Vol. 174, No. 4
JOURNAL OF BACTERIOLOGY, Feb. 1992, p. 1258-1267
0021-9193/92/041258-10$02.00/0 Copyright C 1992, American Society for Microbiology
Reattachment of Surface Array Proteins to Campylobacter fetus Cells LIYING YANG,' ZHIHENG PEI,' SHUJI FUJIMOTO,2 AND MARTIN J. BLASER1,3* Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 372321; Department of Bacteriology, Faculty of Medicine, Kyushu University, Fukuoka, Japan2; and Department of Veterans Affairs Medical Center, Nashville, Tennessee 372123 Received 25 July 1991/Accepted 8 December 1991
Campylobacterfetus strains may be of serotype A or B, a property associated with lipopolysaccharide (LPS) structure. Wild-type C. fetus strains contain surface array proteins (S-layer proteins) that may be extracted in water and that are critical for virulence. To explore the relationship of S-layer proteins to other surface components, we reattached S-layer proteins onto S- template cells generated by spontaneous mutation or by serial extractions of S' cells with water. Reattachment occurred in the presence of divalent (Ba2 Ca2 , Co2 , and Mg2+) but not monovalent (H+, NH4+, Na+, K+) or trivalent (Fe3+) cations. The 98-, 125-, 127-, and 149-kDa S-layer proteins isolated from strains containing type A LPS (type A S-layer protein) all reattached to S- template cells containing type A LPS (type A cells) but not to type B cells. The 98-kDa type B S-layer protein reattached to SAP- type B cells but not to type A cells. Recombinant 98-kDa type A S-layer protein and its truncated amino-terminal 65- and 50-kDa segments expressed in Escherichia coli retained the full and specific determinants for attachment. S-layer protein and purified homologous but not heterologous LPS in the presence of calcium produced insoluble complexes. By quantitative enzyme-linked immunosorbent assay, the S-layer protein copy number per C. fetus cell was determined to be approximately 105. In conclusion, C. fetus cells are encapsulated by a large number of S-layer protein molecules which may be specifically attached through the N-terminal half of the molecule to LPS in the presence of divalent cations. ,
98 to 149 kDa with one form predominating (18, 31); multiple forms of S-layer proteins also are observed in other bacterial species which have more than one layer on the cell surface, and each layer is composed of a single type of polypeptide (24, 25). It is not clear at present whether C. fetus has a single S-layer composed of multiple types of S-layer proteins or multiple layers each composed of a single type of polypeptide. C. fetus strains may shift the size of the predominant S-layer protein produced, which results in antigenic shift, although the mechanism is presently unknown (31, 44). Nine different S-layer protein molecules have been purified and characterized (14, 15, 31), and two different aminoterminal amino acid sequences have been observed, but the biological significance of this variation has not been defined (14, 15, 29). C. fetus may lose its surface layer (becoming an S- mutant without significant change in other bacterial components) during sequential passage on artificial medium (7, 26), a phenomenon also observed in Bacillus thuringiensis (24). When one such C. fetus S- mutant was incubated with free S-layer protein in the presence of calcium, it was agglutinated by antiserum to S-layer protein, and electron microscopy showed an additional structural layer similar in appearance to the surface layer on its S+ parental strain (46); these phenomena suggest that reattachment of the C. fetus S-layer protein can occur. We now have further characterized the S-layer protein of C. fetus by exploring its relationship with other bacterial cell surface components. Emphasis was placed on (i) characterizing factors required for reattachment of S-layer protein to S- cell surfaces, (ii) determining whether reattachment is LPS-type specific, (iii) determining whether reattachment is dependent on the molecular form of the S-layer protein, and (iv) preliminary localization in S-layer protein of the functional domain for reattachment.
Crystalline surface layers have been observed as the outermost component of the bacterial cell envelopes in numerous gram-positive and gram-negative species (38) and are composed of protein subunits assembled over the cell surface into two-dimensional arrays with hexagonal (p6), (p4), or oblique (p2) symmetry (36). For the majority of bacteria, the surface layer is formed from a single species of protein, known as a surface array protein (S-layer protein), which is acidic and generally hydrophobic (36, 38). In gram-negative bacteria, the constituent subunits of most S-layers interact with each other and with the underlying outer membrane components (3, 35, 37, 40), such as outer membrane proteins in an Acinetobacter sp. (41), lipid-lipopolysaccharide (LPS) in Spirillum serpens (11), and LPS in Aeromonas salmonicida (2, 22), and many S-layers require divalent cations for assembly (10, 37, 40). Reattachment of isolated S-layer proteins to bacterial cells that have lost the S-layer proteins (S- cell templates) has been a useful tool to study the relationship of S-layers with other bacterial cell components (37, 40). Campylobacter fetus is an opportunistic bacterial pathogen causing infectious abortion and infertility in ungulates (27) and systemic diseases in immunocompromised humans (5, 9, 17, 20). There are two serotypes of C. fetus (A and B), a property based on LPS characteristics (33). C. fetus S-layer protein has been shown to be a critical virulence factor in resistance to phagocytosis (8, 26) and serum bactericidal activity (6-8) and in the pathogenesis of bacteremia in experimentally infected mice (29). A characteristic of the C. fetus surface layer is that a single bacterial strain may produce up to three different S-layer proteins ranging from square
*
Corresponding author. 1258
REATTACHMENT OF SURFACE ARRAY PROTEINS TO C. FETUS
VOL. 174, 1992
TABLE 1. C. fetus and E. coli strains used in this study Bacterial
strain Isolation site strain
84-32(23D) 84-54(23B) 82-40LP 82-40HP 82-40LP3 85-389 84-112 84-91 84-107 83-88
VC119e
VC119-A{2 VC119-A3e
VC119-B2e TK(LP) TK(HP) pBG1 pBG201 pBH500
Bovine vagina Mutant of 23D Human blood Mutant of 82-40LP Mutant of 82-40LP Reptile
Bovine genital Human blood Human blood Human blood Unknown Mutant of VC119 Mutant of VC119 Mutnat of VC119 Human CSF Mutant of TK(LP) Recombinant E. coli Recombinant E. coli Recombinant E. coli
S-layer
protein'
LPS tye typeb
Serum
susceptibility kill)c
~~~~~~~~(kDa)(logl0 98
98
127 125 149 98 98 135 85 110
130-133
A A A A A A A
B B B
Bf Bf Bf Bf
98
A A
98 65 50
NAM NA NA
JOURNAL OF BACTERIOLOGY, Feb. 1992, p. 1258-1267
0021-9193/92/041258-10$02.00/0 Copyright C 1992, American Society for Microbiology
Reattachment of Surface Array Proteins to Campylobacter fetus Cells LIYING YANG,' ZHIHENG PEI,' SHUJI FUJIMOTO,2 AND MARTIN J. BLASER1,3* Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 372321; Department of Bacteriology, Faculty of Medicine, Kyushu University, Fukuoka, Japan2; and Department of Veterans Affairs Medical Center, Nashville, Tennessee 372123 Received 25 July 1991/Accepted 8 December 1991
Campylobacterfetus strains may be of serotype A or B, a property associated with lipopolysaccharide (LPS) structure. Wild-type C. fetus strains contain surface array proteins (S-layer proteins) that may be extracted in water and that are critical for virulence. To explore the relationship of S-layer proteins to other surface components, we reattached S-layer proteins onto S- template cells generated by spontaneous mutation or by serial extractions of S' cells with water. Reattachment occurred in the presence of divalent (Ba2 Ca2 , Co2 , and Mg2+) but not monovalent (H+, NH4+, Na+, K+) or trivalent (Fe3+) cations. The 98-, 125-, 127-, and 149-kDa S-layer proteins isolated from strains containing type A LPS (type A S-layer protein) all reattached to S- template cells containing type A LPS (type A cells) but not to type B cells. The 98-kDa type B S-layer protein reattached to SAP- type B cells but not to type A cells. Recombinant 98-kDa type A S-layer protein and its truncated amino-terminal 65- and 50-kDa segments expressed in Escherichia coli retained the full and specific determinants for attachment. S-layer protein and purified homologous but not heterologous LPS in the presence of calcium produced insoluble complexes. By quantitative enzyme-linked immunosorbent assay, the S-layer protein copy number per C. fetus cell was determined to be approximately 105. In conclusion, C. fetus cells are encapsulated by a large number of S-layer protein molecules which may be specifically attached through the N-terminal half of the molecule to LPS in the presence of divalent cations. ,
98 to 149 kDa with one form predominating (18, 31); multiple forms of S-layer proteins also are observed in other bacterial species which have more than one layer on the cell surface, and each layer is composed of a single type of polypeptide (24, 25). It is not clear at present whether C. fetus has a single S-layer composed of multiple types of S-layer proteins or multiple layers each composed of a single type of polypeptide. C. fetus strains may shift the size of the predominant S-layer protein produced, which results in antigenic shift, although the mechanism is presently unknown (31, 44). Nine different S-layer protein molecules have been purified and characterized (14, 15, 31), and two different aminoterminal amino acid sequences have been observed, but the biological significance of this variation has not been defined (14, 15, 29). C. fetus may lose its surface layer (becoming an S- mutant without significant change in other bacterial components) during sequential passage on artificial medium (7, 26), a phenomenon also observed in Bacillus thuringiensis (24). When one such C. fetus S- mutant was incubated with free S-layer protein in the presence of calcium, it was agglutinated by antiserum to S-layer protein, and electron microscopy showed an additional structural layer similar in appearance to the surface layer on its S+ parental strain (46); these phenomena suggest that reattachment of the C. fetus S-layer protein can occur. We now have further characterized the S-layer protein of C. fetus by exploring its relationship with other bacterial cell surface components. Emphasis was placed on (i) characterizing factors required for reattachment of S-layer protein to S- cell surfaces, (ii) determining whether reattachment is LPS-type specific, (iii) determining whether reattachment is dependent on the molecular form of the S-layer protein, and (iv) preliminary localization in S-layer protein of the functional domain for reattachment.
Crystalline surface layers have been observed as the outermost component of the bacterial cell envelopes in numerous gram-positive and gram-negative species (38) and are composed of protein subunits assembled over the cell surface into two-dimensional arrays with hexagonal (p6), (p4), or oblique (p2) symmetry (36). For the majority of bacteria, the surface layer is formed from a single species of protein, known as a surface array protein (S-layer protein), which is acidic and generally hydrophobic (36, 38). In gram-negative bacteria, the constituent subunits of most S-layers interact with each other and with the underlying outer membrane components (3, 35, 37, 40), such as outer membrane proteins in an Acinetobacter sp. (41), lipid-lipopolysaccharide (LPS) in Spirillum serpens (11), and LPS in Aeromonas salmonicida (2, 22), and many S-layers require divalent cations for assembly (10, 37, 40). Reattachment of isolated S-layer proteins to bacterial cells that have lost the S-layer proteins (S- cell templates) has been a useful tool to study the relationship of S-layers with other bacterial cell components (37, 40). Campylobacter fetus is an opportunistic bacterial pathogen causing infectious abortion and infertility in ungulates (27) and systemic diseases in immunocompromised humans (5, 9, 17, 20). There are two serotypes of C. fetus (A and B), a property based on LPS characteristics (33). C. fetus S-layer protein has been shown to be a critical virulence factor in resistance to phagocytosis (8, 26) and serum bactericidal activity (6-8) and in the pathogenesis of bacteremia in experimentally infected mice (29). A characteristic of the C. fetus surface layer is that a single bacterial strain may produce up to three different S-layer proteins ranging from square
*
Corresponding author. 1258
REATTACHMENT OF SURFACE ARRAY PROTEINS TO C. FETUS
VOL. 174, 1992
TABLE 1. C. fetus and E. coli strains used in this study Bacterial
strain Isolation site strain
84-32(23D) 84-54(23B) 82-40LP 82-40HP 82-40LP3 85-389 84-112 84-91 84-107 83-88
VC119e
VC119-A{2 VC119-A3e
VC119-B2e TK(LP) TK(HP) pBG1 pBG201 pBH500
Bovine vagina Mutant of 23D Human blood Mutant of 82-40LP Mutant of 82-40LP Reptile
Bovine genital Human blood Human blood Human blood Unknown Mutant of VC119 Mutant of VC119 Mutnat of VC119 Human CSF Mutant of TK(LP) Recombinant E. coli Recombinant E. coli Recombinant E. coli
S-layer
protein'
LPS tye typeb
Serum
susceptibility kill)c
~~~~~~~~(kDa)(logl0 98
98
127 125 149 98 98 135 85 110
130-133
A A A A A A A
B B B
Bf Bf Bf Bf
98
A A
98 65 50
NAM NA NA
