Cell-autonomous targeting of arabinogalactan by host immune factors inhibits mycobacterial growth
- Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, China
- Clinical and Translational Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, China
- Department of Infectious Diseases, National Medical Centre for Infectious Diseases, National Clinical Research Centre for Aging and Medicine, Shanghai Key Laboratory of Infectious Diseases and Biosafety Emergency Response, Huashan Hospital, Fudan University, China
- Abmart Inc, China
- Max Planck Institute for Infection Biology, Germany
- Max Planck Institute for Multidisciplinary Sciences, Germany
- Hagler Institute for Advanced Study, Texas A&M University, United States
- Multitude Therapeutics, China
- School of Life Science, Fudan University, China
- Department of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, China
- Central Laboratory, Shanghai Pulmonary Hospital, Tongji University School of Medicine, China
Figures
Figure 1
Galectin-9 inhibits mycobacterial growth directly.
(A) Profile of Mycobacterium tuberculosis (Mtb) H37Rv (Rv) grown at 37 °C in Middlebrook 7H9 liquid medium with different concentrations 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 hr. (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 after being incubated in Middlebrook 7H9 liquid medium with or without 10 μg/mL Galectin-9 (Gal9) for 30 hr at 37℃. Cultures were grown at 37 °C for 4 weeks for enumeration of CFU. (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 healthy donors (n=40) and active TB patients (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 a colocalization ratio of at least 12 fields in each experiment. (A, 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). 63x magnification.Scar bar, 5μm. (I) Percent of cells with galectin9 positive (gal9+) Mtb H37Rv in total infected THP-1 cells. Symbols indicate a 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.
Figure 2
Carbohydrate recognition is essential for galectin-9-mediated inhibition of Mycobacterium tuberculosis (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 (1 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 (1 μg/mL). (D) Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with 1 μg/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.
Figure 3
Identification of anti-arabinogalactan (AG) antibodies from tuberculosis (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 antibody levels in TB patients (n=25) and healthy BCG-immunized controls (n=17) were 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.
Figure 4
Development of anti-arabinogalactan (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 blue arrow, respectively. (C) Schematic presentation of candidate anti-AG mAbs validation by ELISA. (D) Binding curve of mAb1 and mAb2 to AG was determined by ELISA assay. (E) Confocal microscopy of Mycobacterium tuberculosis (Mtb) H37Rv-GFP (Rv-GFP, green) and anti-AG mAbs (red). 100x oil immersion.Scar bar, 10 μm. (F) Quantification of colocalization between anti-AG mAb and Mtb H37Rv-GFP by calculating Mander’s coefficients in (E). tM2, Mander’s coefficient of red above the autothreshold of green. Data are representative of three independent experiments with similar results (D, E). Data are shown as mean ± SD, n=10 (F). Two-tailed unpaired Student’s t-test (F). p<0.05 was considered statistically significant.
Figure 5
Anti-arabinogalactan (AG) antibody inhibits mycobacterial growth.
(A) Growth profile of Mycobacterium tuberculosis (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=3 biologically independent experiments performed in triplicate (B, D). Two-tailed unpaired Student’s t-test (A–D). p<0.05 was considered statistically significant.
Figure 6
Proteomics profiling of the response of Mycobacterium tuberculosis (Mtb) to anti-arabinogalactan (AG) antibody.
(A) Gene ontology (GO) class of differentially expressed proteins in Mtb H37Rv treated with mAb1 (1 μg/mL) for 30 hr 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).
Figure 7
Mtb cell wall modulation by anti-arabinogalactan (AG) antibodies.
(A) Morphologic characteristics for Mycobacterium tuberculosis (Mtb) H37Rv strain grown in liquid culture with or without anti-AG mAbs (1 μg/mL) observed by 2x magnifier. (B) Bacterial shape of Mtb H37Rv strain treated as in (A) was observed by acid-fast staining under a Leica DM2500 microscope using the 100x oil microscopy. EMB, Ethambutol. Scale bar, 20 μm. (C) Ultrastructural morphology of Mtb H37Rv treated as in (A) analyzed by transmission electron microscopy (TEM). The cell wall was labeled with red arrows. (D) Cell wall thickness of bacteria in (C). (E) 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). Data are means ± SD of 11 bacteria, representatives of three independent experiments (D). Two-tailed unpaired Student’s t-test (D). p<0.05 was considered statistically significant.
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Cell-autonomous targeting of arabinogalactan by host immune factors inhibits mycobacterial growth
eLife 13:RP92737.
https://doi.org/10.7554/eLife.92737.3
