title: Proteolytic Cleavage of the Immunodominant Outer Membrane Protein rOmpA in Rickettsia rickettsii.
abstract: Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, contains two immunodominant proteins, rOmpA and rOmpB, in the outer membrane. Both rOmpA and rOmpB are conserved throughout spotted fever group rickettsiae as members of a family of autotransporter proteins. Previously, it was demonstrated that rOmpB is proteolytically processed, with the cleavage site residing near the autotransporter domain at the carboxy-terminal end of the protein, cleaving the 168-kDa precursor into apparent 120-kDa and 32-kDa fragments. The 120- and 32-kDa fragments remain noncovalently associated on the surface of the bacterium, with implications that the 32-kDa fragment functions as the membrane anchor domain. Here we present evidence for a similar posttranslational processing of rOmpA. rOmpA is expressed as a predicted 224-kDa precursor yet is observed on SDS-PAGE as a 190-kDa protein. A small rOmpA fragment of ∼32 kDa was discovered during surface proteome analysis and identified as the carboxy-terminal end of the protein. A rabbit polyclonal antibody was generated to the autotransporter region of rOmpA and confirmed a 32-kDa fragment corresponding to the calculated mass of a proteolytically cleaved rOmpA autotransporter region. N-terminal amino acid sequencing revealed a cleavage site on the carboxy-terminal side of Ser-1958 in rOmpA. An avirulent strain ofR. rickettsiiIowa deficient in rOmpB processing was also defective in the processing of rOmpA. The similarities of the cleavage sites and the failure ofR. rickettsiiIowa to process either rOmpA or rOmpB suggest that a single enzyme may be responsible for both processing events.IMPORTANCEMembers of the spotted fever group of rickettsiae, includingR. rickettsii, the etiologic agent of Rocky Mountain spotted fever, express at least four autotransporter proteins that are protective antigens or putative virulence determinants. One member of this class of proteins, rOmpB, is proteolytically processed to a passenger domain and an autotransporter domain that remain associated on the rickettsial outer membrane. The protease responsible for this posttranslation processing remains unknown. Here we show that another autotransporter, rOmpA, is similarly processed byR. rickettsiiSimilarities in sequence at the cleavage site and predicted secondary protein structure suggest that all fourR. rickettsiiautotransporters may be processed by the same outer membrane protease.
authors.affiliation: Host-Parasite Interactions Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Nicholas F
authors.initials: NF
authors.lastname: Noriea
authors.affiliation: Host-Parasite Interactions Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Tina R
authors.initials: TR
authors.lastname: Clark
authors.affiliation: Host-Parasite Interactions Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: David
authors.initials: D
authors.lastname: Mead
authors.affiliation: Host-Parasite Interactions Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA Ted_Hackstadt@nih.gov.
authors.forename: Ted
authors.initials: T
authors.lastname: Hackstadt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D016680:Genome, Bacterial; D006168:Guinea Pigs; D012284:Rickettsia rickettsii / Q000235:genetics / Q000378:metabolism*; D012373:Rocky Mountain Spotted Fever / Q000382:microbiology
chemical_list: D001425:Bacterial Outer Membrane Proteins; C080205:rOmpA, Rickettsia
keywords: Rickettsia; autotransporter proteins