The intracellular pathogen Francisella tularensis escapes from adaptive immunity by metabolic adaptation

(A) Schematic of the riboflavin synthetic pathway. The precursor of the MAIT cell antigen presented by MR1 is highlighted in red. (B) Plots of normalized read counts of genes ribAB, ribD, ribH, and ribC in the free-living and pathogenic strains. (C) Comparison of the amino acid sequence of RibD in the pathogenic and free-living strain. Boxed amino acids (red) are the zinc-binding region of the cytidine and deoxycytidylate deaminase domain. Underlined amino acid sequences are catalytic domains. (D) Summary table of amino acids at five defined positions in RibD in free-living (black) and pathogenic (red) strains as found in the KEGG database. (E, F) GFP-reporter activities of cells expressing human (E) and mouse (F) MAIT TCRs after stimulation with escalating amounts (1, 3, 9 μl) of total metabolites from the indicated strains that were cultured in the presence of human and mouse MR1-expressing cells, with or without anti-MR1 antibody (Ab) (10 μg/ml). Data are representative of three independent experiments.

(A) Growth curve of the indicated strains after culture with shaking shows no difference in change in optical density over time. (B) Plots show normalized read counts of genes in the riboflavin synthetic pathway in free-living parental FN or FN∆ribD strains. (C, D, E, F) GFP-reporter activities of cells expressing human (C, E) and mouse (D, F) MAIT TCR after stimulation with escalating amounts (1, 3, 9 μl) (C, D) or 9 μl (E, F) of total metabolites from FN, FN∆ribD, FNribD^FT, FNribD^{FT (56, 61, 62)}, FNribD^{FT (80)}, and FNribD^{FT (254)} strains cultured in the presence of human or mouse MR1-expressing cells. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA, followed by Dunnett’s multiple comparison test. Data are representative of three independent experiments.

(A) Survival rates of mice after intranasal infection with the FNribD^FT or its parental strain FN (n = 15 in each group). Asterisks indicate statistical significance determined by logrank tests (**P < 0.01). (B) Graph shows the changes of bacterial numbers after infection in vitro. After infection with indicated strains to THP-1 cells, bacteria present in the cells were calculated at each time point. (C, D) Histological analysis of inflammation in the lung by immunohistochemistry (C) or H&E staining with quantified data (D) 4 d after intranasal infection. (C, D) Scale bar shows 10 μm (C) or 50 μm (D), respectively. Statistical significance was determined by unpaired two-tailed t tests (*P < 0.05). (E) Bacterial burdens in the indicated tissues 4 d after infection (n = 14 in each group). Statistical significance was determined by unpaired two-tailed t tests (*P < 0.05, **P < 0.01). (F) Numbers of mMR1/5-OP-RU-tet⁺ MAIT cells in the lung 4 d after infection. Statistical significance was determined by unpaired two-tailed t tests (**P < 0.01). (G, H) Flow cytometric analysis of lung 14 d after infection with the indicated strain. (G) mMR1/5-OP-RU-tet⁺ MAIT cells in the lung are shown after gating on total lymphocytes. (H) Plots show the absolute numbers of mMR1/5OP-RU-tet⁺ MAIT cells, mCD11c⁺ DCs, and mF4/80⁺ macrophages. Statistical significance was determined by one-way ANOVA, followed by Dunnett’s multiple comparison test (**P < 0.01, ****P < 0.0001). (I, J) Flow cytometric analysis of lung MAIT cells 9 d after infection with the avirulent strain FN. (I) After gating on mMR1/5OP-RU-tet⁺ cells, IFNγ and IL-17A production was analyzed by intracellular staining. (J) Graph shows frequencies of the indicated populations before and after infection with FN. Statistical significance was determined by unpaired two-tailed t test (****P < 0.0001). Data in (A, E, F, H, J) are combined from three independent experiments. Data in (B, C, D, G, I) are representative of three independent experiments.