Synaptotagmin 1 and Synaptotagmin 7 promote MR1-mediated presentation of Mycobacterium tuberculosis antigens
- Division of Pulmonary, Allergy, and Critical Care Medicine, Oregon Health and Science University, United States
- Department of Molecular Microbiology and Immunology, Oregon Health and Science University, United States
- VA Portland Health Care System, United States
- Department of Microbiology and Molecular Genetics, Michigan State University, United States
Figures
Figure 1
Syt1 and Syt7 specifically mediate MR1 presentation of intracellular Mtb.
(a) Relative gene expression levels of SYT1, SYT7, and GAPDH in BEAS-2B (n=3) and PMA-differentiated THP-1 cells (n=3-4). (b) Genome editing efficiency and percent distribution of individual indels of Syt1 and Syt7 KO BEAS-2B cells as determined by Sanger sequencing and Interference of CRISPR Edits (ICE) (Conant et al., 2022) (n=1). (c) IFN-γ release by T cell clones (MR1- and HLA-B45-restricted) co-cultured with H37Rv Mtb-infected (MOI=8) WT, Syt1 KO, or Syt7 KO BEAS-2B cells, represented as spot forming units (SFU). (d) IFN-γ release by T cell clones (MR1- and HLA-B45-restricted) co-cultured with WT, Syt1 KO, or Syt7 KO BEAS-2B cells in the presence of Msmeg supernatant, CFP102–9 peptide, or (e) 5-A-RU prodrug, represented as SFU. All data are plotted as mean ± SEM and pooled from n=3 independent experiments. For (c–e), the means of technical duplicates were pooled, and non-linear regression analysis of pairwise comparison to WT on best-fit values of top and EC50 was used to calculate p-values by extra sum-of-squares F test. A p value of <0.05 was considered statistically significant.
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Figure 1—source data 1
Source data corresponding to Figure 1.
- https://cdn.elifesciences.org/articles/108318/elife-108318-fig1-data1-v1.xlsx
Figure 2
Syt1 and Syt7 do not affect Mtb uptake and growth.
(a) Gating strategy of BEAS-2B cells infected overnight with auxotrophic strain mEmeraldRFP-AuxMtb (MOI=8) by gating on cells, excluding doublets using forward scatter properties, and selecting Live/Dead Near-IR stain negative cells. (b) Representative gate on GFP+ population to indicate live BEAS-2B cells infected with mEmeraldRFP-AuxMtb. (c) Percent Mtb uptake measured as proportion of live WT, Syt1 KO, or Syt7 KO BEAS-2B cells that are GFP+. (d) Colony-forming units (CFU) of H37Rv Mtb in WT, Syt1 KO, or Syt7 KO BEAS-2B cells after overnight infection (MOI=8). (e) Relative gene expression levels of MR1 and GAPDH in WT, Syt1 KO, or Syt7 KO BEAS-2B cells. All data are plotted as mean ± SEM. (c, d) are pooled from n=3 independent experiments and (e) is pooled from n=2 independent experiments. For (c, d), ordinary one-way ANOVA with Dunnett’s multiple comparisons test was used to analyze significant differences. ns=not significant (p > 0.05).
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Figure 2—source data 1
Source data corresponding to Figure 2.
- https://cdn.elifesciences.org/articles/108318/elife-108318-fig2-data1-v1.xlsx
Figure 3
Syt1 and Syt7 also mediate MR1 presentation of Mtb in THP-1 cells.
(a) Genome editing efficiency and percent distribution of individual indels of Syt1 and Syt7 KO THP-1 cells as determined by Sanger sequencing and ICE analysis (Conant et al., 2022) (n=1). (b) IFN-γ release by MAIT cell clones co-cultured with H37Rv Mtb-infected (MOI=1) WT, Syt1 KO, or Syt7 KO THP-1 cells following PMA differentiation, represented as SFU. (c) IFN-γ release by MAIT cell clones co-cultured with WT, Syt1 KO, or Syt7 KO THP-1 cells following PMA differentiation in the presence of Msmeg supernatant, represented as SFU. For (b, c), the means of technical duplicates were pooled, data were plotted as mean ± SEM and pooled from n=3 independent experiments, and non-linear regression analysis of pairwise comparison to WT on best-fit values of top and EC50 was used to calculate p-values by extra sum-of-squares F test. A p value of <0.05 was considered statistically significant.
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Figure 3—source data 1
Source data corresponding to Figure 3.
- https://cdn.elifesciences.org/articles/108318/elife-108318-fig3-data1-v1.xlsx
Figure 4 with 1 supplement
Syt11 and ER-associated Esyt1 and Esyt2 do not solely affect MR1 presentation of Mtb.
(a) Relative gene expression levels of SYT11 (n=3), ESYT1 (n=3), ESYT2 (n=3), and GAPDH (n=6) in BEAS-2B cells. (b) Knockdown efficiency of Syt11, Esyt1, and Esyt2 after 48 hr of knockdown with missense (Mis) or gene-specific (KD) small-interfering RNA (siRNA). IFN-γ release by T cell clones (MR1- and HLA-B45-restricted) co-cultured with BEAS-2B cells following siRNA knockdown of Syt11 (c), Esyt1 (d), or Esyt2 (e). Cells were either infected overnight with H37Rv Mtb (MOI=8) or incubated with exogenously added antigens (Msmeg supernatant and CFP102–9 peptide). IFN-γ release is represented as SFU. All data are plotted as mean ± SEM and pooled from n=3 independent experiments. Experiments were performed in parallel for (d, e). For (c–e), the means of technical duplicates were pooled, and non-linear regression analysis comparing best-fit values of top and EC50 was used to calculate p-values by extra sum-of-squares F test. A p value of <0.05 was considered statistically significant.
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Figure 4—source data 1
Source data corresponding to Figure 4.
- https://cdn.elifesciences.org/articles/108318/elife-108318-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Esyt2 is important for overall MR1 antigen presentation.
(a) Knockdown efficiency of Esyt2 after 48 hr of knockdown with missense (Mis) or gene-specific (KD) small-interfering RNA (siRNA) (n=3). (b) Missense-treated and Esyt2-knockdown cells were incubated overnight with doxycycline and Ac-6-FP or NaOH (solvent control). Histograms representative of n=3 independent experiments with pooled geometric mean fluorescence intensity (GeoMFI) of surface MR1 and HLA-Ia expression. All data are plotted as mean ± SEM. For statistics, p-values were analyzed by two-way ANOVA with Sidak’s multiple comparisons test. A p value of <0.05 was considered statistically significant.
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Figure 4—figure supplement 1—source data 1
Source data corresponding to Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/108318/elife-108318-fig4-figsupp1-data1-v1.xlsx
Figure 5
Syt1 and Syt7 localize to late endo-lysosomes and MR1 vesicles.
(a) BEAS-2B cells transfected with Syt1- or Syt7-RFP (magenta) plasmids and incubated with CellLight BacMam 2.0 reagents for Rab5a, Rab7a, and LAMP1 (yellow) overnight. Images are representative of n=2 independent experiments (b) Percent co-localization of Rab5a (n=11), Rab7a (n=12), and LAMP1 (n=12) with Syt1 or Syt7. Data are pooled from n=2 independent experiments and plotted as mean ± SEM. Each dot represents one cell. (c) Polyclonal BEAS-2B:TET-MR1GFP (green) cells transfected overnight with Syt1- or Syt7-RFP (magenta) plasmids. Images are representative of n=3 independent experiments. (d) Percent co-localization of Syt1 or Syt7 with MR1 (n=17). Data are pooled from n=3 independent experiments and plotted as mean ± SEM. Each dot represents one cell. Two-way ANOVA with Sidak’s multiple comparisons test (b) and two-tailed unpaired Student’s t-test (d) were used to calculate p-values. A p value of <0.05 was considered statistically significant. All scale bars represent 10 µm.
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Figure 5—source data 1
Source data corresponding to Figure 5.
- https://cdn.elifesciences.org/articles/108318/elife-108318-fig5-data1-v1.xlsx
Figure 6
Absence of Syt1 and Syt7 alters MR1 vesicle size in lysosomal compartments.
Syt1 KO and Syt7 KO were generated in the background of BEAS-2B MR1KO:tetMR1-GFP clone D4 cells. (a) Syt1 and Syt7 KO clones were verified by Sanger sequencing and analyzed using the ICE tool (Conant et al., 2022) (n=1). (b) IFN-γ release by T cell clones (MR1- and HLA-B45-restricted) co-cultured with H37Rv Mtb-infected cells (MOI=8) is represented as SFU. The means of technical duplicates were pooled from n=3-4 independent experiments, and non-linear regression analysis comparing best-fit values of top and EC50 was used to calculate p-values by extra sum-of-squares F test. (c, d) WT, Syt1 KO, and Syt7 KO BEAS-2B MR1KO:tetMR1-GFP cells were incubated overnight with doxycycline and Ac-6-FP or NaOH (solvent control). Images (c) and histograms (d, left) representative of n=3 independent experiments with pooled geometric mean fluorescence intensity (GeoMFI) (d, right) of surface MR1 and HLA-Ia expression. (e) Representative images of WT, Syt1 KO, and Syt7 KO EAS-2B MR1KO:tetMR1-GFP cells incubated overnight with doxycycline and (f) measurement of area of MR1 vesicles, classified into small (1 vesicle) or large (>1 vesicle) vesicles (n=15). Each dot represents one cell. Data are pooled from n=3 independent experiments. (g) Representative images of WT, Syt1 KO, and Syt7 KO BEAS-2B MR1KO:tetMR1-GFP cells incubated overnight with doxycycline and CellLight BacMam 2.0 reagents for Rab5a and LAMP1 (yellow). (h) Percent co-localization of Rab5a (n=16) and LAMP1 (n=16) with MR1 vesicles. Each dot represents one cell. Data are pooled from n=4 independent experiments. For (d, f, h), p-values were analyzed by two-way ANOVA with Dunnett’s multiple comparisons test. All data are plotted as mean ± SEM. A p value of <0.05 was considered statistically significant. All scale bars represent 10 µm.
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Figure 6—source data 1
Source data corresponding to Figure 6.
- https://cdn.elifesciences.org/articles/108318/elife-108318-fig6-data1-v1.xlsx
Figure 7
Syt1 and Syt7 mediate trafficking of MR1 vesicles from the Mtb-containing vacuole.
(a) WT, Syt1 KO, and Syt7 KO BEAS-2B MR1KO:tetMR1-GFP (green) cells were infected with AuxMtb (MOI=5) labeled with Alexa Fluor 555 Succinimidyl Ester (AuxMtb-Alexa Fluor555; magenta). Images representative of n=3 independent experiments are shown. Scale bars represent 10 µm. (b) Number of MR1 vesicles within 1 µm of the center of the AuxMtb surface (n=51–53). (c) Total number (left, n=51–53) and average speed (µm/s) (right, n=14–16) of MR1 vesicles. (d) Overlapped area ratio of MR1 to Mtb surfaces (n=51–53). For (b–d), data are plotted as mean ± SEM and pooled from n=8 independent experiments. Each dot represents one cell. p-values were analyzed by a one-way ANOVA with Dunnett’s multiple comparisons test. (e) WT, Syt1 KO, and Syt7 KO BEAS-2B MR1KO:tetMR1-GFP cells were infected overnight with AuxMtb (MOI=10) labeled with Alexa Fluor 647 Succinimidyl Ester. Mtb-containing vacuoles for each fraction from flow organellometry assay were identified by gating on vesicles, excluding doublets using forward scatter properties, and selecting the AuxMtb+LAMP1+ population (left). Total Lamp1+ and MR1+ populations were gated following the same strategy (right). Representative histograms comparing fractions 40 and 46 are shown. (f) Graphs representative of n=3 independent experiments showing the frequency of total LAMP1+, total MR1+, and AuxMtb+LAMP1+ vesicles of all vesicles from fractions 36–50. (g) Representative histogram and geometric mean fluorescence intensity (GeoMFI) of MR1 in the subcellular fraction with the highest percentage of AuxMtb+LAMP1+ vesicles. Data are pooled from n=3 independent experiments and plotted as mean ± SEM. p-values were analyzed by a one-way ANOVA with Dunnett’s multiple comparisons test. A p value of <0.05 was considered statistically significant.
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Figure 7—source data 1
Source data corresponding to Figure 7.
- https://cdn.elifesciences.org/articles/108318/elife-108318-fig7-data1-v1.xlsx
Additional files
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Supplementary file 1
Microscopy image analysis.
- https://cdn.elifesciences.org/articles/108318/elife-108318-supp1-v1.xlsx
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Supplementary file 2
Plasmid sequences of Syt1-RFP and Syt7-RFP.
- https://cdn.elifesciences.org/articles/108318/elife-108318-supp2-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/108318/elife-108318-mdarchecklist1-v1.docx
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Synaptotagmin 1 and Synaptotagmin 7 promote MR1-mediated presentation of Mycobacterium tuberculosis antigens
eLife 14:RP108318.
https://doi.org/10.7554/eLife.108318.3
