Demonstration of a Heat‐Stable 120‐Kilodalton Protein of Rickettsia japonica as a Spotted Fever Group‐Common Antigen

Abstract

Genomic libraries of Rickettsia japonica were cloned into an expression vector λgt11. A clone expressing a protein reactive with antiserum against 120‐kilodalton (kDa) proteins, a mixture of heat‐modifiable and heat‐stable polypeptides, was selected and designated as λRj120‐1. The expressed protein has a molecular mass of 180 kDa. Western immunoblotting demonstrated that the expressed protein was a fusion protein with β‐galactosidase. The antiserum against 120‐kDa proteins was absorbed by the induced lysogen, resulting in the removal of reactivity to the heat‐stable 120‐kDa polypeptide. The antiserum against the expressed protein reacted with heat‐stable 120‐ to 130‐kDa polypeptides of spotted fever group (SFG) rickettsiae in addition to R. japonica. The findings indicated that the protein expressed from the cloned gene of R. japonica possessed the antigenicity group‐common to SFG rickettsiae. Primers designed from the gene coding for R. conorii heat‐stable 120‐kDa protein (Schuenke, K.W., and Walker, D.H., Infect. Immun. 62: 904‐909, 1994) and λgt11 lacZ gene amplified the λRj120‐1 DNA by the polymerase chain reaction (PCR). Analysis of restriction fragment length polymorphism (RFLP) of the PCR‐amplified products revealed that the cloned DNA corresponds to a portion of the gene coding for the heat‐stable 120‐kDa protein of R. conorii with 2,519 nucleotides beginning at nucleotide 190 of the open reading frame. RFLP demonstrated that the cloned gene was highly homologous to the corresponding gene of R. conorii.

Phylogeny of Rickettsia spp. inferred by comparing sequences of 'gene D', which encodes an intracytoplasmic protein.

“…The sequencing data also showed that ' gene D ' had no significant homology with any previously sequenced genes. Later, a major part of the corresponding ' gene D ' of Rickettsia japonica was cloned and recombinant protein was expressed in Escherichia coli (Uchiyama et al, 1996). Antisera against the recombinant protein were found to react with the heat-stable ' PS120 ' proteins of R. japonica (Uchiyama et al, 1996).…”

“…Later, a major part of the corresponding ' gene D ' of Rickettsia japonica was cloned and recombinant protein was expressed in Escherichia coli (Uchiyama et al, 1996). Antisera against the recombinant protein were found to react with the heat-stable ' PS120 ' proteins of R. japonica (Uchiyama et al, 1996). These proteins are distinct from the rOmpB outer-membrane protein (Carl et al, 1990) and, by immunoelectron microscopy, are found outside the electron-lucent nucleoid-like region in the cytoplasm of R. japonica (Schuenke & Walker, 1994 ;Uchiyama, 1997).…”

Adherence to and Invasion of Host Cells by Spotted Fever Group Rickettsia Species

“…In light of subsequent studies revealing the humoral immune-recognition of other high molecular weight Scas, including Sca1, Sca2, and Sca4 (Schuenke and Walker, 1994; Uchiyama et al, 1996; Uchiyama, 1997, and our unpublished data), it cannot be ruled out that the protective antibody responses attributed to rOmpA and rOmpB might also be associated with other less characterized rickettsial antigens. Notably, the humoral responses generated from a prior rickettsial infection have been seen to be long-lived and can be detected years following an infection (Bengtson and Topping, 1942; Fox et al, 1957).…”

Phenotypic and Genotypic Homogeneity of the Strains of Rickettsia japonica Isolated from Patients with Oriental Spotted Fever

“…The genes coding for PS 120 (rps120) of R. japonica and R. conorii were cloned, sequenced, and expressed in Escherichia coil (10,17,21). PCR amplification is successfully performed by using a primer pair designed in the rps120 gene of R. conorii and templates of various species and strains of SFG rickettsiae, suggesting the high homology of the gene among strains and species (10).…”

“…protein which locates in the cytoplasm of the organisms possessing common antigenicity among SFG rickettsiae (13,17,21). In the case of R. japonica, PS 120 is deduced to be made of 1,019 amino acids (aa) (unpublished data) and comigrates with one of the major outer membrane proteins, rOmp B, at the 120-kDa position on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) when the organisms are solubilized without heat denaturation before electrophoresis (19).…”