THE LATE CHLAMYDIAL INCLUSION MEMBRANE IS NOT DERIVED FROM THE ENDOCYTIC PATHWAY AND IS RELATIVELY DEFICIENT IN HOST PROTEINS

Chlamydiae are obligate intracellular parasites which multiply within infected cells in a membrane-bound structure termed an inclusion. Newl y internalized bacteria are surrounded by host plasma membrane; howeve r, the source of membrane for the expansion of the inclusion is unknow n. To determine if the membrane for the mature inclusion was derived b y fusion with cellular organelles, we stained infected cells with fluo rescent or electron-dense markers specific for organelles and examined inclusions for those markers. We observed no evidence for the presenc e of endoplasmic reticulum, Golgi, late endosomal or lysosomal protein s in the inclusion. These data suggest that the expansion of the inclu sion membrane, beginning 24 h postinoculation, does not occur by the a ddition of host proteins resulting from either de novo host synthesis or by fusion with preexisting membranes. To determine the source of th e expanding inclusion membrane, antibodies were produced against isola ted membranes from Chlamydia-infected mouse cells. The antibodies were demonstrated to be solely against Chlamydia-specified proteins by bot h immunoprecipitation of [S-35] methionine-labeled extracts and Wester n blotting (immunoblotting). Techniques were used to semipermeabilize Chlamydia-infected cells without disrupting the permeability of the in clusion, allowing antibodies access to the outer surface of the inclus ion membrane. Immunofluorescent staining demonstrated a ring-like fluo rescence around inclusions in semipermeabilized cells, whereas Triton X-100-permeabilized cells showed staining throughout the inclusion. Th ese studies demonstrate that the inclusion membrane is made up, in par t, of Chlamydia-specified proteins and not of existing host membrane p roteins.