Serological reactivity and biochemical characterization of methylated and unmethylated forms of a recombinant protein fragment derived from outer membrane protein B of Rickettsia typhi.
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.
Chao
Chien-Chung
CC
Viral and Rickettsial Diseases Department, Infectious Diseases Directorate, Naval Medical Research Center, Silver Spring, MD 20910, USA.
Zhang
Zhiwen
Z
Wang
Hui
H
Alkhalil
Abdulnaser
A
Ching
Wei-Mei
WM
D000907:Antibodies, Bacterial / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000097:blood / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008745:Methylation; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012286:Rickettsia typhi / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D008239:Lysine