Two Protein Lysine Methyltransferases Methylate Outer Membrane Protein B from Rickettsia

Abstract

c Rickettsia prowazekii, the etiologic agent of epidemic typhus, is a potential biological threat agent. Its outer membrane protein B (OmpB) is an immunodominant antigen and plays roles as protective envelope and as adhesins. The observation of the correlation between methylation of lysine residues in rickettsial OmpB and bacterial virulence has suggested the importance of an enzymatic system for the methylation of OmpB. However, no rickettsial lysine methyltransferase has been characterized. Bioinformatic analysis of genomic DNA sequences of Rickettsia identified putative lysine methyltransferases. The genes of the potential methyltransferases were synthesized, cloned, and expressed in Escherichia coli, and expressed proteins were purified by nickelnitrilotriacetic acid (Ni-NTA) affinity chromatography. The methyltransferase activities of the purified proteins were analyzed by methyl incorporation of radioactively labeled S-adenosylmethionine into recombinant fragments of OmpB. Two putative recombinant methyltransferases (rRP789 and rRP027-028) methylated recombinant OmpB fragments. The specific activity of rRP789 is 10-to 30-fold higher than that of rRP027-028. Western blot analysis using specific antibodies against trimethyl lysine showed that both rRP789 and rRP027-028 catalyzed trimethylation of recombinant OmpB fragments. Liquid chromatographytandem mass spectrometry (LC/MS-MS) analysis showed that rRP789 catalyzed mono-, di-, and trimethylation of lysine, while rRP027-028 catalyzed exclusively trimethylation. To our knowledge, rRP789 and rRP027-028 are the first biochemically characterized lysine methyltransferases of outer membrane proteins from Gram-negative bacteria. The production and characterization of rickettsial lysine methyltransferases provide new tools to investigate the mechanism of methylation of OmpB, effects of methylation on the structure and function of OmpB, and development of methylated OmpB-based diagnostic assays and vaccine candidates. Rickettsial OmpBs belong to the outer membrane protein autotransporter family and contain large passenger domains. Rickettsial OmpBs have been shown to participate in adhesion to mammalian cells in vitro (8,36). Ectopically expressed rickettsial OmpB in Escherichia coli has been demonstrated to be sufficient in mediating bacterial attachment and invasion of cultured mammalian cells (8,24). Unlike the ␣-helical transmembrane proteins in plasma membranes, the autotransporter domains of these outer membrane proteins have structural characteristics of ␤-barrel integral membrane proteins (3, 33). As the immunodominant antigenic surface protein, native OmpB induces strong humoral and cellular immune responses in animal models and in patients (6,12,15,16,23). Methylation of OmpA of Rickettsia canadensis and ompB of Rickettsia typhi is known, but whether this is important for any other pathogenic rickettsia is unknown.One important but not well understood structural feature of rickettsial OmpB is the methylation at ε-amino groups of lysine residues (28). Postt...

Multimethylation of Rickettsia OmpB Catalyzed by Lysine Methyltransferases

“…This table shows that the total numbers of PSM of trimethyllysine-containing peptides in native OmpB from Madrid E, RP22, and Breinl are 0, 271, and 139, respectively (supplemental Table S5). The absence of trimethyllysine in R. prowazekii Madrid E is consistent with the fact that the gene encoding RP027-028 is interrupted by a frameshift mutation, which generates the inactive RP027 and RP028 fragments (17,27). Fig.…”

“…The MTs were prepared by the same method as previously described (27). No methyllysines in recombinant MTs were found as determined by LC-MS/MS.…”

“…The constructs were confirmed by sequencing. Expression and purification was performed as described previously (27).…”

“…We have recently characterized two rickettsial lysine methyltransferases (MTs) (27). They are RP789 (PKMT1) and RP027-028 (PKMT2) from Rickettsia prowazekii.…”

The METTL20 Homologue from Agrobacterium tumefaciens Is a Dual Specificity Protein-lysine Methyltransferase That Targets Ribosomal Protein L7/L12 and the β Subunit of Electron Transfer Flavoprotein (ETFβ)

“…Of these, PrmA, which targets ribosomal protein RpL11, has been studied in the most detail and appears to be present in all bacteria (36). Pseudomonas and certain other species of ␥-proteobacteria and firmicutes possess a 7BS MTase, EftM (EF-Tu-modifying enzyme), which methylates Lys-5 in translation elongation factor EF-Tu, thus promoting bacterial infectivity (42) Similarly, two related 7BS MTases, PKMT1 and PKMT2, that methylate the outer membrane protein OmpB on several Lys residues in Rickettsia subspecies, are also important for bacterial virulence (43,44). Thus, bacterial KMTs seem to primarily target components of the translational machinery as well as determinants of bacterial virulence.…”