strain, determination of the LPS type (19) and serum susceptibility (5), and characterization of the S-layer protein (16) were done as previously described. All strains had been frozen at -700C in brucella broth (BBL Microbiology Systems, Cockeysville, Md.) containing 15% glycerol until used in these studies. Production of monoclonal antibodies. BALB/c mice were immunized intraperitoneally with water-extracted S-layer proteins emulsified in incomplete Freund's adjuvant.
* Corresponding author. 4979
4980
J. BACTE RIOL.
WANG ET AL. TABLE 1. Characteristics of the C. fetus and E. coli strains studied Serum
LPS
kill'
L type typ
~
A A A A A A A B B B A
Isolation site
Strain
Major
M-lajor Saer prtei
82-40HP 23B 82-40LP 23D 84-86 81-173 85-388 84-91 84-104 84-107 82-40LP3
Mutant of 82-40LP Mutant of 23D Human blood Bovine vagina Human blood Human cerebrospinal fluid Reptile feces Human blood Monkey blood Human blood Mutant of 82-40LP
80-109 84-112 85-389 pBG1
Human blood Bovine vagina Reptile feces E. coli with cloned sapA producing 97kDa S-layer protein E. coli with truncated cloned sapA gene E. coli with truncated cloned sapA gene E. coli with cloned S-layer protein encoded by sapAl
.0.05 0.06 .0.05 2.03
A A A A
97 97 97 97 96 97 97 97 97 127 127 149 149 97
3.32 3.34 3.49
A A A
80 50 97
a
b
of S-layer Recognition by monoclonal antibody:
i~~~~D1
2E11
-
-
-
-
+ + + + + + + + + + + + + +
+ + + + + + + + +
+ + + +
+ + + + + +Wb +W +W + -
+ + +
-
+ + +
(k~~~~a)
1.02 3.38
