Identification, cloning, and expression of the CAMP-like factor autotransporter gene (cfa) of Bartonella henselae

Abstract

The CAMP reaction was first described by Christie et al. (R. Christie, N. E. Atkins, and E. Munch-Petersen, Aust. J. Exp. Biol. 22:197-200, 1944) as the synergistic lysis of sheep red blood cells by Staphylococcus aureus sphingomyelinase and CAMP factor (cohemolysin), a secreted protein from group B streptococci. We observed a CAMP-like reaction when Bartonella henselae was grown in close proximity to S. aureus on 5% sheep blood agar. This study describes the cloning, sequencing, and characterization of a CAMP-like factor autotransporter gene (cfa) from B. henselae. A cosmid library of B. henselae ATCC 49793 was constructed using SuperCos1 in Escherichia coli XL1-Blue MR. Cosmids were screened for the CAMP reaction, and a quantitative cohemolysis microtiter assay was developed using purified sphingomyelinase. Cosmid clones with the strongest cohemolytic reaction had similar restriction enzyme patterns. A DNA fragment that expressed the cohemolysin determinant was subcloned in a 7,200-bp StuI-BamHI fragment which contained a 6,024-bp open reading frame. The deduced amino acid sequence showed homology to the family of autotransporters. The autotransporters are a group of proteins that mediate their own export through the outer membrane. They contain an N-terminal passenger region, the alpha-domain, and a C-terminal transporter region, the beta-domain. The alpha-domain contained four, nearly identical 42-amino-acid repeats and showed homology to the family of RTX (repeat in toxin) hemolysins. The concentrated supernatant of the recombinant strain expressed a protein with a molecular mass of 180 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis consistent with the calculated molecular weight of the secreted alpha-domain. In conclusion, we have characterized a novel secreted cohemolysin autotransporter protein of B. henselae.

FIG. 1.

CAMP test with B. henselae and E. coli XL1-Blue MR recombinant strains. Strains were grown perpendicular to the S. aureus streak (b). The diffusion zone of the sphingomyelinase produced by S. aureus is indicated with two arrows (a). The B. henselae strain shows the CAMP effect with increased hemolysis next to the S. aureus streak (lane 1). The group B streptococcus control strain shows a large distinct zone with complete hemolysis next to the indicator strain (lane 2). The Listeria monocytogenes control strain shows a mild CAMP reaction (lane 3). The E. coli XL1-Blue MR strain containing the SuperCos1 cosmid vector shows no CAMP reaction (lane 4). The E. coli XL1-Blue strain containing the SuperCos1 cosmid pCML75 with the 29.7-kb insert of B. henselae chromosomal DNA shows a mild CAMP effect (lane 5). The E. coli XL1-Blue strain containing plasmid pCML76 with the 7.2-kb subclone with the entire cfa gene and promoter shows a distinct zone of complete hemolysis next to the S. aureus streak (lane 6).

FIG. 2.

Restriction map of the region near the cfa gene and positions of clones. The arrows represent the cfa gene with the arrowhead showing the direction of transcription. The diagonal hatch marks at the arrowhead represent the area of homology of the translated protein with the β-barrel region of proteins in the family of autotransporters. The grey area and vertical bars in the stem of the arrow represent homology of the translated protein with RTX (repeat in toxin) hemolysins. The vertical bars represent the four tandem nearly identical repeats in the amino acid sequence. Plasmid pCML77 contains the StuI-BamHI insert from pCML76 (solid dark line) with an internal 5.6-kb EcoRV-EcoRV deletion (open line) of the coding sequence of cfa.

FIG. 3.

(A) Homology between the amino-terminal region of Cfa and RTX hemolysins of Nitrosomonas europea and Chromobacterium violaceum. (B) Homology between the carboxy-terminal region of Cfa and the autotransporters YchA of E. coli and Vag8 of B. pertussis. The numbers in parentheses indicate the position in the unprocessed protein of the first amino acid listed. Conserved amino acids between two proteins are indicated by colons, and substitutions of functionally similar amino acids are marked by periods.

FIG. 4.

(A) Microtiter assay of the hemolysin activity of various clones in E. coli XL1-Blue MR. Twofold serial dilutions of supernatants (except for row B) were added sequentially to columns 1 to 8 (1:2 to 1:256) in microtiter plates to a final volume of 100 μl with 1% (vol/vol) washed sheep erythrocytes in KRT. Row A contains supernatant of L. monocytogenes. Row B contains sheep erythrocytes with no added bacterial supernatant; row C contains supernatant of E. coli containing SuperCos1; row D contains supernatant of pCML77; row E contains supernatant of pCML75; row F contains supernatant of pCML76. (B) Microtiter assay of the cohemolysin activity of various clones in E. coli XL1-Blue MR. Same experiment as that for panel A, except that sheep erythrocytes were pretreated with 0.025 U/ml of sphingomyelinase for 30 min at 37°C prior to the addition of the bacterial supernatants. A and B are representative of at least three independent experiments.

FIG. 5.

(A) SDS-PAGE of secreted, outer membrane, and cytoplasmic proteins stained with Coomassie blue. Lane 1, 10× concentrated supernatant of E. coli containing SuperCos1; lane 2, 10× concentrated supernatant of E. coli containing pCML76 (cfa clone); lane 3, outer membrane protein, E. coli containing SuperCos1; lane 4, outer membrane protein, E. coli containing pCML76; lane 5, cytoplasmic protein, E. coli containing SuperCos1; lane 6, cytoplasmic protein, E. coli containing pCML76. The numbers on the left indicate the positions of protein standards (in kilodaltons). The black arrow indicates the position of the 180-kDa protein. (B) Western blot transfer of panel A probed with a sera pool from 68 patients with cat scratch disease (IgG IFA titer ranging from 1:512 to 1:4,096 for B. henselae). The white arrow indicates the position of the 180-kDa immunogenic protein. (C) Western blot transfer of lanes 1 and 2 of panel A probed with patient serum negative for B. henselae antibodies (IgG IFA titer, <1:64).