Galectin-9 inhibits mycobacterial growth directly.

A. Profile of Mtb H37Rv (Rv) grown at 37°C in Middlebrook 7H9 liquid medium with different concentration of Galectin-9 (Gal9, 0, 0.01, 0.1, 1, 10 μg/mL). Growth curve was measured using a Bioscreen Growth Curve Instrument. Optical density was measured at absorbance at 600 nm every 2 h.

B. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with10 μg/mL galectin-9 (Gal9) or inactivated galectin-9 (Gal9 HK, heat killed at 95 ℃ for 5 min).

C. CFU of Mtb H37Rv (Rv) on Middlebrook 7H10 solid medium with or without 10 μg/mL Galectin-9 (Gal9). Cultures were grown at 37°C for 4-8 weeks.

D. Growth profile of Mycobacterium smegmatis (MS) in Middlebrook 7H9 liquid medium with different concentrations of Galectin-9 (Gal9, 0, 0.01, 0.1, 1 μg/mL).

E. Concentrations of galectin-9 in sera of tuberculin skin test negative healthy donors (TST-, n = 20), tuberculin skin test positive healthy donors (TST+, n = 20) and active TB patients (Active TB, n = 40).

F. Confocal microscopy of M. bovis BCG-DsRed (BCG-DsRed, red) and Galectin-9 (Anti-Gal9, green) in THP-1 cells. Nuclei was stained with DAPI (blue).

G. Percent of cells with galectin-9 positive (Gal9+) BCG in total infected THP-1 cells. Symbols indicate colocalization ratio of at least 12 fields in each experiment.

H. Confocal microscopy of Mtb H37Rv-GFP (Rv-GFP, green) and Galectin-9 (Anti-Gal9, red) in THP-1 cells. Nuclei were stained with DAPI (blue).

I. Percent of cells with galectin9 positive (Gal9+) Mtb H37Rv in total infected THP-1 cells. Symbols indicate colocalization ratio of at least 12 fields in each experiment.

Data are shown as mean ± SD, n = 3 biologically independent experiments performed in triplicate (A-D). Data are representative of three independent experiments with similar results (F and H). Two-tailed unpaired Student’s t test (A-D, G, and I) or Mann-Whitney U test (E). P < 0.05 was considered statistically significant.

Carbohydrate recognition is essential for galectin-9-mediated inhibition of Mtb growth.

A. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with or without galectin-9 (Gal9, 10 μg/mL) and lactose (10 μg/mL).

B. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with or without galectin-9 (Gal9, 10 μg/mL) and D-glucose (10 μg/mL).

C. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with or without galectin-9 (Gal9, 10 μg/mL) and AG (10 μg/mL).

D. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with 1μg/mL CRD1 or CRD2 of galectin-9.

Data are shown as mean ± SD, n = 3 biologically independent experiments performed in triplicate (A-D). Two-tailed unpaired Student’s t test (A-D). P < 0.05 was considered statistically significant.

Identification of anti-AG antibodies from TB patients.

A. Schematic presentation of ELISA assay for detecting anti-AG IgG antibodies in the serum of TB patients.

B. Linear correlation between OD and serum dilution ratio determined by ELISA assay.

C. Anti-AG IgG antibodies levels in TB patients (n = 25) and healthy BCG-immunized controls (n = 17) determined via ELISA.

Data are representative of three independent experiments with similar results (B). Mann-Whitney U test (C). P < 0.05 was considered statistically significant.

Development of anti-AG mAbs.

A. Schematic presentation of mAb screening for AG specificity.

B. Representative image of chip hybridization for mAb screening. Bright spots in the bottom mark the end line of each array block. Other spots represent AG binding to mAbs. CL010746 (mAb1) and CL046999 (mAb2) were labeled with red arrow and yellow arrow, respectively.

C. Schematic presentation of candidate anti-AG mAbs validation by ELISA.

D. Binding curve of mAb1 and mAb2 to AG determined by ELISA assay.

E. Confocal microscopy of Mtb H37Rv-GFP (Rv-GFP, green) and anti-AG mAbs (red).

anti-AG antibody inhibits mycobacterial growth.

A. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with or without mAb1/mAb2 (1 μg/mL).

B. CFU of Mtb H37Rv (Rv) on Middlebrook 7H10 solid medium with or without mAb1/mAb2 (1 μg/mL). Cultures were grown at 37°C for 4-8 weeks.

C. Growth profile of Mycobacterium smegmatis (MS) in Middlebrook 7H9 liquid medium with or without mAb1/mAb2 (1 μg/mL).

D. CFU of Mycobacterium smegmatis (MS) on Middlebrook 7H10 solid medium with or without mAb1/mAb2 (1 μg/mL). Cultures were grown at 37°C for 5-10 days.

Data are shown as mean ± SD, n = 3 (A, C) and n = 2 biologically independent experiments performed in triplicate (B, D). Two-tailed unpaired Student’s t test (A-D). P < 0.05 was considered statistically significant.

Proteomics profiling of the response of Mtb to anti-AG antibody.

A. GO class of differentially expressed proteins in Mtb H37Rv treated with mAb1 (1 μg/mL) for 30 h followed by proteomics analysis. IgG was set as control.

B. Functional enrichment of differentially expressed proteins in Mtb H37Rv in (A).

C. Protein domain of differentially expressed proteins in Mtb H37Rv in (A).

D. KEGG class of differentially expressed proteins in Mtb H37Rv in (A).

E. Upregulation or downregulation genes in Mtb H37Rv in (A).

Mtb cell wall modulation by anti-AG antibodies.

A. Morphologic characteristics for Mtb H37Rv strain grown in liquid culture with or without anti-AG mAbs (1 μg/mL) observed by × 2 magnifier.

B. Bacterial shape of Mtb H37Rv strain treated as in (A) observed by acid fast staining under a Leica DM2500 microscope using the 100× oil microscopy. EMB, Ethambutol. Scale bar, 20μm.

C. Ultrastructural morphology of Mtb H37Rv treated as in (A) analyzed by transmission electron microscopy (TEM).

D. Schematic presentation of Mtb growth arrest by Galectin-9 or anti-AG antibodies.

Data are representative of three independent experiments with similar results (A, B and C, left panel). Data are means ± SD of five bacteria, representatives of three independent experiments (C, right panel). Two-tailed unpaired Student’s t test (C, right panel). P < 0.05 was considered statistically significant.