Multiple Phospholipid N-Methyltransferases with Distinct Substrate Specificities Are Encoded in Bradyrhizobium japonicum

ABSTRACT Phosphatidylcholine (PC) is the major phospholipid in eukaryotic membranes. In contrast, it is found in only a few prokaryotes including members of the family Rhizobiaceae. In these bacteria, PC is required for pathogenic and symbiotic plant-microbe interactions, as shown for Agrobacterium tumefaciens and Bradyrhizobium japonicum. At least two different phospholipid N-methyltransferases (PmtA and PmtX) have been postulated to convert phosphatidylethanolamine (PE) to PC in B. japonicum by three consecutive methylation reactions. However, apart from the known PmtA enzyme, we identified and characterized three additional pmt genes (pmtX1, pmtX3, and pmtX4), which can be functionally expressed in Escherichia coli, showing different substrate specificities. B. japonicum expressed only two of these pmt genes (pmtA and pmtX1) under all conditions tested. PmtA predominantly converts PE to monomethyl PE, whereas PmtX1 carries out both subsequent methylation steps. B. japonicum is the first bacterium known to use two functionally different Pmts. It also expresses a PC synthase, which produces PC via condensation of CDP-diacylglycerol and choline. Our study shows that PC biosynthesis in bacteria can be much more complex than previously anticipated.

Autoreactive CD4+ T-cell clones to β2-glycoprotein I in patients with antiphospholipid syndrome: preferential recognition of the major phospholipid-binding site

Autoreactive CD4+ T cells to β2-glycoprotein I (β2GPI) that promote antiphospholipid antibody production were recently identified in patients with antiphospholipid syndrome (APS). To further examine antigen recognition profiles and T-cell helper activity in β2GPI-reactive T cells, 14 CD4+ T-cell clones specific to β2GPI were generated from 3 patients with APS by repeated stimulation of peripheral blood T cells with recombinant β2GPI. At least 4 distinct T-cell epitopes were identified, but the majority of the β2GPI-specific T-cell clones responded to a peptide encompassing amino acid residues 276 to 290 of β2GPI (KVSFFCKNKEKKCSY; single-letter amino acid codes) that contains the major phospholipid-binding site in the context of the DRB4*0103 allele. Ten of 12 β2GPI-specific T-cell clones were able to stimulate autologous peripheral blood B cells to promote anti-β2GPI antibody production in the presence of recombinant β2GPI. T-cell helper activity was exclusively found in T-cell clones capable of producing interleukin 6 (IL-6). In vitro anti-β2GPI antibody production induced by T-cell clones was inhibited by anti-IL-6 or anti-CD40 ligand monoclonal antibody. In addition, exogenous IL-6 augmented anti-β2GPI antibody production in cultures of the T-cell clone lacking IL-6 expression. These results indicate that β2GPI-specific CD4+ T cells in patients with APS preferentially recognize the antigenic peptide containing the major phospholipid-binding site and have the capacity to stimulate B cells to produce anti-β2GPI antibodies through IL-6 expression and CD40-CD40 ligand engagement. These findings are potentially useful for clarifying the pathogenesis of APS and for developing therapeutic strategies that suppress pathogenic antiphospholipid antibody production in these patients.