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Parkinson’s disease kinase LRRK2 coordinates a cell-intrinsic itaconate-dependent defence pathway against intracellular Salmonella

Nature Microbiology volume 8, pages 1880–1895 (2023)Cite this article

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Abstract

Cell-intrinsic defences constitute the first line of defence against intracellular pathogens. The guanosine triphosphatase RAB32 orchestrates one such defence response against the bacterial pathogen Salmonella, through delivery of antimicrobial itaconate. Here we show that the Parkinson’s disease-associated leucine-rich repeat kinase 2 (LRRK2) orchestrates this defence response by scaffolding a complex between RAB32 and aconitate decarboxylase 1, which synthesizes itaconate from mitochondrial precursors. Itaconate delivery to Salmonella-containing vacuoles was impaired and Salmonella replication increased in LRRK2-deficient cells. Loss of LRRK2 also restored virulence of a Salmonella mutant defective in neutralizing this RAB32-dependent host defence pathway in mice. Cryo-electron tomography revealed tether formation between Salmonella-containing vacuoles and host mitochondria upon Salmonella infection, which was significantly impaired in LRRK2-deficient cells. This positions LRRK2 centrally within a host defence mechanism, which may have favoured selection of a common familial Parkinson’s disease mutant allele in the human population.

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Fig. 1: LRRK2 is a component of the RAB32-dependent host defence pathway against Salmonella.
Fig. 2: The kinase activity of LRRK2 is required for its contribution to the RAB32-dependent pathogen restriction pathway.
Fig. 3: LRRK2 scaffolds the formation of the RAB32–IRG1 complex.
Fig. 4: Intimate association of the SCV with the mitochondria observed by cryo-ET.
Fig. 5: LRRK2 is required for establishing a close association between the SCV and the mitochondria.
Fig. 6: Salmonella deploys its type III secretion system at the SCV–mitochondria interface.

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Data availability

Subtomogram-average density maps and the raw cryo-ET tilt series have been deposited in EMDB (deposition ID numbers: EMD-41046, EMD-41047 and EMPIAR-11577). The rest of the data are available in the main text, supplementary materials and auxiliary files. Source data are provided with this paper.

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Acknowledgements

We thank T. Lam and W. Wang from the WM Keck Foundation Biotechnology Resource Laboratory at the Yale University School of Medicine for assistance with the itaconate measurements. We also thank M. Shao (Yale University) for assistance with the cryo-ET experiments. F.S. was partially supported by a fellowship from the Human Frontiers Science Program (LT000056/2020-C). This work was supported by National Institutes of Health grants R01AI152421 and R01AI087946 to J.L. and R01AI114618 and R01AI055472 to J.E.G. and a pilot grant from the Parkinson’s Foundation (PF-RCE-1946). The Proteomics Resource of the WM Keck Foundation Biotechnology Resource Laboratory was partially supported by CTSA grant number UL1TR001863 from the National Center for Advancing Translational Sciences (of the National Institutes of Health).

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Author notes

  1. Huan Lian

    Present address: Department of Laboratory Medicine, Zhongnan Hospital of Wuhan University, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China

  2. Donghyun Park

    Present address: Department of Integrative Structural and Computational Biology, Scripps Research, La Jolla, CA, USA

  3. Meixin Chen

    Present address: Institute of Infectious Diseases, Shenzhen Bay Laboratory, Shenzhen, China

  4. These authors contributed equally: Huan Lian, Donghyun Park.

Authors and Affiliations

  1. Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT, USA

    Huan Lian, Donghyun Park, Meixin Chen, Florian Schueder, Maria Lara-Tejero, Jun Liu & Jorge E. Galán

  2. Microbial Science Institute, Yale University School of Medicine, New Haven, CT, USA

    Donghyun Park & Jun Liu

  3. Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA

    Florian Schueder

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Contributions

H.L. performed the functional and biochemical experiments. D.P. conducted all the cryo-ET experiments with the assistance of H.L. and M.C. and under the direction of J.L. F.S. performed the DNA-PAINT imaging experiments; M.L.-T. performed the liquid chromatography–tandem mass spectrometry experiments and coordinated the animal experiments. J.E.G. conceived and directed the project and wrote the manuscript with comments from all the authors.

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Correspondence to Jorge E. Galán.

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Nature Microbiology thanks Clare Bryant, Elizabeth Villa, Siyu Chen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 LRRK2 is required for efficient itaconate delivery to the Salmonella-containing vacuole.

(a and b) LRRK2 is required for efficient itaconate delivery to the Salmonella-containing vacuole. Parental (control) and Lrrk2-/- Raw264.7 cells were infected with S. Typhi (MOI = 6) encoding an eGFP-based itaconate biosensor and the number of cells expressing eGFP was determined 20 hours after infection. Each square and circle represents the mean of an individual experiment experiments in which at least 200 infected cells were examined (b). The p value (unpaired two-tailed Student’s t test) of the indicated comparison is shown. Infected cells were fixed, stained with DAPI (blue) to visualize nuclei, and stained with an anti-Salmonella LPS antibody along with Alexa 594-conjugated anti-rabbit antibody (red) to visualize all bacteria. Representative fields of infected cells are shown (a) (scale bar = 5 µm). (c-g) Absence of LRRK2 does not influence the uptake of Salmonella into phagocytic cells. Raw264.7 or DC2.4 parental (control) and Lrrk2-/- cells, as well as bone marrow-derived macrophages (BMDM) derived from C57BL/6 and Lrrk2-/- mice were infected with either wild-type S. Typhi (MOI = 6) or a S. Typhimurium ∆gtgE ∆sopD2 mutant strain (MOI = 3) (as indicated) and the number of CFU was determined 1 hr after infection. Each square or circle represents the CFU in an independent measurement. The mean ± SD and p values (unpaired two-tailed Student’s t test) of the indicated comparisons are shown (n = 6 for each category).

Source data

Extended Data Fig. 2 Salmonella infection results in LRRK2 activation.

DC2.4 cells were treated with LPS or infected with the indicated bacterial strains for the indicated times. The activation of LRRK2, assessed by its phosphorylation at S935, was then analyzed by immunoblotting with the indicated antibodies.

Source data

Extended Data Fig. 3 LRRK2 scaffolds the formation of RAB32 and IRG1 complex.

(a and b) LRRK2 interacts with RAB32 and IRG1. HEK293T cells were transiently co-transfected with a plasmid expressing GFP-LRRK2 and a plasmid expressing either FLAG-RAB32 (a) or FLAG-IRG1 (b). Twenty hours after transfection cells were infected with S. Typhi (MOI = 6) and 4 hs after infection, cell lysates were analyzed by immunoprecipitation and immunoblotting with antibodies against the FLAG epitope and GFP. (c-e) The kinase activity of LRRK2 is not required to form a complex with RAB32 and IRG1. (c and d) Raw264.7 (c) or DC2.4 (d) cells stably expressing FLAG-RAB32 or FLAG-IRG1 were pre−treated with the LRRK2 kinase inhibitor GSK2578215A for 90 min, infected with the S. Typhimurium ∆gtgE ∆sopD2 mutant strain (MOI = 3) (c) or treated with LPS (d). Eighteen hours after infection or 5 or 20 hs after LPS treatment, cell lysates were analyzed by immunoprecipitation and immunoblotting with the indicated antibodies. (e) HEK293T cells were transiently co-transfected with plasmids expressing GFP-RAB32, FLAG-Irg1, and the indicated forms of LRRK2: wild type (WT), kinase defective (3XKD = LRRK2K1906A/D1994A/D2017A), and constitutively active (LRRK2G2019S). Twenty hours after transfection, cell lysates were analyzed by immunoprecipitation and immunoblotting with the indicated antibodies. The quantification of the intensity of the RAB32 band relative to the intensity of the IRG1 band is shown in. Each circle, square, or triangle represents a measurement in an independent experiment. The mean ± SD and p values (unpaired two-tailed Student’s t test) of the indicated comparisons are shown (n = 3 for each category). (f and g) HEK293T parental or LRRK2-/- cells were transfected with GFP-RAB32 and FLAG-IRG1 for 20 hs. Cell lysates were then analyzed by immunoprecipitation with anti-FLAG and immunoblotting with anti-GFP antibody. The quantification of the intensity of the RAB32 band relative to the intensity of the IRG1 band is shown (f). Each circle or square represents a measurement in an independent experiment. The mean ± SD and p values (unpaired two-tailed Student’s t test) of the indicated comparisons are shown (n = 3 for each category). (g) Raw264.7 parental or Lrrk2-/- cells stably expressing FLAG-RAB32 were left untreated, treated with LPS, or infected with S. Typhimurium ∆gtgE ∆sopD2 mutant strain (MOI = 3) for 18 hs. Cell lysates were then analyzed by immunoprecipitation with anti-FLAG and immunoblotting with the indicated antibodies.

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Extended Data Fig. 4 Localization of LRRK2, RAB32, and IRG1.

(a and b) LRRK2, RAB32, and IRG1are associated with the mitochondria accessible to protease digestion. DC2.4 cells stably expressing RAB32 (a), or DC2.4 parental (control) and Lrrk2-/- cells (b) were treated with LPS for 18 hs, mitochondria were purified and treated with proteinase K or left untreated, and subsequently analyzed by immunoblotting with the indicated antibodies. (c) Two color DNA-PAINT super-resolution image demonstrating that IRG1 does not co-localize with the mitochondrial matrix protein Cox IV. The top panel presents a HeLa cell expressing GFP-tagged IRG1 (green). Cells were fixed and stained with nanobodies to the GFP epitope, and primary and secondary antibodies to Cox IV (magenta). Nanobodies and secondary antibodies were labeled with a single stranded DNA oligomer acting as a docking site for DNA-PAINT super-resolution microscopy. First and second zoom levels show that Cox IV and IRG1 are spatially excluded from each other. The yellow arrows in zoom level two highlight examples of the spatial exclusion of Cox IV and IRG1. Scale bars 2 µm (top panel), 400 nm (zoom level 1) and 100 nm (zoom level 2). (d and e) Three-plex DNA-PAINT super-resolution image showing proximity of RAB32, LRRK2, and IRG1. (d) Hek293T cells expressing GFP-tagged LRRK2 (purple – DNA-PAINT), FLAG-tagged Rab32 (green – DNA-PAINT), and M45-tagged IRG1 (yellow – DNA-PAINT) were infected with S. Typhi carrying plasmid encoding an mCherry-based itaconate reporter (red – diffraction limited image). Cells were fixed and stained with nanobodies to the GFP epitope, and M45 and FLAG tags were labeled with primary antibodies and secondary antibodies conjugated to a single stranded DNA oligomer acting as a docking site for DNA-PAINT super-resolution microscopy. (e) The zoom in shows the spatial proximity of the three proteins in the proximity of S. Typhi expressing the itaconate reporter. The white arrows in the zoom-in highlight examples of the proximity cluster of the three proteins. Scale bars 5 µm (d), 1 µm (e).

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Extended Data Fig. 5 Inhibition of the mitochondrial tricarboxylate transporter SLC25A1 impairs itaconate delivery to the Salmonella-containing vacuole.

(a and b) HeLa cells stably expressing EGFP-tagged IRG1 were pre-treated with the SLC25A1 transporter inhibitor CTPI-2 for 3, 6, or 18 hs (as indicated), and then infected with wild-type S. Typhi (MOI = 6) encoding a luciferase-based itaconate biosensor. The levels of luciferase activity in the cell lysates were then measured 3 hs after infection. Each circle or square represents a single luciferase measurement. The mean ± SD and p values (unpaired two-tailed Student’s t test) of the indicated comparisons are shown (n = 6 for each category). (a) and (b) show results of two independent experiments. (c and d) Inhibition of the mitochondrial tricarboxylate transporter SLC25A1 does not impair IRG1 expression or overall itaconate biosynthesis. (c) HeLa cells stably expressing EGFP-tagged IRG1 were pre-treated with the SLC25A1 transporter inhibitor CTPI-2 for 3, 6, or 18 hs (as indicated), and then infected with wild-type S. Typhi (MOI = 6) encoding a luciferase-based itaconate biosensor as indicated in Extended Data Fig. 10. The levels of IRG1 3, 6 or 18 hours after CTPI-2 treatment were evaluated by western immunoblot with the indicated antibodies. (d) HeLa cells stably expressing EGFP-tagged IRG1 were pre-treated with the SLC25A1 transporter inhibitor CTPI-2 for 18 hs, and the levels of itaconate were measured as indicated in Materials and Methods. Each square represents a single measurement and the mean and SD are shown (n = 3 for each category).

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Extended Data Fig. 6 Tomographic slices of S. Typhi infected cells at different times after infection.

HeLa cells expression IRG1 (a and b) or BMDMs obtained from C57BL/6 mice (c and d) were infected with S. Typhi and 1 (a and c) and 3 (b and d) hs after infection were processed for cryo-ET imaging. Shown are representative tomographic slices showing that the appearance of S. Typhi within cells over time. Bacteria within HeLa-IRG1 cells 1 hr after infection appear normal, with many ribosomes and an intact bacterial envelope. However, bacteria within HeLa-IRG1 cells 3 hs post-infection or within BMDMs at 1 and 3 hs post infection exhibit altered morphology. Mi: mitochondria.

Extended Data Fig. 7 Visualization of tethers at the SCV-mitochondria interface.

(a-e) 3D renderings of the SCV-mitochondria interfaces shown in Figs. 4f, j, k, o, and p, respectively. Magenta, yellow, and green represent bacterial, vacuolar, and mitochondrial membranes, respectively. Intermembrane tethers are depicted in white. Please refer to the main Fig. 4 figure legend for experimental details. (f-j) Top-down views of the corresponding interfaces in Panels (a-e), revealing vacuolar membrane surfaces decorated with intermembrane tethers.

Extended Data Fig. 8 Itaconate delivery and bacterial growth in cells used for cryo-ET analysis.

(a) HeLa cells stably expressing IRG1 or BMDMs from C57BL/6 mice treated with LPS (200 ng/ml) for 3 hours were infected with S. Typhi (MOI = 10), and the number of CFU was determined 1 and 3 hs after infection. Each circle represents the CFU in an independent measurement; the mean ± SEM of all the measurements and p values of the indicated comparisons (two-sided Student’s t test) are shown. ns, not significant. ****p < 0.0001 (n = 6 for each category). (b and c) HeLa cells stably expressing IRG1 (b) or BMDMs from C57BL/6 mice treated with LPS (200 ng/ml) for 3 hs (c) were infected with S. Typhi (MOI = 10) carrying a plasmid encoding the itaconate nanoluciferase biosensor. One and three hours after infection, the levels of nanoluciferase were measured in lysates of the infected cells. Each circle represents a single luciferase measurement. The mean ± SD and p-values of the indicated comparisons (two-sided Student’s t-test) are shown. ****p < 0.0001 (n = 6 for each category). (d-g) BMDMs obtained from C57BL/6 (WT) or Hps4−/− were infected with S. Typhi (MOI = 10) carrying a plasmid encoding the itaconate nanoluciferase biosensor, and the number of CFU was determined 1 (a) or 3 (c) hs after infection. Alternatively, the levels of nanoluciferase were measured in lysates of the infected cells (b and d). Each circle represents the CFU in independent measurements (a and c) or a single luciferase measurement (b and d). Shown are the mean ± SEM of all the measurements (n = 6 for each category); p values of the indicated comparisons (two-sided Student’s t test) are shown. **p < 0.01 and ***p < 0.001, ****p < 0.0001. (h and i) Itaconate delivery and intracellular growth of S. Typhi expressing gtgE in cells used for cryo-ET analysis. (h and i) HeLa cells stably expressing IRG1 were infected with S. Typhi or S. Typhi- expressing gtgE (MOI = 10) carrying a plasmid encoding the itaconate nanoluciferase biosensor. The number of CFU (a) or the level of luciferase activity (b) was determined 1 hour or 3 hours after infection. Each circle represents a single measurement. Values are the mean ± SEM of all the measurements and p values of the indicated comparisons (two-sided Student’s t test) are shown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 6 for each category).

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Extended Data Fig. 9 Expression of the S. Typhimurium effector protein GtgE in S. Typhi does not prevent SCV-mitochondria association and tethering.

(a) Tomographic slice showing S. Typhi strain expressing GtgE within its replication vacuole and surrounding mitochondria (Mi) intimately interacting with the vacuolar membrane (VM). (b) 3D-rendering of the tomogram shown in panel (a)(z = 86 slices). Mitochondria is depicted in green, the SCV membrane in yellow, bacterial envelope in blue, inter membrane tethers in white, type III secretion machines in light blue, and bacterial ribosomes in grey (see close ups of the SCV-mitochondria interface in Fig. S14).

Extended Data Fig. 10 Model for the role of LRRK2 in itaconate delivery to the Salmonella containing vacuole.

LRRK2 may coordinate the close apposition between the Salmonella-containing vacuole (SCV) and the mitochondria (not depicted in this model) as it has been proposed to do with other intracellular organelles (64). In addition, as depicted in this model, through its ability to scaffold a complex between RAB32, IRG1, and SLC25A1, LRRK2 may coordinate the localized synthesis of itaconate at the mitochondria/SCV interface.(generated with the help of Biorender (www.biorender.com).

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Supplementary Video 1

Tethering of the SCV with the mitochondria observed by cryo-ET.

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Lian, H., Park, D., Chen, M. et al. Parkinson’s disease kinase LRRK2 coordinates a cell-intrinsic itaconate-dependent defence pathway against intracellular Salmonella. Nat Microbiol 8, 1880–1895 (2023). https://doi.org/10.1038/s41564-023-01459-y

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