Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

Abstract

The interplay between host and pathogen relies heavily on rapid protein synthesis and accurate protein targeting to ensure pathogen destruction. To gain insight into this dynamic interface, we combined Click chemistry with pulsed stable isotope labelling of amino acids in cell culture to quantify the host proteome response during macrophage infection with the intracellular bacterial pathogen Salmonella enterica Typhimurium. We monitored newly synthesized proteins across different host cell compartments and infection stages. Within this rich resource, we detected aberrant trafficking of lysosomal proteases to the extracellular space and the nucleus. We verified that active cathepsins re-traffic to the nucleus and that these are linked to cell death. Pharmacological cathepsin inhibition and nuclear targeting of a cellular cathepsin inhibitor (stefin B) suppressed S. enterica Typhimurium-induced cell death. We demonstrate that cathepsin activity is required for pyroptotic cell death via the non-canonical inflammasome, and that lipopolysaccharide transfection into the host cytoplasm is sufficient to trigger active cathepsin accumulation in the host nucleus and cathepsin-dependent cell death. Finally, cathepsin inhibition reduced gasdermin D expression, thus revealing an unexpected role for cathepsin activity in non-canonical inflammasome regulation. Overall, our study illustrates how resolution of host proteome dynamics during infection can drive the discovery of biological mechanisms at the host–microbe interface.

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Fig. 1: Newly synthesized proteome enrichment detects diverse host responses during STm infection.
Fig. 2: STm infection induces distinct host proteome responses compared to LPS across time and space.
Fig. 3: Lysosomal proteases are enriched in the nuclear fraction following STm infection.
Fig. 4: STm SPI-2 promotes nuclear cathepsin activity.
Fig. 5: Cathepsin activity promotes STm-induced caspase-11-dependent cell death.
Fig. 6: Cathepsin activity promotes GSDMD expression and its nuclear activity is required for cell death.

Data availability

Proteomic pSILAC-AHA data have been deposited at ProteomeXchange (http://www.proteomexchange.org/) under the identifiers PXD010179 (pSILAC-AHA) and PXD016086 (2D-TPP). Source data for Figs. 1–6 and Extended Data Figs. 1–7 and 10 are provided with the paper.

Code availability

The code and pipelines used for data analysis are available upon request.

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Acknowledgements

We thank the EMBL Flow Cytometry, Proteomics and ALMF Core Facilities; N. Kurzawa for 2D-TPP data analysis; S. Helaine (Imperial College) for the pDiGc plasmid; O. Wagih (EBI) for R scripts; K.-M. Noh (EMBL) for the H3.cs1 antibody; and members of the Typas and Krijgsveld laboratories for discussions. We acknowledge funding from EMBL for this research. J.S., N.L. and H.I. were supported by Marie Skłodowska-Curie Actions COFUND grant no. 291772, and A.M. by grant no. 664726. E.L. was supported by the Deutsche Forschungsgemeinschaft (German Research Foundation) under Germany’s Excellence Strategy (nos. EXC2151 and 390873048) and the ERC consolidator grant InflammAct. N.L. is now supported by the National Key Research and Development Programme of China (no. 2018YFA0902703), the Shenzhen Science and Technology Innovation Committee (no. JCYJ20170818164014753) and the National Natural Science Foundation of China (nos. 31800694 and 31971354). N.K.-J. and B.T. were supported by the Slovene Research Agency (no. P1-0140).

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Contributions

Conceptualization was carried out by J.S., J.K. and A.T. Investigation was done by J.S., N.L., A.M., G.S., M.S. and J.K. (proteomics), and by J.S., J.B., A.S., M.S., J.M. and M.Z. (molecular biology, biochemistry and cell biology). Resources were provided by B.I.F., H.S.O. (cathepsin reactive probes), M.M., E.L. (iMACs), N.K.-J., B.T. (stefin B construct), A.H. and W.-D.H. (BMDMs). Data analysis was performed by J.S., M.M., A.S., B.E.D., N.L., H.I. and A.M. The article was written by J.S. and A.T. with input from all authors. The figures were made by J.S., except for 2D-TPP (A.M.), bacterial growth curves (A.S.) and iMAC data (M.M.), with input from J.K. and A.T. The study was supervised by A.T., J.K., M.S., E.L. and P.B. Funding acquisition was carried out by A.T. and J.K.

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Correspondence to Jeroen Krijgsveld or Athanasios Typas.

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Extended data

Extended Data Fig. 1 Increasing intracellular STm load over time co-occurs with dynamic proteome changes.

a) RAW264.7 cells infected with STm constitutively expressing mCherry (pFCcGi) at MOI 100:1 and incubated for the indicated times were analysed by CellProfiler. SPI-1 OFF bacteria were chosen to mimic the pathogen-host interaction at systemic sites. After bacterial internalization, cells were treated with gentamicin to kill non-internalised bacteria, synchronise the infection and avoid re-infection cycles. The intracellular bacterial load was quantified at the single-cell level to ensure sampling times spanned distinct phases of intracellular STm proliferation states i.e. pre-proliferation (4h), initial (8h) and extensive (20h) proliferation post uptake. Images were captured at 20x objective and analysis was conducted with CellProfiler to segment infected from uninfected cells and quantify bacterial load per cell based on mCherry fluorescence per infected cell. CellProfiler quantification of bacterial count per infected cell (only infected cells) are displayed as a histogram and shows increasing bacterial load with time. Data contains combined counts from two biological replicate experiments, whereby each replicate received reverse SILAC labels (see Methods). 2-5 fields of view (technical replicates) were acquired per biological replicate and per time point. The combined total of infected host cells quantified is indicated (n=). The dotted red line indicates distribution median. Representative images of quantified cells are displayed to the right of histograms. Scale bars represent 50 μm. The % of infected cells per field of view (FOV) at the beginning of the experiment (i.e. immediately post gentamicin 100 μg/ml treatment, t=0) is displayed as a boxplot on the far right. Box plots are depicted as in Fig. 3C. n denotes FOV, amounting to 1224 cells in total (infected & non-infected). b) Replicate correlation of infected versus uninfected samples for the indicated time points and different subcellular fractions - cytoplasmic and solubilised organelles within the Tx-100 soluble fraction (lysatome; upper), nuclear enriched fraction in the Tx-100 insoluble fraction (nucleome-middle) and proteins secreted into the culture supernatant (secretome-lower). Two biological replicates containing reversed SILAC labels were obtained for each cell fraction and each time point (See Supplementary Table 1). We observed concordant biological replicate correlation (r = Pearson’s correlation) across all time points, and within each subcellular fraction (mean r = 0.886). Data is from n=2 biologically independent samples. A red dotted line represents a linear model by robust regression and r refers to the Pearson’s correlation coefficient. Source data

Extended Data Fig. 2 Quantification of cytokine and chemokine release from RAW264.7 cells during STm infection.

After RAW264.7 cells were infected with STm for 20 hours, the conditioned media was removed and indicated cytokines and chemokines were quantified using a custom array (Biocat, GmbH). n denotes the combined data from 2 independent experiments (batches), each batch containing 2 biological replicates per condition. Box boundaries indicate the upper and lower IQR, the median is depicted by the middle boundary and whiskers represent 1.5x IQR. Relative infected/uninfected Δ (Log2) from the cytokine array are depicted within the plot for each protein. Corresponding pSILAC-AHA infected/uninfected (Log2) ratios are presented below the corresponding protein for the 20hr post-infection time-point (with the exception of TNFa, which we could only detect at 8 hpi). Non-hits and hits match perfectly between the 2 methods. Fold effect differences between pSILAC-AHA and array-based quantification are most likely due to the fact that the cytokine assay measures steady-state protein levels and pSILAC-AHA newly synthesized proteins. As expected, for more abundant proteins, measuring steady-state levels masks changes in expression dynamics. A two-sided unpaired t-test was used to calculate p.. Source data

Extended Data Fig. 3 Nuclear cathepsin translocation occurs already from 8 hpi, is independent of cathepsin activity and does not result in increased Histone 3 cleavage.

a) Time course of nuclear cathepsin activity. Nuclei and STm were enriched by sequential 50 x g and 8,000 x g centrifugation steps, respectively. RAW264.7 cells were infected with wildtype STm 14028s and treated with DCG04-Bodipy-FLike (5μM) for 4 hours prior to harvesting. Samples were separated by SDS-PAGE and visualised using a fluorescent scanner (Excitation 405 nm/Emission 520 nm), followed by immunoblotting for the soluble cytoplasmic protein GAPDH, the bacterial protein RpoD and Coomassie staining for histones as a loading control for nuclear extracts. L = lysatome, N = nucleome, STm = STm enriched fraction. Experiment was performed once. b) RAW264.7 cells were infected with wildtype STm at MOI 100:1 and harvested at 20 hpi. Whole cell lysates were immunoblotted with the CtsL specific cleavage product of H3 (H3.cs1)16 and histone 3 (H3) as loading control. The experiment was performed in biological duplicate per condition and were analysed in adjacent lanes. c) Nuclear extracts from RAW264.7 cells either mock infected, infected with wildtype STm or heat killed wildtype (STm-HK) for 20 hours. CA-074-Me was present throughout the infection experiment at the indicated concentrations. Nuclear extracts were analysed by immunoblot for CtsB (1:2,000), Lamin A (1:5,000) and histones by Coommassie stain (see Supplementary Information (SI) for second replicate data). Source data

Extended Data Fig. 4 Nuclear cathepsin activity is enhanced by wildtype STm and mildly by an ssaV mutant and heat killed bacteria.

Nuclei were extracted from RAW264.7 cells treated with DCG04-Boclicky-TAMRA (5 μM) for 2 hours prior to harvest at 20 hpi with wildtype (Wt), a SPI-2 mutant (ΔssaV) or heat killed wildtype bacteria (Wt HK) (MOI = 100:1). Samples were processed as described in Fig. 5a. Data is combined from two independent experiments (batches). n denotes the number of biologically independent samples. A two-sided unpaired t-test was used to calculate p. Boxplots are depicted as in Fig. 3C. Source data

Extended Data Fig. 5 STm growth is unaffected by the cathepsin inhibitor CA-074-Me in batch culture conditions.

STm 14028s growth was measured in the presence of the selective cathepsin inhibitor CA-074-Me in LB 6, 12.5 and 25 µM and DMSO solvent controls. Drug concentrations used are indicated. Experiment was performed in biological triplicate. Source data

Extended Data Fig. 6 CA-074-Me inhibits cathepsins and reduces expression of proinflammatory proteins.

a) 2D-TPP of RAW264.7 cells infected with wildtype STm 14028s for 20 hours in the presence of increasing concentrations of CA-074-Me (1-100 µM). To assess CA-074-Me specificity, we applied 2-dimensional Thermal Proteome Profiling (2D-TPP). 2D-TPP enables proteome assessment of protein ligand binding based on the principle that ligand-bound proteins are more thermally stable than proteins not bound to a ligand20,21, and in parallel provides information of proteome-wide protein abundance. We subjected RAW264.7 cells infected with wildtype STm to increasing doses of CA-074-Me and compared proteome-wide thermostabilisation relative to solvent controlled cells (see methods). Increased (blue) or decreased fold changes are calculated by normalising the abundance of each protein relative to the abundance in the DMSO vehicle per temperature. Protein stabilization (red framed) is demonstrated by increasing (blue) fold changes with respect to increasing temperature (top to bottom) as well as with increasing drug concentrations (left to right). Changes in protein expression (green framed) can be detected if abundance changes are already evident from low temperatures i.e. before proteins melt. All cathepsins detected by 2D-TPP are displayed. CtsA and CtsD are serine and aspartic-acid proteases, respectively, and are therefore not targeted by CA-074-Me and serve as negative controls. Contrary to evidence demonstrating CA-074-Me targets CtsL35, we failed to detect its stabilisation in our experiment. Cathepsins are arranged in the top row, followed by caspases and other cellular proteases in descending order. Note Caspase-1 stability was not affected by CA-074-Me, in line with previous reports22. Additionally, CA-074-Me reduced IL-1b expression as previously shown35,36. 2D-TPP data can be found in Supplementary Table 6. b) Caspase-11-like proteolysis activity was assessed using purified active Caspase-11 and measuring cleavage of the fluorescent substrate AcLEHD-afc as previously described37. Caspase-11-like proteolytic activity was assessed in the presence of indicated concentrations of CA-074-Me and the Caspase-11 inhibitor Z-LEHD-FMK as a positive control. Activity is expressed as the % of AcLEHD-afc cleavage relative to the DMSO solvent control. Data is combined from n = 3 biologically independent experiments; error bars depict standard deviation and the center value denotes the mean. c) 2D-TPP profiles of inflammation related proteins reduced in abundance upon CA-074-Me treatment. Data presented as in (a). Source data

Extended Data Fig. 7 Rapid cell death induced by SPI-1 ON STm is independent of cathepsin activity.

Wildtype and Caspase-1/11 -/- BMDMs were infected with wildtype STm and a SPI-1 mutant (ΔprgK), late-exponential growing STm (SPI-1 ON) in the presence of CA-074-Me at the indicated concentrations. LDH release was measured 1.5 hpi. Boxplots are depicted as in Fig. 3C. n denotes the number of biologically independent samples. Source data

Extended Data Fig. 8

LPS transfection promotes nuclear cathepsin activity. RAW264.7 cells were transfected with purified LPS for 20 hours. 2 hours prior to harvest cells were treated with DCG04-Boclicky-TAMRA (5 µM) to quantify nuclear cathepsin activity. Nuclei were extracted and analysed by flow cytometry as per Fig. 5A. The percentage of cathepsin positive (TAMRA+) nuclei is indicated and a second biological replicate is located in the supplementary material replicate data section.

Extended Data Fig. 9

CA-074-Me does not affect Caspase-11 expression or processing. Wildtype BMDMs were infected with wildtype STm for 20 hours in the presence of CA-074-Me (25 µM) or DMSO control. Lysates were probed for Caspase-11 by immunoblot and using GAPDH as a loading control. Experiment was performed once.

Extended Data Fig. 10 Characterisation of iMACs expressing Stefin B targeted to the nucleus or expressed in the cytoplasm.

a) Expression of Stefin B fusions targeted to either the nucleus (Nuc) or expressed in the cytoplasm (Cyto) were verified in iMAC lysates by immunoblot using anti-FLAG (M2) and anti-tubulin as a loading control. Cells were FACS sorted into high (Hi) and low (Lo) Stefin B expressing populations prior to use. Cells treated in parallel expressing mCitrine alone were used as negative controls. Experiment was repeated twice with similar results (see Supplementary Information for replicate data). b) Expression and localisation of Stefin B was assessed by microscopy in cells expressing nuclear-targeted (Nuc-Lo and Nuc-Hi) or cytoplasmic (Cyto-Lo and Cyto-Hi) Stefin B or mCitrine alone as a control. Scale bar denotes 25 µm. Experiment was repeated twice with similar results (see Supplementary Information for replicate data). c) iMACs expressing nuclear or cytoplasmic Stefin B were treated with 1 mM LLoMe or untreated and LDH release was quantified and plotted as described previously in Fig. 5b. Each data point is the combined average of biological replicate wells from three independent experiments. Boxplots are depicted as in Fig. 3c. d) iMACs expressing nuclear or cytoplasmic Stefin B were untreated, LPS treated or LPS and nigericin treated and LDH release was quantified and plotted as described previously in Fig. 5b. Each data point is the combined average of biological replicate wells from three independent experiments. Box plots are depicted as in Fig. 3c. Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–6.

Reporting Summary

Supplementary Tables 1–6

Supplementary Table 1 contains replicate data of reversed pSILAC-AHA-labelled RAW264.7 cells during STm infection; Supplementary Table 2 contains GO term enrichment analysis of proteome changes during STm infection of RAW264.7 cells; and Supplementary Table 3 contains merged pSILAC-AHA proteomic data from cells stimulated with LPS or STm infection. Supplementary Table 4 contains STm-infected lysatome pSILAC-AHA-labelled RAW264.7 cell data after subtraction of LPS signal8; Supplementary Table 5 contains GO term enrichment of STm-infected lysatome, nucleome and secretome pSILAC-AHA data after subtraction of LPS signal8; and Supplementary Table 6 contains 2D-TPP of RAW264.7 cells infected with wild-type STm for 20 h and treated with CA-074-Me.

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Selkrig, J., Li, N., Hausmann, A. et al. Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection. Nat Microbiol (2020). https://doi.org/10.1038/s41564-020-0736-7

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