Evidence for proteolytic cleavage of the 120-kilodalton outer membrane protein of rickettsiae: identification of an avirulent mutant deficient in processing

Abstract

The 120-kDa rickettsial outer membrane protein (rOmpB) is encoded by a gene with the capacity to encode a protein of approximately 168 kDa. The carboxy-terminal end of the molecule is apparently cleaved to yield 120and 32-kDa products. Both polypeptides are surface exposed and remain associated with the outer membrane of intact rickettsiae. All species of rickettsiae examined display similar cleavage of rOmpB. Comparison of diverse species of rickettsiae demonstrate a conserved N terminus of the 32-kDa fragment, with a predicted procaryotic secretory signal peptide immediately upstream of the proposed cleavage site. Coprecipitation of the 120-kDa rOmpB protein and the 32-kDa peptide by monoclonal antibodies specific for the 120-kDa portion of the molecule suggests that the two fragments remain noncovalently associated on the surface of rickettsiae. Analysis of an avirulent mutant of Rickettsia rickettsii revealed reduced amounts of the 120and 32-kDa fragments, but with a correspondingly larger rOmpB protein that displayed properties expected of the putative precursor. This avirulent mutant grows intracellularly but fails to cause the lysis of infected cells that is typical of R. rickettsii. DNA sequence analysis of the region of the gene encoding the cleavage site of the avirulent strain revealed no difference from the sequence obtained from virulent R. rickeffsii. The 168-kDa putative precursor of the avirulent strain of R. rickeffsii was not extracted from the surfaceby dilute buffers, as is the 120-kDa protein of virulent R. rickefusii or R. prowazekii. These latter results suggest that the 32-kDa C-terminal region of the molecule may serve as a membrane anchor domain.

Progress in Rickettsial Genome Analysis from Pioneering of Rickettsia prowazekii to the Recent Rickettsia typhi

“…1,2,19 The precursor of OmpB is a 168-kDa protein that is post-translationally processed to the 135-kDa mature OmpB by cleavage of a 32-kDa β-peptide. 20 The amino acid sequences of the β-peptides of all five rickettsial autotransporters are highly homologous and share homology with autotransporters of distantly related bacteria. 17 The diversity among the passenger domains that are coupled with the conserved βdomain suggests that the autotransporters are either derived originally from a single gene or resulted from recombination of unrelated proteins with the β-domains.…”

Replication of Coxiella burnetii Is Inhibited in CHO K‐1 Cells Treated with Inhibitors of Cholesterol Metabolism

“…burnetii (Nine Mile strain in phase II) was propagated and harvested as previously described. 7 Chinese hamster ovary (CHO K-1) epithelial cells (CRL-9618, American Type Culture Collection) were maintained in DMEM/F-12 supplemented with 5% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) and incubated at 37°C in 5% CO 2 . Semi-confluent CHO K-1 monolayers were infected with C. burnetii at a multiplicity of infection of 10 for 2 h at room temperature.…”

Species-specific BALB/c mouse antibodies to rickettsiae studied by Western blotting

“…Immunoblotting assays with sera of different laboratory animals (mice, guinea pigs, rabbits) show that each SFG rickettsia contains high * Corresponding author. molecular mass species-specific protein antigens (SPA) in the outer membrane and low molecular mass group specific lipopolysaccharide-like (LPS) antigens [3,4].…”