title: Pore-forming activity of Coxiella burnetii outer membrane protein oligomer comprised of 29.5- and 31-kDa polypeptides. Inhibition of porin activity by monoclonal antibodies 4E8 and 4D6.
abstract: Envelopes of large-cell variant Coxiella burnetii, the agent of Q fever, were the starting material for purification of an outer membrane protein (OMP) oligomer with aggregate molecular mass of approximately 2 x 10(4) kDa. The oligomer was resistant to trypsin and dissociation by SDS at 100 degrees C. Reducing agents dissociated the oligomer into monomers of 29.5 and 31 kDa, which migrated as a doublet during SDS-polyacrylamide gel electrophoresis. Both monomers were reactive in an immunoblot assay with monoclonal antibodies (mAbs) 4E8 and 4D6, which were previously selected for their reactivity with purified and SDS-denatured 29.5 kDa protein. Proteoliposomes were functional in an equilibrium assay at pH 7 and a swelling assay at pH 7 and 4.5. The pores in proteoliposomes allowed the passage of arabinose, glucose, and sucrose, but restricted stachyose. Polyclonal antibodies to C. burnetii cells and the mAbs were able to bind C. burnetii at pH 7 and 4.5. The uptake of 14C-glucose at pH 4.5 was inhibited by polyclonal antibodies and mAbs after binding to cells at pH 7. The mAbs did not inhibit 14C-glucose uptake at pH 4.5 after binding to cells at pH 4.5. Although the mAbs bind C. burnetii porin epitopes before and after acid activation, the mAbs bound under acidic conditions were unable to inhibit porin function. The inhibition of porin channel function by mAbs confirms the role of porin as a permeability barrier for the subsequent active transport of glucose by C. burnetii. In another study, we showed that the 29.5 kDa OMP antigen induced active immunity against virulent challenge. This information, combined with the recent confirmation that porins are important antigens in the induction of specific protective immune responses against infection by gram-negative bacteria, suggests that humoral immunity directed against C. burnetii porins might play an important role in immunity against Q fever (human infection) and coxiellosis (animal infection), global enzootic diseases.
authors.affiliation: Bacteriology Division, United States Army Medical Research Institute of Infectious Diseases Fort Detrick Frederick, Maryland 21702, USA.
authors.forename: N
authors.initials: N
authors.lastname: Banerjee-Bhatnagar
authors.forename: C R
authors.initials: CR
authors.lastname: Bolt
authors.forename: J C
authors.initials: JC
authors.lastname: Williams
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001089:Arabinose / Q000378:metabolism; D001692:Biological Transport; D015153:Blotting, Western; D002462:Cell Membrane / Q000502:physiology; D002642:Chick Embryo; D002645:Chickens; D002852:Chromatography, Ion Exchange; D016997:Coxiella burnetii / Q000254:growth & development / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D018272:Porins / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D011510:Proteolipids / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D009844:Oligosaccharides; D018272:Porins; D011510:Proteolipids; C020485:proteoliposomes; C005695:stachyose; D013395:Sucrose; D001089:Arabinose; D005947:Glucose