A comparative view of Rickettsia tsutsugamushi and the other groups of rickettsiae.

Recent researches on the rickettsial group microorganisms are summarized in their comparative aspects of morphology, cultivation and multiplication, susceptibility to chemotherapeutics, chemical structure of envelopes, nucleic acid, protein constitution, and gene structures. From this overview, Rickettsia tsutsugamushi seems to have different properties from the others and should be reclassified into a new genus, and a new species name as Orientia tsutsugamushi is proposed.

Purification and partial characterization of a type-specific antigen of Rickettsia tsutsugamushi.

A type-specific antigen (54- to 56-kilodalton polypeptide) in the envelope of Rickettsia tsutsugamushi was purified from each of three prototype strains (Gilliam, Karp, and Kato) by a combination of mild anionic detergent treatment, gel filtration, and reverse-phase high-performance liquid chromatography. The purified antigens from the three strains were shown to have similar amino acid compositions: primarily aspartic acid, glutamic acid, and glycine, with lesser amounts of cysteine, methionine, and tyrosine. The N-terminal amino acid sequences of the antigens were 74.3% homologous among the three strains.

Deficiency of peptidoglycan and lipopolysaccharide components in Rickettsia tsutsugamushi.

Analyses of chemical composition in whole cells of Rickettsia tsutsugamushi were performed and compared with those of the other rickettsiae and gram-negative bacteria. The results indicated that R. tsutsugamushi does not contain detectable amounts of 3-deoxy-D-mannooctulosonic acid, heptose, muramic acid, or glucosamine (less than 2, less than 2, less than 3, and less than 3 nmol/mg, respectively). The microorganism was found to contain four kinds of fatty acids (16:0, 18:0, 18:1, and 18:2), but not hydroxy fatty acids. Furthermore, in analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver or Coomassie blue staining, lipopolysaccharide bands were not detected in preparations treated with proteinase K. It is concluded that R. tsutsugamushi has little or no peptidoglycan or lipopolysaccharide.

Identification and partial characterization of Rickettsia tsutsugamushi major protein immunogens.

Strains of Rickettsia tsutsugamushi so far examined have either three or four quantitatively predominant proteins, which apparently are surface proteins and which range in size between 50 and 63 kilodaltons. These polypeptides also were the major immunogens detected by polyacrylamide gel electrophoresis of extracted rickettsial proteins which had been precipitated by hyperimmune rabbit sera. The major proteins from different rickettsial strains share some epitopes, as evidenced by the lack of strain specificity of the rabbit sera in the immunoprecipitation tests. However, similar experiments with a limited number of monoclonal antibodies showed that strain-specific determinants also are associated with at least the 58/60-kilodalton polypeptide. A lack of strain-specific epitopes on the rickettsial surface was indicated by our inability to detect binding of heterologous antisera to the rickettsial surface by immunoferritin labeling. Because the three major proteins of the Karp and Gilliam strains are accessible to antibody in unextracted organisms, it is possible that the exteriorly exposed epitopes of these three polypeptides are strain specific and that their common determinants are normally buried in the membrane or otherwise inaccessible. Attempts to absorb out specific antibody with intact rickettsiae gave equivocal results; however, when immune complexes formed before rickettsial extraction were examined by electrophoresis, antibody appeared to have bound strain specifically with at least the 60-kilodalton protein.

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

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 Gilliam, 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 followed 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.