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.
Litwin
Christine M
CM
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
Johnson
Joel M
JM
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