Identification, cloning, and expression of the CAMP-like factor autotransporter gene (cfa) of Bartonella henselae.
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.
Section of Clinical Immunology, Microbiology and Virology, Department of Pathology, 50 N. Medical Dr., University of Utah, Salt Lake City, Utah 84132, USA. Christine.Litwin@path.utah.edu
Christine M
CM
Litwin
Joel M
JM
Johnson
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D018416:Bartonella henselae / Q000235:genetics* / Q000472:pathogenicity; D002415:Cats; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data
D001426:Bacterial Proteins; D006460:Hemolysin Proteins