SMURF2 inhibits autophagic control of Mycobacterium tuberculosis in macrophages
Priscila C. Campos, Kathryn C. Rahlwes, Victoria A. Ektnitphong, Beatriz R.S. Dias, Kubra F. Naqvi, Samuel Alvarez-Arguedas, Michael U. Shiloh
Priscila C. Campos, Kathryn C. Rahlwes, Victoria A. Ektnitphong, Beatriz R.S. Dias, Kubra F. Naqvi, Samuel Alvarez-Arguedas, Michael U. Shiloh
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Research Article Immunology Infectious disease Microbiology

SMURF2 inhibits autophagic control of Mycobacterium tuberculosis in macrophages

Abstract

Autophagy is a critical host defense mechanism that restricts intracellular pathogens such as Mycobacterium tuberculosis. A key step in this process is the ubiquitination of M. tuberculosis or M. tuberculosis–associated structures. The E3 ligase Smad-specific E3 ubiquitin protein ligase 1 (SMURF1) catalyzes K48-linked ubiquitination, promoting bacterial clearance. However, the function of its homolog, SMURF2, in host defense remains undefined. Here, we demonstrate that Smurf2 deletion in murine macrophages increases SMURF1 levels, enhances LC3B lipidation, augments K48 ubiquitination of M. tuberculosis–associated structures, and reduces intracellular M. tuberculosis replication. These effects are reversed by Smurf1 deletion, supporting a role for SMURF1 in SMURF2-dependent control of M. tuberculosis. Mice with myeloid-specific Smurf2 deletion exhibit modestly prolonged survival after aerosol M. tuberculosis infection. In human macrophages, SMURF2 knockdown or its pharmacological inhibition with the HECT E3-ligase inhibitor Heclin reduces M. tuberculosis replication. Together, our findings identify SMURF2 as a negative regulator of macrophage control of M. tuberculosis and support further investigation of SMURF2 as a potential target for host-directed therapy in tuberculosis.

Authors

Priscila C. Campos, Kathryn C. Rahlwes, Victoria A. Ektnitphong, Beatriz R.S. Dias, Kubra F. Naqvi, Samuel Alvarez-Arguedas, Michael U. Shiloh

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Figure 9

Inhibition of SMURF2 by Heclin reduces M. tuberculosis intracellular growth in human macrophages.

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Inhibition of SMURF2 by Heclin reduces M. tuberculosis intracellular gro...
(A) Luminescence from M. tuberculosis–pLUX in hMDMs exposed to either 0.1% DMSO (vehicle control) or increasing concentrations of Heclin, as indicated (see Methods for details). Luminescence was measured at different time points (mean ± SD of a representative experiment out of 2 independent experiments, each performed in 12 replicates, ns, nonsignificant, *P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001; 2-way ANOVA). (B) Luminescence from M. tuberculosis–pLUX in BV2-WT and Smurf2-KO cells exposed to either 0.1% DMSO (vehicle control) or increasing concentrations of Heclin. Luminescence was measured at different time points (mean ± SD of a representative experiment out of 2 independent experiments, each performed in quintuplicate. ns, nonsignificant, *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001; 2-way ANOVA, with multiple comparisons). (C) Representative immunofluorescence images of M. tuberculosis–mCherry (red) and LC3B (green) in human macrophages exposed to either 0.1% DMSO (vehicle control) or 10 μM Heclin. (D) Quantification of M. tuberculosis–mCherry colocalization with LC3B shown in C (mean ± SD of 3 independent experiments performed in triplicate, **P ≤ 0.01; unpaired t test). Scale bar: 5 μm.