Multimethylation of Rickettsia OmpB catalyzed by lysine methyltransferases

Abstract

Methylation of rickettsial OmpB (outer membrane protein B) 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 (i) recombinantly expressed fragments of Rickettsia typhi OmpB exposed in vitro to trimethyltransferases of Rickettsia prowazekii RP027-028 and of R. typhi RT0101 and to monomethyltransferases of R. prowazekii RP789 and of R. typhi RT0776, and (ii) native OmpBs purified from R. typhi and 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), whereas 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 uncovering the link between OmpB methylation and rickettsial virulence.

Keywords: Bacteria; Cell Surface Protein; Enzyme Catalysis; Mass Spectrometry (MS); Protein Methylation.

FIGURE 1.

The overall schemes of LC-MS/MS analysis (A) and OmpB and recombinant OmpB(AN) and OmpB(K) (B). A, the state, location, and normalized fraction of methylation in native OmpB and in enzymatically methylated rOmpB fragments catalyzed by rickettsial MTs were determined by LC-MS/MS analysis according to the outlined scheme. See “Materials and Methods” for details. B, the full-length R. typhi OmpB precursor consists of a signal peptide (amino acids 1–32) in red, a passenger domain (amino acids 33–1353) in green, and an autotransporter domain (amino acids 1354–1645) in blue. The corresponding residue numbers in R. typhi OmpB for recombinant OmpB(AN) and OmpB(K) are shown.

FIGURE 2.

Time courses and normalized fractions of methylation in OmpB(AN) and OmpB(K) catalyzed by four rickettsial methyltransferases. A and B, time courses of RT0101- (A) and RT0776-catalyzed (B) methylation of 2 μm OmpB(AN) (●) or 1 μm OmpB(K) (■) in the presence of 0.16 mm [methyl-3H]AdoMet (34 mCi/mmol in A and 68 mCi/mmol in B), and 8.3 mm sodium phosphate (pH 8.0) were monitored using the radioactivity assay as described under “Materials and Methods.” The reaction was initiated by the addition of RT0101 or RT0776 to a final concentration of 2 μm. The control that contained MT alone (♦) is also shown. C, the normalized fractions of trimethylation at indicated lysine residues in OmpB(AN) and (K) that are catalyzed by RT0101 (open bars) and RP027-028 (solid bars) at 2 μm each are shown. The enzymatically methylated OmpB(AN) and (K) were prepared, and the methylated OmpB(AN) and (K) were analyzed using LC-MS/MS as described under “Materials and Methods.” The PSM values were combined from three independent trials each for RT0101 and RP027-028 and shown in supplemental Table S1. The correlation coefficient of the normalized fractions of trimethylations catalyzed by RT0101 and RP027-028 is 0.65. D, the normalized fractions of monomethylation at lysine residues in OmpB(AN) and (K) catalyzed by RT0776 (open bars) and RP789 (closed bars) at 2 μm are shown. Normalized fractions were determined as described for Fig. 2C. The PSM values were combined from three independent trials each for RT0776 and RP789 and shown in supplemental Table S2. The correlation coefficient of normalized fractions of monomethylation catalyzed by RT0776 and RP789 is 0.45.

FIGURE 3.

The normalized fractions of RT0776ΔN- and RP789ΔN-catalyzed monomethylation at lysine residues of OmpB(AN) and (K). The normalized fractions of monomethylation at indicated Lys residues in OmpB(AN) and (K) that are catalyzed by RT0776ΔN (A, solid bars) and full-length RP0776 (A, open bars) and RP789ΔN (B, solid bars) and full-length RP789 (B, open bars) are shown. The enzymatically methylated OmpB(AN) and (K) were prepared and analyzed using LC-MS/MS as described under “Materials and Methods.” The PSM numbers were combined from three independent trials each for RP789ΔN and one trial for RT0776ΔN and shown in supplemental Table S3. The correlation coefficient of the normalized fractions of monomethylation catalyzed by RT0776 and RT0776ΔN is 0.68, and that by RP789 and RP789ΔN is 0.75.

FIGURE 4.

Locations of trimethylation in native OmpB purified from R. typhi Wilmington coincide with those in RT0101-catalyzed trimethylation in rOmpB. A, normalized fractions of mono- (red), di- (green), and trimethylations (purple) at indicated lysine residues in native OmpB from R. typhi are shown. Native OmpB protein (2 μg) from R. typhi Wilmington was prepared and analyzed by LC-MS/MS as described under “Materials and Methods.” The PSM values were combined from two independent trials and are shown in supplemental Table S4. B, the locations and normalized fractions of trimethylation in native OmpB purified from R. typhi (solid bars, panel B) are compared with those of the trimethylation in OmpB(AN) and (K) catalyzed by RT0101 (open bars; as in Fig. 2C). The correlation coefficient of the normalized fractions of trimethylation in native OmpB and those catalyzed by RT0101 is 0.76.

FIGURE 5.

Methylation in native OmpBs purified from R. prowazekii strains Breinl, RP22, and Madrid E. The locations and normalized fractions of tri- (A), di- (B), and monomethylation (C) in native OmpBs from R. prowazekii strains Breinl (blue), RP22 (red), and Madrid E (green) are shown. Native OmpBs purified from respective strains (2 μg each) were prepared and analyzed by LC-MS/MS as described under “Materials and Methods.” The PSM numbers of unmethylated and mono-, di-, and trimethyllysine-containing peptides in OmpBs from R. prowazekii strains Breinl, RP22, and Madrid E are shown in supplemental Table S5. The PSM numbers were obtained from two, two, and three independent trials of OmpBs from Breinl, RP22, and Madrid E, respectively.

FIGURE 6.

Trimethylation in native OmpB of R. prowazekii strain Madrid E catalyzed by trimethyltransferases. Native OmpB of R. prowazekii Madrid E (2 μg) was methylated using 2 μm RT0101 or RP027-028. The methylated proteins were prepared and analyzed using LC-MS/MS as described under “Materials and Methods.” The locations and the normalized fractions of trimethylation catalyzed by RT0101 (solid bars) and RP027-028 (open bars) are shown.