Cloning and Characterization of an Ehrlichia canis Gene Encoding a Protein Localized to the Morula Membrane

Abstract

ABSTRACT A gene encoding a 23.5-kDa ehrlichial morula membrane protein designated MmpA was cloned by screening an Ehrlichia canis expression library with convalescent dog sera, which resulted in three positive clones. Sequence analysis of the insert DNAs from all three clones indicated an open reading frame with a size of 666 bp that encodes MmpA. The structural analysis of MmpA indicated that it is a transmembrane protein with extreme hydrophobicity. Southern blot analysis of the Hin dIII-digested chromosomal DNA demonstrated the presence of a single copy of the mmpA gene in E. canis and Ehrlichia chaffeensis but not in the human granulocytic ehrlichiosis agent. The mmpA gene was amplified, cloned, and expressed as a fusion protein. Polyclonal antibodies to the recombinant protein (rMmpA) were raised in rabbits. Western blot analysis of E. canis and E. chaffeensis lysates with the anti-rMmpA serum resulted in the presence of an MmpA band only in E. canis , not in E. chaffeenesis . Sera from dogs which were either naturally or experimentally infected with E. canis recognized the recombinant protein. Double immunofluorescence confocal microscopy studies demonstrated that MmpA was localized mainly on the morula membrane of E. canis . Since the morula membrane is the interface between the ehrlichial growing environment and the host cytoplasm, MmpA may play a role in bacterium-host cell interactions.