Serological Reactivity and Biochemical Characterization of Methylated and Unmethylated Forms of a Recombinant Protein Fragment Derived from Outer Membrane Protein B of Rickettsia typhi

Abstract

Rickettsia typhi, an obligate intracellular bacterium that causes murine typhus, possesses a heavily methylated outer membrane protein B (OmpB) antigen. This immunodominant antigen is responsible for serological reactions and is capable of eliciting protective immune responses with a guinea pig model. Western blot analysis of partially digested OmpB with patient sera revealed that most of the reactive fragments are larger than 20 kDa. One of these fragments, which is located at the N terminus (amino acids 33 to 273), fragment A (At), has been expressed in Escherichia coli. The expressed protein (rAt) was purified by chromatography and properly refolded by sequential dialysis. The refolded rAt protein was recognized by at least 87% of the typhus group patient sera as determined by enzyme-linked immunosorbent assay (ELISA). However, the titers were lower than those obtained with OmpB of R. typhi. Since native OmpB is hypermethylated at lysine residues, we chemically methylated the lysine residues in rAt. The methylation was confirmed by amino acid composition analysis, and the methylation pattern of the methylated rAt (mrAt) protein was similar to that of native At from OmpB, as revealed by liquid chromatography-mass spectrometry analysis. Both rAt and mrAt were evaluated in an ELISA for their serological reactivity with patient sera. Among patient sera tested, 83% exhibited higher titers with mrAt than with rAt. These results suggest that rAt, with or without methylation, can potentially replace rickettsia-derived OmpB or whole-cell antigen for the diagnosis of R. typhi infection.Rickettsiae are classified into two groups: the spotted fever group and the typhus group. The typhus group of rickettsiae includes Rickettsia typhi and R. prowazekii. R. typhi, a gramnegative, obligate intracellular bacterium, is the causative agent of murine typhus (endemic typhus). While R. typhi can be transmitted to the mammalian host by the bite of an infected flea or louse (the rat flea Xenopsylla cheopis, the cat flea Ctenocephalides felis, or the rat louse Polyplax spinulosa), the more important mechanism of transmission is by inoculation of feces from the vector. Organisms in the feces enter the host through irritated, abraded skin. The bacterium then hematogenously spreads and ultimately invades endothelial cells (1). Transmission can also occur via inhalation of aerosolized fecal particles. To invade the host cell, R. typhi induces phagocytosis by an unknown mechanism. Once within the cell, the organisms rapidly escape the phagosome, multiply within the cytoplasm, and then exit the host cell by burst lysis, allowing subsequent spreading to other cells (18).Infection with R. typhi causes fever, headache, and myalgia and if not treated in time will lead to disseminated, multisystem disease, including infection of the brain, lung, liver, kidney, and heart endothelia, lymphohistiocytic vasculitis of the central nervous system, diffuse alveolar damage and hemorrhage, interstitial pneumonia, pulmonary edema, interstitial myocarditis...

Two Protein Lysine Methyltransferases Methylate Outer Membrane Protein B from Rickettsia

“…Recombinant OmpB fragments, including rOmpB(A) (33 to 273), rOmpB(AN) (33 to 744), and OmpB(K) (745 to 1353) based on the genomic sequence of R. typhi were prepared as previously described (10). Briefly, the bacterially expressed proteins in the inclusion body were dissolved in 8 M urea and 1% deoxycholate, purified by ion-exchange chromatography on DEAE-cellulose in 6 M urea, and refolded.…”

“…Proteins were obtained by slow dialysis at stepwise, decreasing concentrations of urea. SET7 methyltransferase and the histone peptide substrate H3 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) were from New England Biolabs. N,N=-diallyltartardiamide (DATD) was from Bio-Rad.…”

“…In practical clinical applications, enzymatic methylation of OmpB in vitro may provide methylated OmpB for the detection of patient antibodies against virulent rickettsiae and for developing improved vaccine candidates. As a first step toward these goals, we searched for methyltransferases from R. prowazekii that can enzymatically methylate OmpB (10). Genes encoding hypothetical proteins and putative methyltransferases in the genome of R. prowazekii were examined by bioinformatics.…”

Epidemiology of Spotted Fever Group and Typhus Group Rickettsial Infection in the Amazon Basin of Peru

“…Human, canine, and feline sera were tested by ELISA for IgG antibodies against SFGR and TGR. Microtiter plates were coated at 4°C for 2 days with appropriate antigen diluted in 1X phosphate buffered saline (PBS) (for SFGR 0.15 μg fragment X and 0.3 μg fragment Y from the OmpA gene product of R. rickettsii ; for TGR 0.15 μg fragment AN and 0.15 μg fragment K from the Omp B gene product of R. typhi 28 ). Following coating, plates were blocked in 10% milk and 0.1% Tween 20 in 1 X PBS for 1 hour at room temperature.…”

Analysis of the Cross-Reactivity of Various 56 kDa Recombinant Protein Antigens with Serum Samples Collected after Orientia tsutsugamushi Infection by ELISA

“…The experiment was performed as described. 40 Briefly, coated plates were first rinsed three times with 1× phosphate-buffered saline (PBS) containing 0.1% Tween 20 (1× PBST), blocked with 200 µL/well of 10% milk in 1× PBST, and incubated for one hour at room temperature. Primary antibody (mouse serum samples for 12 strains and human serum samples for 2 strains) was serial diluted by a factor of 4 (1:100, 1:400, 1:1,600) in 5% milk in 1× PBST.…”