Ubiquitin-mediated targeting of intracellular bacteria to the autophagy pathway is a key innate defence mechanism against invading microbes, including the important human pathogen Mycobacterium tuberculosis. However, the ubiquitin ligases responsible for catalysing ubiquitin chains that surround intracellular bacteria are poorly understood. The parkin protein is a ubiquitin ligase with a well-established role in mitophagy, and mutations in the parkin gene (PARK2) lead to increased susceptibility to Parkinson’s disease. Surprisingly, genetic polymorphisms in the PARK2 regulatory region are also associated with increased susceptibility to intracellular bacterial pathogens in humans, including Mycobacterium leprae and Salmonella enterica serovar Typhi, but the function of parkin in immunity has remained unexplored. Here we show that parkin has a role in ubiquitin-mediated autophagy of M. tuberculosis. Both parkin-deficient mice and flies are sensitive to various intracellular bacterial infections, indicating parkin has a conserved role in metazoan innate defence. Moreover, our work reveals an unexpected functional link between mitophagy and infectious disease.
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We thank N. Mizushima, S. Cherry, and K. Huynh for mice and reagents. We are grateful to S. Johnson for use of his microscope, members of the Schneider laboratory for assistance with fly work and D. Portnoy, R. Vance and S. Virgin for helpful discussions. This work was supported by National Institutes of Health grants R01 AI081727, P01 AI063302 and R01 AI099439, and NINDS P30NS069496 to K.N.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Quantification of parkin co-localization and effect of LRSAM1 knockdown in BMDMs.
a, Quantification of parkin-positive M. tuberculosis in BMDMs from wild-type and Park2−/− mice, from Fig. 1a. b, BMDMs from LC3–GFP transgenic mice were transduced with lentivirus expressing either a scrambled shRNA control (Ctrl) or shRNAs targeting either LRSAM1 or parkin. Lentiviral transduced cells were then infected with mCherry-expressing M. tuberculosis and the co-localization of GFP–LC3 and ubiquitin was quantified by immunofluorescence. *P < 0.014, **P < 0.008 by Student’s t-test c, Quantitative PCR with reverse transcription (RT–qPCR) expression of LRSAM1 and parkin transcripts in lentiviral transduced cells from a. Data shown are expressed relative to actin expression. *P < 0.033, **P < 0.0035 by Student’s t-test.
a, Wild-type BMDMs were transduced with lentivirus expressing HA-tagged constructs of wild-type ubiquitin (WT), ubiquitin with all lysine residues mutated to arginine except for lysine 63 (K63), or ubiquitin with all lysine residues mutated to arginine except for lysine 48 (K48). Transduced cells were then infected with mCherry-expressing M. tuberculosis and immunostained using anti-HA antibodies 4 h post-infection. b, Quantification of HA-ubiquitin co-localization with M. tuberculosis from a. **P < 0.001 by Student’s t-test.
a, Cartoon model showing digitonin differential permeabilization of macrophages and antibody accessibility to phagosomes. b, Microscopy images of wild-type BMDMs were infected with mCherry-expressing M. tuberculosis. Cells were immunostained by digitonin permeabilization alone or digitonin permeabilization with Triton X-100 treatment. c, Quantification of ubiquitin co-localization with M. tuberculosis from b. N.D., not determined.
Extended Data Figure 4 Immunohistochemistry analysis of parkin within human patients with active tuberculosis.
Lung biopsy samples were obtained from three different human patients with active tuberculosis. Immunohistochemistry was performed on specimens using either anti-parkin, anti-M. tuberculosis or an IgG control antibody. Positive cells were visualized by DAB staining. Scale bar, 100 μm.
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Manzanillo, P., Ayres, J., Watson, R. et al. The ubiquitin ligase parkin mediates resistance to intracellular pathogens. Nature 501, 512–516 (2013). https://doi.org/10.1038/nature12566
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