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.

FIG. 1.

(A) SDS-PAGE and Western blotting (WB) of trypsin-digested OmpB fragments. Trypsin digest of OmpB was separated on SDS-PAGE and transferred onto a polyvinylidene fluoride membrane for Western blot analysis using a standard procedure. Fragment A was the most prominent band on the WB and is indicated by the arrow. (B) Diagram of fragments of full-length OmpB reactive with patient sera. The full-length OmpB and different fragments which reacted with patient sera were labeled A, B, and C. The proteases used to partially digest OmpB and the molecular sizes (MW) of the resultant fragments are also shown.

FIG. 2.

The purity of fragment A eluted from the DEAE column. The fragment was purified using the DEAE column as described in Materials and Methods. The fractions were collected and analyzed by SDS-PAGE to determine the purity of each fraction. The fractions containing fragment A are more than 95% pure, as indicated by a single band.

FIG. 3.

CD spectra of rAt (A) and mrAt (B). Approximately equal amounts of rAt (0.24 mg/ml) and mrAt (0.25 mg/ml) were used for the measurement of secondary structures using a Jasco 700 CD spectrophotometer. The molar ellipticity (degrees cm²/dmol) was plotted on the x axis and wavelength (nm) on the y axis. The spectra were then deconvoluted into five individual components of secondary structure as described in Materials and Methods.

FIG. 4.

Western blot analysis of rAt and mrAt using patient sera. rAt and mrAt were loaded on an SDS-PAGE gel, and Western blotting was performed using normal serum, patient serum no. 1, and patient serum no. 2. Pure mrAt was loaded in lanes labeled A, and rAt was loaded in lanes labeled B. Two lanes, labeled MW, were loaded with molecular size markers.

FIG. 5.

Identification of peptides from rAt, mrAt, and OmpB and the methylated lysine residues in mrAt and OmpB by LC-MS. The sequence of fragment A (amino acids 33 to 273) of R. typhi OmpB is shown with all nine lysine residues in bold. The upper panel shows the identified sequence of rAt (underlined, 97.1% coverage). The middle panel shows the identified sequence of mrAt (underlined, 95% coverage). Among the eight lysine residues detected, K118 of mrAt (black lettering on yellow background) was not methylated. Both K149 and K157 of mrAt (magenta) were either tri-, di-, or monomethylated. K205 and K226 of mrAt (red) were either mono- or trimethylated. K131, K222, and K232 of mrAt (blue) were trimethylated. The lower panel represents fragment A of OmpB, with identified peptides underlined (51.5% coverage). Among the six identified lysine residues, K273 (black lettering on yellow background) was not methylated. K118 (magenta) was either tri-, di-, or monomethylated. Both K149 and K232 (blue) were trimethylated. K205 (green) was monomethylated, and K131 (magenta) was either mono- or dimethylated.