2′-O-methylation at tRNA position G18 is responsible for non-immunostimulatory character of E. coli Nissle 1917. (A) Human PBMCs were stimulated with 2, 0.4 and 0.1 µg/ml of purified tRNA from E. coli BW25113– and E. coli BW25113–derived knockout mutants for trmA (uracil-5-methyltransferase, m5U54), trmB (guanine-7-methyltransferase, m7G46), and trmH (Gm18-2′-O-methyltransferase). PBMCs were incubated with RNA for 20 h with subsequent IFN-α detection by ELISA. Representative experimental data from one out of four PBMC preparations are shown (n = 2 ± SD). (B) HPLC analysis of P1 nuclease and phosphatase-treated tRNA from E. coli BW25113, E. coli ΔtrmH, and E. coli ΔtrmH methylated in vitro with T. thermophilus trmH. (C) SDS-PAGE of purified recombinant T. thermophilus trmH protein expressed in E. coli BL21. (D) Purified tRNA from E. coli ΔtrmH was incubated with ¹⁴C-labeled SAM and with or without T. thermophilus trmH. tRNA was further visualized after PAGE by ethydium bromide (EtBr) staining and autoradiography (AR). For B–D, one representative experiment (n ≥ 2) is shown. (E) Human PBMCs were stimulated with purified tRNA (2 µg/ml) from A. lwoffii, T. thermophilus, E. coli ΔtrmH, and E. coli ΔtrmH methylated in vitro with recombinant trmH and T. thermophilus. PBMCs were incubated with RNA for 20 h with subsequent IFN-α detection by ELISA. (F) Human enriched monocytes were stimulated with 100 ng/ml LPS, 1 µM CpG2216, 1 µg/ml RNA40, nuclease P1, and 2 µg/ml tRNA from T. thermophilus and A. lwoffii ± nuclease P1 digestion. After incubation for 20 h, IL-6 production was measured by ELISA. Representative experimental data from one out of four PBMCs (E) or four monocyte (F) preparations are shown (n = 2 ± SD).