Analysis of polypeptide composition and antigenic components of Rickettsia tsutsugamushi by polyacrylamide gel electrophoresis and immunoblotting

Abstract

Polyacrylamide gel electrophoresis of lysates of purified Rickettsia tsutsugamushi revealed as many as 30 polypeptide bands, including major bands corresponding to molecular sizes of 70, 60, 54 to 56, and 46 to 47 kilodaltons. Compared with the polypeptide composition of the rickettsiae of Gifliam, Karp, and Kato strains and a newly isolated Shimokoshi strain, the major polypeptide in the Kato strain (54-56K) and in the Karp strain (46-47K) migrated a little faster and slower, respectively, than the corresponding polypeptides in the other strains. The largest major polypeptide (54-56K) was digestible by the treatment of intact rickettsiae with trypsin and variable in content in separate preparations, suggesting that the polypeptide exists on the rickettsial surface and is easily degraded during the handling of these microorganisms. Several surface polypeptides of rickettsiae, including the 54-56K and 46-47K polypeptides, were detected by radioiodination of intact rickettsiae foliowed by polyacrylamide gel electrophoresis of the lysate; however, the 70K and 60K polypeptides were not labeled. Immunoblotting experiments with hyperimmune sera prepared in guinea pigs against each strain demonstrated that the 70K, 54-56K, and 46-47K polypeptides showed antigenic activities. The 54-56K polypeptide appeared to be strain specific, whereas the 70K and 46-47K polypeptides cross-reacted with the heterologous antisera.on July 15, 2020 by guest http://iai.asm.org/ Downloaded from

Phylogenetic analysis of Orientia tsutsugamushi strains based on the sequence homologies of 56-kDa type-specific antigen genes

“…However, other strain types were found in Thailand [4], and it was also reported that Shimokoshi [5], Kawasaki [6], and Kuroki [7] strains, which were isolated from patients in Japan, were antigenically distinguished from the prototype strains of Gilliam, Karp and Kato. This antigenic variation depends largely on the diversities of the immunodominant 56-kDa type-speci¢c antigen located on the surface of this microorganism [8], and typing of newly isolated strains can be carried out using immuno£uorescent (IF) testing using strain-or type-speci¢c hyperimmune sera or monoclonal antibodies which recognize 56-kDa antigen, or by restriction fragment length polymorphism (RFLP) of 56-kDa protein genes ampli¢ed by polymerase chain reaction (PCR). Many newly isolated strains from patients and natural hosts in Japan and in Taiwan were tested using IF tests with monoclonal antibodies and RFLP analyses, and the isolates were classi¢ed not only into types but also into further subtypes [1,2].…”

Production and Characterization of Monoclonal Strain‐Specific Antibodies against Prototype Strains of Rickettsia tsutsugamushi

“…Until now, since in our own (16) and other studies (4, 11) on R. tsutsugamushi polyclonal antisera raised in animals were used, it was difficult to make solve the problems regarding antigenic identity at the strain or type level. Monoclonal strain-specific antibodies to prototype strains of R. tsutsugamushi produced by the hybridoma technique (20) appear to be useful for both serotyping (4,11,16) and antigenic analysis (3,6,7,15) of the rickettsiae. The present study deals with the production of murine monoclonal strain-specific antibodies against Gilliam, Karp, and Kato strains of R. tsutsugamushi and the characterization of the antibodies prepared.…”

“…SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis (SDS-PAGE) was performed by a modification of the Laemmli method (10) as described previously (15). The stacking gel contained 4.5% acrylamide, 0.12% bisacrylamide, and 0.1 % SDS in 0.125 M tris hydrochloride buffer (pH 6.8), and the separation gel contained 10% polyacrylamide, 0.26% bisacrylamide, and 0.1 % SDS in 0.375 M tris hydrochloride buffer (pH 8.8).…”

“…Immunoblotting. Immunoblotting was performed by modifications of the technique of Towbin et al (18) and Burnette (2) as described previously (15). Briefly, the rickettsial antigens separated by SDS-PAGE were transferred to a nitrocellulose sheet by electrophoresis.…”

Sequence Analysis of the Hypervariable Regions of the 56 kDa Immunodominant Protein Genes of Orientia tsutsugamushi Strains in Malaysia

“…The 56 kDa protein is the immunodominant antigen detected in the sera of scrub typhus patients (15,21). DNA sequence analysis of 56 kDa immunodominant protein genes from six O. tsutsugamushi strains revealed that the sequences may be divided into four conserved and four variable domains (8,14).…”