Multi-methylation in Rickettsia OmpB Catalyzed by Lysine Methyltransferases [Molecular Bases of Disease]

February 4th, 2014 by Abeykoon, A., Wang, G., Chao, C.-C., Chock, P. B., Gucek, M., Ching, W.-M., Yang, D. C. H.

Methylation of rickettsial outer membrane protein B (OmpB) has been implicated in bacterial virulence. Rickettsial methyltransferases, RP789 and RP027-028, are the first biochemically characterized methyltransferases to catalyze methylation of outer membrane protein (OMP). Methylation in OMP remains poorly understood. Using semiquantitative integrated liquid chromatography-tandem mass spectroscopy, we characterize methylation of (1) recombinantly expressed fragments of R. typhi OmpB exposed in vitro to trimethyltransferases of R. prowazekii RP027-028 and of R. typhi RT0101, and to monomethyltransferases of R. prowazekii RP789 and of R. typhi RT0776, and (2) native OmpBs purified from R. typhi, R. prowazekii strains Breinl, RP22 and Madrid E. We found that in vitro trimethylation occurs at relatively specific locations in OmpB with consensus motifs, KX(G/A/V/I)N and KT(I/L/F), while monomethylation is pervasive throughout OmpB. Native OmpB from virulent R. typhi contains mono- and trimethyllysines at locations well correlated with methylation in recombinant OmpB catalyzed by methyltransferases in vitro. Native OmpBs from highly virulent R. prowazekii strains Breinl and RP22 contain multiple clusters of trimethyllysine in contrast to a single cluster in OmpB from mildly virulent R. typhi. Furthermore, OmpB from the avirulent strain Madrid E contains mostly monomethyllysine and no trimethyllysine. The native OmpB from Madrid E was minimally trimethylated by RT0101 or RP027-028, consistent with a processive mechanism of trimethylation. This study provides the first in-depth characterization of methylation of an OMP at the molecular level and may lead to uncover the link between OmpB methylation and rickettsial virulence.