Spatiotemporal Proteomics Uncovers Cathepsin-Dependent Host Cell

bioRxiv preprint doi: https://doi.org/10.1101/455048; this version posted November 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

1 Spatiotemporal proteomics uncovers cathepsin-dependent host 2 cell death during bacterial infection

3 Joel Selkrig1, Nan Li1,2,3, Jacob Bobonis1,4, Annika Hausmann5, Anna Sueki1,4, Haruna 4 Imamura6, Bachir El Debs1, Gianluca Sigismondo3, Bogdan I. Florea7, Herman S. 5 Overkleeft7, Pedro Beltrao6, Wolf-Dietrich Hardt5, Jeroen Krijgsveld3‡, Athanasios Typas1‡. 6 7 1 European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Meyerhofstrasse 1, 69117 8 Heidelberg, Germany. 9 2 Institute of Synthetic Biology (iSynBio), Shenzhen Institutes of Advanced Technology (SIAT), 10 Chinese Academy of Sciences (CAS), 1068 Xueyuan Avenue, Shenzhen University Town, Shenzhen, 11 China. 12 3 German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany. 13 4 Collaboration for joint PhD degree between EMBL and Heidelberg University, Faculty of Biosciences 14 5 Institute of Microbiology, ETH Zurich, CH-8093 Zurich, Switzerland. 15 6 European Bioinformatics Institute, European Molecular Biology Laboratory, Wellcome Trust Genome 16 Campus, Hinxton, Cambridge, CB10 1SD 17 7 Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Einsteinweg 18 55, 2333 CC Leiden, the Netherlands. 19 20 21 ‡ to whom correspondence should be addressed: 22 [email protected] & [email protected] 23 24 SUMMARY 25 Immune cells need to swiftly and effectively respond to invading pathogens. This response 26 relies heavily on rapid protein synthesis and accurate cellular targeting to ensure pathogen 27 destruction. In return, pathogens intercept this response to ensure their survival and 28 proliferation. To gain insight into this dynamic interface, we combined click-chemistry with 29 pulsed stable isotope labeling of amino acids (pSILAC-AHA) in cell culture to quantify the 30 newly synthesised host proteome during macrophage infection with the model intracellular 31 bacterial pathogen, Salmonella enterica Typhimurium (STm). We monitored newly 32 synthesised proteins across different host cell compartments and infection stages, and used 33 available proteomics data in response to lipopolysaccharide to deconvolute the STm-specific 34 response. Within this rich resource, we detected aberrant trafficking of lysosomal proteases 35 to the extracellular space and the nucleus, the latter of which correlated with signatures of 36 cell death. Pharmacological cathepsin inhibition suppressed Caspase-11 dependent 37 macrophage cell death, thus demonstrating an active role for cathepsins during STm 38 induced pyroptosis. Our study illustrates that resolving host proteome dynamics during 39 infection can drive the discovery of biological mechanisms at the host-microbe interface.

40 INTRODUCTION 41 Bacterial pathogens can survive inside mammalian immune cells by injecting toxic effector 42 proteins to co-opt the endogenous host cell machinery and promote their intracellular 43 survival. Prior to proliferation, a successful pathogen must avoid detection by host 44 cytoplasmic pattern recognition receptors (PRRs), which constantly surveil the host 45 cytoplasm for microbial ligands. Upon detection of intracellular pathogens, activated 46 cytoplasmic PRRs trigger the assembly of a large cytoplasmic protein scaffold called the 47 inflammasome, which drives an inflammatory form of cell death termed pyroptosis. 48 Pyroptosis is an effective host strategy to remove a pathogens’ replicative niche and subject 49 it to further destruction by the immune system. To avoid triggering pyroptosis, pathogens 50 can maintain physical separation from PRRs by residing within membrane bound organelles 51 sculpted from the host endosomal compartment, whose function is to otherwise destroy 52 pathogens. Thus, triumph of either host or pathogen is fought through active engagement in 53 a dynamic molecular interplay spanning time and space. This complex process remains 54 poorly understood even for well-studied pathogens. 55 56 Pyroptosis is driven by the canonical inflammasome cysteine proteases Caspase-1 (mouse 57 and human) and the non-canonical Caspase-11 (mouse) and Caspases-4 and 5 (human)1. 58 During canonical inflammasome activation, microorganism-associated molecular patterns 59 (MAMPs) or damage-associated molecular patterns (DAMPs) are recognised by cytoplasmic 60 sensors (e.g. NLRP3 and NLRC4), which then initiate inflammasome assembly 2. This 61 macromolecular scaffold activates Caspase-1, which cleaves the proinflammatory cytokines 62 pro-IL-1β and pro-IL-18. In contrast, the non-canonical inflammasome does not rely on 63 cytoplasmic sensors for its activation. Instead, Caspase-11 appears to be both a sensor and 64 an activator by directly binding to cytoplasmic lipopolysaccharide (LPS) derived from Gram- 65 negative bacteria 3. Upon activation, Caspase-11 cleaves gasdermin (GSDMD) into an 66 active form that then punctures holes in the plasma membrane, ultimately leading to the 67 extracellular release of cytoplasmic contents and cell death 4,5. Mice lacking key 68 inflammasome components (e.g. Caspase-1, Caspase-11, NLRP3 and NLRC4) are more 69 susceptible to bacterial infection 6,7, illustrating the importance of pyroptotic cell death in 70 clearance of intracellular pathogens. 71 72 Subcellular compartmentalisation of molecules that trigger inflammasome activation 73 prevents premature activation and control pyroptotic cell death. For example, lysosomes are 74 membrane-bound organelles containing approximately 50 different hydrolytic enzymes that 75 degrade various forms of internalised material, including microbes, within a low-pH

76 environment 8. Disrupting lysosome integrity with chemical destabilisers leads to 77 inflammasome activation and cell death 9,10, both of which can be abrogated by the addition 78 of cathepsin inhibitors 9–14 . This implicates lysosomal cathepsins as critical effectors of 79 lysosome instability and cell death, although how this occurs, particularly during bacterial 80 infection, remains poorly understood. 81 82 Remarkably, the intracellular pathogen Salmonella enterica subsp. enterica serovar 83 Typhimurium (STm) evades degradation via the lysosomal pathway by residing within a 84 specialized cellular compartment called the Salmonella-containing vacuole (SCV). STm 85 sculpts the SCV via a secretion system (SPI-2), which delivers effector proteins into the host 86 cytosol to hijack various components of the host cell machinery 15,16. STm actively prevents 87 the delivery of toxic lysosomal hydrolases to the SCV by inhibiting retrograde trafficking of 88 mannose-6-phosphate receptors via the SPI-2 effector protein SifA 17. This ultimately leads 89 to the export of immature lysosomal contents outside the host cell 17. To safeguard against 90 intracellular pathogens with such capabilities, interferon-induced guanylate-binding proteins 91 (GBPs) lyse bacteria-containing vacuoles, thereby spilling pathogen-derived LPS into the 92 cytoplasm. This activates Caspase-11 and initiates pyroptosis 18. Thus, control of lysosomal 93 trafficking is critical to both host and pathogen survival. Despite intense research into 94 understanding how STm manages to evade triggering host cell death in the vacuole before 95 replication has taken place, many facets of this interface remain unknown. 96 97 To shed light onto this multi-faceted interface, we leveraged a method recently developed in 98 our lab designed to enrich, identify and quantify newly synthesised host secretory proteins in 99 serum-containing media 19. In the present study, we extended this approach to include 100 sampling of three host cell compartments (i.e. secretome, lysatome and the nucleome) from 101 STm infected macrophages spanning three distinct stages of the infection cycle. We 102 detected aberrant subcellular distribution of lysosomal proteases during the later stages of 103 infection, and demonstrated their active role during STm-induced cell death via the non- 104 canonical inflammasome Caspase-11. Combined, our findings exemplify the treasure trove 105 of functional biology that can be uncovered by spatiotemporally resolving a host-pathogen 106 interface.

107 RESULTS 108 Dynamic proteome mapping unravels diverse host responses during STm infection. 109 To model the intracellular STm infection cycle, we infected mouse-derived macrophages 110 with SPI-1 OFF STm. SPI-1 OFF bacteria were chosen to mimic the pathogen-host 111 interaction at systemic sites. After bacterial internalization, the cells were treated with 112 gentamicin to kill non-internalised bacteria, synchronise the infection and avoid re-infection 113 cycles (Figure 1A). The intracellular bacterial load was quantified at the single-cell level to 114 ensure sampling times spanned distinct phases of intracellular STm proliferation states i.e. 115 pre-proliferation (4h), initial (8h) and extensive (20h) proliferation post uptake (Figure S1A). 116 In order to determine the subcellular distribution of host proteins throughout an infection 117 cycle, we utilised our recently developed proteomic methodology that allows specific 118 enrichment and quantification of the newly synthesised host proteome 19. 119 120 To quantify the subpopulation of host proteins synthesised within a specific timeframe, we 121 simultaneously pulsed cells with SILAC amino acids and an azide-containing analogue of 122 methionine called azidohomoalanine (AHA). Intermediate (I) or heavy (H) SILAC labels 123 facilitate robust protein quantification relative to uninfected controls, whereas AHA enabled 124 enrichment of the newly synthesised proteome. These amino acid labels can be used as 125 substrates by the hosts’ endogenous translational machinery and are thereby incorporated 126 into newly synthesised proteins (pSILAC-AHA). A three-hour pulse window is sufficient to 127 allow protein synthesis and subsequent subcellular trafficking to occur 20. After pSILAC-AHA 128 labeling, the azide moiety of AHA was utilized to couple proteins to an alkyne-activated resin 129 via a click-chemistry reaction, thus allowing for selective capture of proteins containing 130 incorporated AHA, while serum and other pre-existing background proteins were removed 131 by stringent washing conditions. Infected and uninfected samples were then combined in a 132 1:1 ratio, followed by LC-MS/MS analysis (Figure 1A). 133 134 We quantified the newly synthesised host proteome (4978 proteins in total) by sampling 135 three distinct subcellular locations from macrophages infected with intracellular STm; 215 136 proteins in the secretome (conditioned media), 4640 in the lysatome (Tx-100 soluble; this 137 contains cytoplasmic and organelle content) and 1283 from the nucleome (Tx-100 insoluble) 138 (Table S1). We observed concordant biological replicate correlation across all time points, 139 and within each subcellular fraction (mean r = 0.886; Figure S1B). GO term enrichment

140 analysis for host proteins displaying >1.5 ±Log2 fold change revealed a total of 879 enriched 141 GO terms (p = ≤0.05, right-sided hypergeometric test, Bonferroni corrected), with 832

142 upregulated and 47 were downregulated (Table S2). Consistent with the lysatome containing

143 the majority of quantified proteins, 693 enriched GO terms were detected in the lysatome

144 fraction, whereas 97 and 87 GO terms were enriched in the nucleome and secretome

145 samples respectively. 146 147 In general terms, dynamic changes occurring at distinct time-points of the infection time 148 course were more frequent in the subcellular compartments, whereas the lysatome was 149 dominated by responses that were observed from the first time-point post infection (4h) and 150 remained stable across time (Figure 1B). Such early and stable responses included many 151 GO terms related to infection and adaptation to immune stimulation. For example, and as 152 expected, we detected a strong response to bacteria at all time points in the lysatome, as 153 well as cytokine stimulus (GO:0071345), interferon (GO:0035456), an extrinsic apoptotic 154 pathway (GO:0097191) and a progressive decrease in cellular responses to 155 lipopolysaccharide (LPS; GO:0071222). The latter behaviour is consistent with macrophage 156 adaptation to TLR activation by a highly immunogenic stimulus, such as LPS, in order to 157 dampen detrimental impacts from a hyperactive pro-inflammatory response 21. A strong and 158 constant response to interferon-γ (GO: 0071346) and virus (GO:0009615) were also visible, 159 with the antiviral response at 20 hours consisting of 33 proteins (Table S2). This is in 160 agreement with the recently discovered role of classic antiviral responses during bacterial 161 infection 22. 162 163 We also detected an early induction of the I-kappaB kinase/NF-kappaB signaling response, 164 which was sustained throughout infection in the lysatome fraction (Figure 1B). For instance, 165 consistent with an early NF-κB response that relies on NF-κB1 translocation to the nucleus, 166 we observed strong enrichment of NF-κB1 in the nucleome at 4 hrs post infection (+2.12 -5 167 Log2 Fold change, adjusted p = 6.72x10 ) (Table S3). Furthermore, NF-κB1 expression 168 peaked at 8 hrs in the lysatome, followed by a relative decrease at 20 hrs (Table S3). This 169 suggested an overall blunting of the NF-κB response over time by reducing nuclear NF-κB1 170 translocation from and abundance within the lysatome. In addition, Nfkbiz (NF-κB inhibitor

171 zeta [IκBiζ]), was also significantly enriched in the nucleome at 4 hrs (+1.7 Log2 Fold change, 172 adjusted p = 1.25x10-4), followed by a strong increase in nuclear abundance over time (20 h; -2 173 +4.5 Log2 fold change, adjusted p = 3.8x10 ). In addition to its role as an inhibitor, IκBiζ can 174 promote transcription of the siderophore LCN2 23. Consistently, LCN2 expression in the 175 lysatome followed a similar pattern in infected cells (Table S3). These findings highlight the 176 spatiotemporal dynamics of NF-κB signaling during STm infection, whereby early nuclear 177 NF-κB translocation is followed by a second wave of regulation via IκBiζ. 178

179 Moreover, several broader responses occurred at specific time-points, including an early 180 reduction in the abundance of DNA binding proteins Uhrf1, Mki67, Tmpo and Zfp3 in the 181 lysatome (GO:0003677; 4h) (Figure 1B). At later time-points, a decrease in protein involved 182 in the DNA replication fork (GO:0005657; 8h) preceded a decrease in the nucleic acid 183 metabolic process (GO:0090304; 20h), suggesting multi-level remodeling of DNA replication, 184 firstly by suppressing the DNA replication fork machinery, followed by metabolic reduction in 185 DNA building blocks to control the host cell cycle. This may indicate a host response aimed 186 at suppressing pathogen proliferation by limiting vital nucleotide building blocks essential for 187 pathogen survival 24–26. 188 189 In the nucleome, significant enrichment of several nuclear pore proteins was observed by 190 STm infection. Specifically, the nuclear pore scaffold proteins Nup205, Nup188, Nup155 and 191 Nup93 increased at 20 h post infection. This suggests the nuclear pore is remodeled during 192 infection to modify protein trafficking across the nuclear envelope. Furthermore, in 193 secretome samples, lysosomal proteins (GO:0005764) displayed enhanced secretion at 20 194 h post infection (Table S2). The lysosomal trafficking protein Kxd1 was also exported at the 195 same time, suggesting that it may contribute to modifying vacuole transport during STm 196 infection. Finally, similarly to the secretome, we observed an unexpected increase in 197 lysosome components in the nuclear fraction (GO:0005764, Figure 1B), consisting of many 198 lysosomal proteases e.g. Cathepsin A (CtsA), Cathepsin B (CtsB), Cathepsin D (CtsD), 199 Cathepsin L1 (CtsL1), Cathepsin S (CtsS), Cathepsin Z (CtsZ), Legumain (Lgmn). Although 200 cathepsins have previously been observed in the nucleus 27–29, this has not been reported to 201 occur during infection. 202

203 STm infection induces distinct host proteome responses compared to LPS 204 As macrophage-like cells are programmed to respond to MAMPs, we anticipated that many 205 of the observed proteomic-wide responses in our experimental design would be attributable 206 to the highly immunogenic LPS present on the STm cell surface. To test this, we directly 207 compared our lysatome samples from STm infected cells with previously published data 208 from pSILAC-AHA labelled RAW264.7 cells stimulated with LPS 20 (Table S3). The two time 209 points analysed span two distinct phases of the STm intracellular life cycle, namely i) pre- 210 SPI-2 dependent proliferation (3-4 hrs) and ii) active SPI-2 dependent growth (8 hrs). 211 Consistent with our expectations, STm infection and LPS stimulation showed a strong 212 positive correlation at both 4 h (r = 0.65) and 8 h (r = 0.635) (Figure 2A); note that equivalent 213 20h samples of pSILAC-AHA labelled RAW264.7 cells stimulated with LPS were not

214 available. Thus, much of the proteome-response of STm infected cells can be explained by 215 innate immune responses to LPS alone. 216 217 Beyond the overall similarity, this comparison enabled us to identify host responses specific 218 to STm infection by filtering out the LPS response from STm infected samples (Figure 2A & 219 Table S4). We then performed GO term enrichment analysis to identify the STm-infection 220 specific responses. A number of infection-related GO terms, including response to virus 221 (GO:0009615) and defence response (GO:0006952) were significantly enriched at both 222 time-points (Figure 2A & Table S6). The antiviral response includes proteins (Stat1, Oas2, 223 Tlr7, Tllr13, Lgals8, Lgals9, Samhd1 and Eif2ak2), which may provide insights into the 224 molecular players involved in this novel but seemingly universal response towards 225 intracellular bacterial pathogens 22. The remaining defence response, even after normalizing 226 out the LPS response, points to the production of these proteins being significantly higher 227 during STm infection. This could be due to i) relative differences in LPS doses and/or 228 structure between the two conditions (free E. coli O111:B vs. STm bound), ii) the nature of 229 the LPS presentation to the host cell, or more interestingly, iii) a possible STm-dependent 230 amplification of the host defence response. 231 232 Despite the overall innate immune response to LPS and STm being similar, we detected a 233 number of STm-specific deviations. First, there were LPS-induced responses that were 234 silenced by STm, such as lysozyme Lyz2 (Figure 2B & Table S4). STm presumably 235 suppresses this part of the innate immune defence to avoid cell lysis, in line with previous 236 observations 30,31. Second, we detected an even larger number of core cellular processes 237 that were specifically activated during STm-infection. At 8 h, there was a significant 238 upregulation of the apoptotic process (GO:0006915; Figure 2B & Table S6), which contained 239 Caspase-1 (Casp1), Caspase-11 (human ortholog of Caspase-4 32), Caspase-8 (Casp8) and 240 death domain-associated protein (Daxx). Activation of the cell death pathway at 8 h is 241 congruent with the timing of delayed Caspase-11 dependent cell death induced by STm SPI- 242 2 during macrophage infection 7, which initiates ~8-10 hours post infection. Second, the IMP 243 metabolic process, which plays an important role in purine metabolism, was downregulated 244 at 8 h (GO:0046040; Figure 2B & Table S6). This response may hint to an interesting host- 245 microbe antagonism revolving around purines, as their synthesis appears to be a strong 246 limiting factor for STm infection 33,34. Third, we observed down regulation of cell cycle control 247 proteins at 8h (KEGG:04110; Figure 2B & Table S6), which included Bub1b, Cdc20, Cdk6, 248 Mcm2, Mcm4, Mcm5, Mcm6, Mcm7, Rbl1. It has recently been shown that promotion of 249 G1/S transition enhances STm intracellular proliferation, and that the STm effector protein 250 SpvB facilitates G2/M arrest in host cells, which is more conducive to STm intracellular 7

251 proliferation 35. The suppression of host cell pathways important for DNA synthesis and 252 replication may signify a host-driven response to counter STm G2/M arrest. Together, these 253 findings demonstrate modulation of core cellular processes spanning from DNA replication 254 to cell death specifically during STm infection however, the functional significance of many of 255 these proteins will require further study. 256 257 In addition to changes in protein expression, we noticed a considerable number of 258 extracellular proteins being less abundant at 8 h after STm infection (e.g. Lpl, Plau, C3, Olr1, 259 Serpine1, Tjp2) (Figure 2B, Table S4). This suggested a potential STm-specific 260 downregulation of biological processes at the cell surface and extracellular space. To 261 examine this in more detail, we compared the secretome of LPS and STm treated cells 262 (Figure 2C, Table S5). Similar to the lysatome, STm infection and LPS stimulation induced 263 broadly similar protein secretion dynamics over time (Figure 2B, Table S5), although clear 264 discrepancies were apparent. For example, we observed incongruent regulation of the 265 chemokine Osteopontin (Ssp1) with STm infection preventing its secretion and LPS 266 stimulation inducing it (Figure 2B). Previous reports have indicated Osteopontin is 267 detrimental to the host during Pneumococci or viral infection 36,37, and its intracellular form 268 induces the antiviral response by stabilising TRAF3 38. Our findings indicate Osteopontin is 269 specifically regulated during STm infection; its functional relevance in this context remains to 270 be elucidated. 271 272 The most profound difference between the extracellular response to LPS and STm infection 273 was the enhanced secretion of lysosomal hydrolases, such as Cathepsin C (CtsC), CtsL1, 274 CtsD, CtsZ, CtsA and Lgmn, in the latter (Figure 2C). This secretion is specific and is not 275 due to pyroptosis, as this would create a general increase of cytosolic contents in the 276 external milieu. Interestingly, STm was previously shown to enhance the secretion of 277 immature cathepsin D, which was linked to SifA-mediated inhibition of mannose-6- 278 phosphate receptor recycling to the ER, ultimately depriving lysosomes of their hydrolytic 279 enzymes and promoting STm proliferation 17. Our findings suggest that STm-induced 280 lysosomal detoxification is more general than for Cathepsin D alone. 281 282 Lysosomal proteases are enriched in the nuclear fraction upon STm infection. 283 Reminiscent of STm-induced secretion of lysosomal proteases observed in the secretome, 284 we also detected a strong and significant increase in several lysosomal hydrolases at 20 h 285 post-infection in the nucleome (Figure 3A). As the expression levels of these proteins 286 remained relatively unchanged in the lysatome samples (Figure 3B-C), their selective 287 increase in the nucleus is presumably due to their increased trafficking to this compartment. 8

288 We excluded that this is due to introduced artefacts from our partial lysis fractionation 289 method, as our fractionation selectively solubilised lysosomal and not nuclear membranes - 290 corroborated by the lysosomal markers Lamp1 and Lamp2 being reproducibly detected in 291 the lysatome but not nucleome samples (Table S1 & S3). We did however notice that not 292 only lysosomal hydrolases, but also membrane proteins, such as V-type proton ATPase 293 subunits, were also enriched in the nuclear fraction. Comparison of lysosomal lumen 294 proteins and membrane proteins demonstrated two distinct populations within the nuclear 295 fraction, suggesting cathepsin enrichment (lumen proteins) cannot be generally explained by 296 increased levels of lysosomal membrane proteins in the nucleome alone (Figure 3C). These 297 findings strongly suggest that newly synthesised lysosomal proteins, in addition to being 298 secreted, are also trafficked to the nucleus during STm infection. Overall, these findings 299 imply that lysosomal trafficking during infection is more complex than previously appreciated. 300 We hypothesised that this organized re-trafficking of lysosomal proteases during STm 301 infection has a key intracellular role during the infection process and therefore, we set out to 302 examine its significance in more detail. 303 304 STm SPI-2 is required for nuclear cathepsin activity. 305 To examine cathepsin localisation and activity, we added a cell permeable cathepsin 306 reactive probe, DCG04-Bodipy-FLike (DCG04-FL), to live cells during STm infection. Such 307 inhibitor-based probes covalently link to the reactive cysteine in the catalytic site of 308 endolysosomal proteases, thus the amount of bound probe directly corresponds to cathepsin 309 activity, which is visualised via the appended fluorophore on SDS-PAGE or by microscopy 310 39. Endosomal organelles containing active lysosomal cysteine proteases were effectively 311 solubilised with the non-ionic detergent Tx-100, as evidenced by the presence of highly 312 active mature cathepsins in cells treated with the cathepsin probe 488nm DCG04-Bodipy- 313 FLike (Figure 4A left panel), and not in DMSO treated cells, thus demonstrating specificity of 314 the DCG04-Bodipy-FLike probe. Cathepsin activity was elevated in the Tx-100 soluble 315 fraction of infected relative to uninfected cells, particularly for CtsB and CtsZ (Figure 4A left 316 panel). Analysis of nuclear extracts from these same samples exhibited a striking increase in 317 nuclear cathepsin activity upon infection with wildtype STm compared to uninfected (Figure 318 4A, right panel). The high reactivity of the cathepsin probe in nuclear fractions of infected 319 cells demonstrated that the multiple nuclear enriched cathepsins are active. Nuclear 320 cathepsins were also of a higher molecular weight relative to their endolysosomal 321 counterparts (Figure 4A), which are generally known to be processed to more active mature 322 forms 40. However, consistent with previous reports describing higher molecular weight 323 nuclear forms of cathepsin CtsB and CtsL in human and murine cell lines 27,28,41, we found 324 these larger forms to be also proteolytically active 27,41, since our probes bind only active 9

325 enzymes. We eliminated the possibility that this nuclear cathepsin activity was an artefact of 326 cross-reactivity with STm proteins, whereby reactivity to the DCG04-FL probe was detected 327 only in nuclear enriched fractions and not in STm enriched fractions (Figure S2). 328 Interestingly, this increase in nuclear cathepsin activity was not followed by an increase in 329 histone H3 cleavage (Figure S3), which is mediated by nuclear CtsL during stem cell 330 differentiation 42. Thus, newly synthesised cathepsins are re-trafficked to the nucleus during 331 STm infection, yielding active enzymes of higher molecular weight compared to their 332 endolysosomal counterparts. 333 334 As the SPI-2 secretion system of STm has been implicated in modifying cellular trafficking of 335 lysosomal contents 17, and nuclear cathepsin activity coincides with SPI-2 dependent 336 proliferation (Figure S2), we examined whether SPI-2 secretion is required for nuclear 337 cathepsin activity. Cathepsin activity in Tx-100 soluble lysates was comparable between 338 RAW264.7 cells infected with wildtype, SPI-1 (ΔprgK) or SPI-2 (ΔssaV) secretion system 339 mutants (Figure 4B left panel). In contrast, nuclear cathepsin activity was reduced in cells 340 infected with the SPI-2 mutant, but not the SPI-1 mutant infected cells (Figure 4B right 341 panel). Thus, SPI-2 does not only control export of lysosomal contents to the extracellular 342 space 17, but also nuclear cathepsin re-trafficking and activation. 343 344 To ensure our observations of SPI-2 dependent nuclear cathepsin activity by SDS-PAGE 345 were not artefacts of biochemical fractionation or reduced bacterial load in the SPI-2 mutant, 346 we used fluorescence microscopy to quantify in situ nuclear cathepsin activity from single 347 cells (Figure 4C-D, Figure S4). After live cell labeling with the cathepsin probe DCG04- 348 Boclicky-TAMRA, cathepsin activity overlaying with nuclear DNA was quantified in 349 uninfected bystanders and infected cells containing similar loads of wildtype or SPI-2 mutant 350 STm. Increased nuclear cathepsin activity was observed in wildtype infected cells relative to 351 uninfected bystanders (unpaired Wilcoxon rank sum, p = 7.86e-11) (Figure 4D). 352 Furthermore, nuclear cathepsin activity in cells infected with the SPI-2 deficient mutant was 353 significantly lower compared to wildtype-infected cells (unpaired Wilcoxon rank sum, p = 354 7.2e-3). Finally, confocal microscopy demonstrated increased cathepsin activity inside the 355 nucleus (i.e. cathepsin activity within the nuclear boundary defined by Hoechst stained host 356 nuclei) and as well as in the perinuclear region of wildtype-infected cells, but not uninfected 357 bystanders, uninfected naïve or those infected with a SPI-2 deficient mutant (Figure 4E). 358 Cells displaying elevated nuclear cathepsin activity also exhibited a strong increase in 359 cathepsin activity throughout the cell body. Thus, STm SPI-2 elevates cathepsin activity 360 throughout the cell, as well as driving it towards the nucleus during infection. 361 10

362 Nuclear cathepsin activity correlates with signatures of cell death 363 To further examine the consequences of nuclear cathepsin activity, we extracted nuclei from 364 cells treated with the cathepsin probe DCG04-Boclicky-TAMRA and analysed by flow 365 cytometry. In uninfected cells, 0.47 % of nuclei were positive for cathepsin activity, in 366 contrast to 5.6 % of nuclei from cells exposed to wildtype STm and 3.6 % exposed to the 367 ssaV mutant (Figure 5A). This suggested that only a small fraction of cells undergo nuclear 368 cathepsin delivery at any given time and this partially depends on the SPI-2 secretion 369 system. Interestingly, a substantial fraction of the cathepsin positive nuclei from only the 370 wildtype infected cells were detected in the sub-nuclear region of the FACS plot (Figure 5A, 371 red box), indicative of nuclear DNA fragmentation and cell death. This implied that nuclear 372 cathepsin activity correlates with cell death, which prompted us to examine further the role of 373 cathepsin activity in cell death. 374 375 To determine the role of specific cathepsins during STm induced cell death, we used the 376 widely reported cathepsin B and L specific inhibitor, CA-074-Me, and measured LDH release 377 as a more direct readout of delayed pyroptotic cell death, which can be effectively modelled 378 in RAW264.7 cells 43. We infected RAW264.7 cells with wildtype STm, SPI-2 deficient 379 mutant and the effector mutant ΔsifA, which has a strong replication defect and readily 380 escapes into the cytoplasm, promoting Caspase-11 dependent cell death 44 (Figure 5B). 381 Both wildtype STm and the sifA mutant induced considerable levels of cell death after 19 382 hours of infection (the latter exhibiting higher cell death, as expected). In wildtype-infected 383 cells, cell death was partially inhibited by the addition of CA-074-Me in a dose dependent 384 manner. As expected, cell death was largely dependent on the expression of a functional 385 SPI-2 (ΔssaV; Figure 5B). Unexpectedly, the elevated cell death induced by a sifA mutant 386 was largely insensitive to inhibition by CA-074-Me. These findings suggest that cathepsin 387 dependent cell death requires an active SPI-2, and intravacuolar STm. 388 389 Cathepsin activity is required for STm induced Caspase-11 dependent cell death 390 To further understand the host molecules at play, and to ensure our observations were 391 generalisable to cells other than RAW264.7 cells which lack key inflammasome components 392 45, we sought to replicate our findings using BMDMs from rodent knockout lines. Consistent 393 to previous reports 7,46, LDH release started ~10 h post-infection (Figure 6A). This LDH 394 release was dose-dependently suppressed with the cathepsin B/L specific inhibitor CA-074- 395 Me (Figure 6A), similarly to our observations in RAW264.7 cells. 396 397 Delayed lytic cell death of STm infected macrophages depends on the cytoplasmic LPS 398 sensor Caspase-11 3. We therefore tested whether cathepsin dependent cell death requires

399 the presence of caspase-11. Cell death was low in cells lacking Caspase-11, and cathepsin 400 inhibitor addition did not change this, demonstrating the critical importance of Caspase-11 in 401 mediating cathepsin-dependent cell death during STm infection (Figure 6B). Cell death 402 occurred to a large degree independently of the inflammasome activators NLRP3 and 403 NLRC4, and the cathepsin inhibitor also blocked cell death in these mutants (Figure 6B). 404 405 Cathepsin inhibitors may rescue Caspase-11-mediated cell death because they inhibit the 406 activity of cathepsins or because they inhibit STm growth inside macrophages. To exclude 407 the latter scenario, we grew STm in the presence of E-64 and CA-074-Me and observed no 408 effect on STm growth (Figure S5). Furthermore, we hypothesised that if the suppression of 409 STm-induced host cell death by cathepsin inhibition can be explained by cathepsin inhibition 410 in the SCV and thereby SPI-2 activation, then the addition of cathepsin inhibitors subsequent 411 to the initiation of SPI-2-dependent replication should largely abrogate their rescuing effect 412 on host cell death. However, addition of inhibitor at 8 h post infection rescued cathepsin- 413 dependent cell death induced by STm in BMDMs equally well as addition of the inhibitor 414 from the beginning of infection (Figure 6C). Therefore, it is the late cathepsin activity that 415 promotes the Caspase-11-mediated cell death. Since cathepsins relocate to the nucleus or

416 the extracellular milieu at this time point, it is possible that these activities trigger cell death.

417 DISCUSSION 418 We selectively enriched and quantified the host pool of newly synthesised proteins, enabling 419 unprecedented proteome-wide spatiotemporal resolution of a host-pathogen interaction. This 420 dataset provides a rich resource for infection biology, and could serve as a basis for a 421 plethora of hypotheses. We used it to reveal a novel role for cathepsins in activating STm- 422 induced pyroptosis via the non-canonical inflammasome. This proof-of-principle example 423 illustrates how the selective quantification of the newly synthesised host proteome within 424 different cellular compartments can illuminate mechanisms that would otherwise remain 425 hidden using conventional proteomic approaches. 426 427 The host proteome during STm infection has been mapped before: whole proteome analysis 428 during infection of RAW264.7 macrophages (at the time, the authors could identify in total 429 ~1,000 host proteins) 47, and mapping the secretome of human monocytes (THP-1) during 430 early stages of infection - before intracellular SPI-2-dependent proliferation 48. In contrast to 431 prior studies, our approach has three clear advantages: i) we monitor dynamic host 432 proteome responses, collecting samples at different phases of infection and quantifying the 433 newly synthesized proteins at each stage; ii) we use subcellular fractionation and pSILAC- 434 AHA to assess protein redistribution between cellular compartments, quantifying 4978 newly 435 synthesized host proteins in total from the cytoplasm/organelles, the nucleus and the 436 extracellular milieu (lysatome, nucleome and secretome); and iii) we deconvolute the STm- 437 specific response by directly comparing our data with similarly acquired data on the 438 macrophage response to LPS 20. To our knowledge, this level of comprehensive proteomic 439 analysis has never been performed before for any host-pathogen interface. 440 441 The benefits of the different angles of our approach are reflected by the multiple facets of 442 infection biology that each can uncover. First, although a large fraction of the host proteome 443 response to STm is due to LPS, there is distinct STm specific proteome regulation compared 444 to LPS stimulation. For example, proteins typically associated with antiviral defense 445 including Tmem173, Oas2, Stat2 and Tlr7 shown distinct regulation during STm infection 446 compared to LPS stimulation. Induction of Oas2 expression in cells infected with 447 Mycobacterium leprae promotes intracellular bacterial survival 49. Tlr7 recognises RNA 448 within bacterial containing vacuoles 50, and has previously been implicated in the recognition 449 of STm in BMDMs 51. The functional relevance of antiviral proteins during STm infection 450 remains to be elucidated 49. Second, through temporal sampling, we detected the increased 451 abundance of proteins relevant for host surveillance of symbiont-containing vacuolar 452 membrane (GO:0020005) in the lysatome at both 8 and 20 hours post infection i.e. Gbp2,

453 Gbp2b, Gbp6 and Gbp7. Furthermore, we detected down-regulation of the nucleic acid 454 metabolic process (GO:0090304) only at 20 hours post infection, which consisted of 149 455 proteins. Third, by monitoring the subcellular proteome, we found a nuclear enrichment of 456 NF-κB at early stages of infection, which was followed by nuclear re-localization of the NF- 457 κB inhibitor IκBi-ζ. This suggests that blunting of the NF-κB signaling response after LPS 458 stimulation 52 may be mediated by nuclear enrichment of IκBiζ. In addition, Spp1 secretion 459 was reduced during STm infection and instead increased inside host cells. As intracellular 460 Spp1 plays an important role in immune modulation 53 including the antiviral response 38, and 461 Spp1 mRNA is elevated in STm infected cells 54, we hypothesise that Spp1 plays an as yet 462 undefined role during STm infection. Last, we detected an increase in lysosomal proteases 463 in the secretome and the nuclear fraction during the later stages of STm infection. This 464 unexpected observation led us to unravel a novel functional role for cathepsins during STm 465 induced pyroptosis. 466 467 Our findings that lysosomal proteases are rewired in their cellular trafficking during STm 468 infection builds on a large body of evidence implicating altered trafficking of lysosomal 469 proteases during STm infection. It is now well appreciated that the SCV contains reduced 470 levels of vacuolar hydrolases, including cathepsins, likely due to fusion of the SCV with 471 lysosomes whose hydrolytic activity has been deactivated by the STm effector protein SifA 472 17. Consistent with our observations in RAW264.7 cells, STm infection of HeLa cells resulted 473 in enhanced CtsD secretion in a SPI-2 dependent manner 17. In addition to CtsD, we 474 detected eight additional lysosomal proteases (i.e. CtsC, CtsB, CtsS, CtsL1, CtsZ, CtsA, 475 Psap and Lgmn) showing elevated secretion during infection (Figure 2C). This implies that 476 STm induced rewiring of cathepsin trafficking is a more general phenomenon, not specific to 477 CtsD. Furthermore, recent observations that STm elevates vacuolar pH in both infected and 478 bystander cells 55, and given that secretion of CtsD pro-forms play an active role in paracrine 479 signaling 56, it is plausible that cathepsin secretion induced by STm infection may affect 480 lysosomal function in uninfected bystander cells. 481 482 In addition to cathepsin secretion, we noticed a pronounced enrichment for lysosomal 483 proteases in the nucleus during STm infection. Although cathepsins and lysosomal 484 hydrolases have previously been shown to be targeted to the nucleus during cellular stress, 485 in cancer cells, or different phases of the cell cycle 28,29,41,42,57–60, to our knowledge this is the 486 first report of active cathepsin delivery to the nucleus during infection. Despite the 487 considerable overlap in the lysosomal proteases enriched in both the secretome and the 488 nucleome upon infection, CtsB and CtsS displayed pronounced enrichment only in the 489 nucleus. This distinct cathepsin enrichment profile between these two cellular compartments 14

490 suggests that STm-induced changes to subcellular cathepsin targeting are not identical for 491 nuclear and extracellular targeted proteins. 492 493 Analogous to the previously reported SPI-2 dependent cathepsin secretion 17, cathepsin 494 delivery to the nucleus required SPI-2. Furthermore, nuclear cathepsins were of higher 495 molecular weight compared to lysosomal forms found in the Tx-100 soluble fraction, akin to 496 previously reported SPI-2 dependent CtsD secretion 17. However in this instance, high 497 molecular weight nuclear cathepsins are active. Nuclear cathepsins often exist as higher 498 molecular weight pro-forms 28,29,41,57 and have also been reported to be active in thyroid 499 carcinoma cells and during the S-phase of the cell cycle 27,41. This suggests a non-canonical 500 form of protein trafficking delivers cathepsins to the nucleus during STm infection, possibly 501 as a result of alternate splicing events leading to signal-peptide-devoid N-termini as 502 previously observed for CtsL and CtsB 27,61, followed by a subsequent pH-independent 503 maturation and/or activation (currently, cathepsin S is the only known example that remains 504 as active at neutral pH 62). We can exclude that the nuclear cathepsins are simply proteins 505 derived from ruptured vacuoles, as these are newly synthesised proteins and have not 506 undergone maturation in the low pH of a lysosome. Taken together, nuclear cathepsins 507 represent a branch of cathepsin trafficking distinct from those destined for the 508 endosome/lysosomal compartment however, the precise mechanism for nuclear cathepsin 509 targeting and activation during infection will require further investigation. 510 511 Once in the nucleus, cathepsins have been shown to induce transcriptional changes through 512 the cleavage of proteinaceous DNA regulatory elements 29,42. The most extensively 513 characterised nuclear cathepsins are CtsL and CtsB. Once in the nucleus, CtsL cleaves the 514 N-terminal tail of histone 3 (H3), and is thought to play a role in cellular differentiation by 515 altering the transcriptional landscape 42. This specific mechanism is unlikely to occur in our 516 system, as we detected less CtsL specific histone cleavage products 42 (Fig S3). Instead, 517 parallels exist between the here observed nuclear cathepsin activity and the mechanisms 518 underlying Neutrophil Extracellular Traps (NETs) production. Nuclear targeting of the 519 lysozyme-derived serine protease neutrophil elastase (NE) promotes partial degradation of 520 several histones, including H2B and H4, and thereby induces chromatin condensation and 521 NET production 63. Reminiscent of this, nuclear cathepsins increased at 20 h post infection, 522 at which point H4 and H3 reduced in abundance (Figure 3A). In addition, we also saw 523 reduced Mki67 levels, which is a commonly used cell proliferation marker, recently shown to 524 play an important role in dispersal of mitotic chromosomes 64. This reduction in nuclear 525 proteins critical for maintaining chromatin structure co-occurs with nuclear cathepsin activity,

526 providing a link between these proteins. Whether histone H3/H4 and Mki67 are direct 527 substrates of nuclear cathepsin remains to be tested. 528 529 Consistent with previous reports, we observe that pyroptotic cell death induced by SPI-1 530 OFF STm is strongly Caspase-11 dependent 7,44. At this point we are unable to conclude 531 whether cathepsin activity lies upstream or downstream of Caspase-11 in promoting cell 532 death. Cathepsins play an undisputed role in promoting cell death 65. For example, 533 Cathepsin B can directly cleave inflammatory Caspases-1/11 66,67. Cathepsins, in particular 534 CtsB and CtsC, mediate sterile forms of cell death via lysosomal destabilisation 9,13,14,68, or 535 with purified bacterial components such as flagellin 69. Some of these cathepsin-dependent 536 effects are mediated by NLRP3 9 and NLRC4 69, whereas others are NLRP3 independent 13. 537 We show that CA-074-Me mediated cathepsin inhibition during STm infection suppresses 538 cell death via Caspase-11, but independently of NLRP3 and NLRC4. This is consistent with 539 reports describing that Caspase-11 dependent cell death occurs independently of NLRP3 540 and NLRC4 during bacterial infection 44. However, it remains plausible that cathepsins might 541 still function via both NLRP3 and NLRC4, which would remain undetected in our assays due 542 to their functional redundancy 6. 543 544 Additional support for nuclear cathepsins playing an active role in cell death comes from the 545 observation that CtsB is delivered to the nucleus during bile salt-induced apoptosis, whereby 546 addition of the CtsB/L inhibitor CA-074-Me, or silencing ctsB expression, abrogates cell 547 death 70. Cathepsin B can also induce nuclear apoptosis in digitonin-permeabilized cells 67. 548 Moreover, deletion of the endogenous cathepsin inhibitor Stefin-B, which interacts with CtsL 549 in the nucleus 58,71, leads to enhanced Caspase-11 transcription in BMDMs 72. Albeit in the 550 absence of infection, these findings provide strong support that nuclear cathepsins play an 551 active role during cell death. Consistently, we observe increased nuclear localization of 552 CtsA, CtsB, CtsL1, CtsS ,CtsD, CtsZ upon STm infection, which correlates with signs of cell 553 death, such as DNA fragmentation. Furthermore, we could inhibit pyroptosis by adding CA- 554 074-Me at 8 h after infection when cathepsin activity is present in the nucleus, consistent 555 with nuclear cathepsin activity playing a functional role in STm-induced cell death. At this 556 stage we cannot rule out that endosomal cathepsin activity or cathepsin leakage into the 557 cytosol may also play an active role in STm-induced cell death, as the available cathepsin 558 inhibitors are not cell compartment-selective. Vacuolar cathepsin leakage into the cytosol 559 could directly cleave Caspase-11 66 and/or GSDMD and lead to pyroptosis as seen 560 previously in neutrophils 73. However, in our case the cathepsin inhibitors were less able to 561 rescue cell death in an STm sifA mutant, in which SCVs rupture more readily, releasing 562 cathepsins into the cytosol. 16

563 564 In summary, we used a novel proteome-wide approach to selectively enrich and quantify the 565 newly synthesised host proteome during infection with an intracellular bacterial pathogen, 566 unmasking a hidden layer of the mammalian innate immune response. This rich tapestry of 567 regulated proteins, resolved throughout time and space offers a proteome-wide resource 568 that can now be used to formulate new hypotheses governing host-pathogen interactions. 569 Here, we provide a proof-of-principle by further interrogating the re-trafficking of cathepsins 570 outside of the cell and into the nucleus during STm infection. This highlights a new role for 571 cathepsins during infection, which actively participate in STm-induced cell death via 572 Caspase-11.

573 ACKNOWLEDGEMENTS 574 We thank Dr. Malte Paulsen and Dr. Diana Ordonez from the EMBL Flow Cytometry core 575 facility for performing and analysing flow cytometry experiments; members of the Proteomics 576 Core Facility (PCF), especially Mandy Rettel and Per Haberkant for helping in sample 577 preparation, data acquisition and analysis; and the EMBL ALMF Core Facility for providing 578 expertise in image acquisition and data analysis using CellProfiler. We also thank Dr. Sophie 579 Helaine (Imperial College) for the pDiGc plasmid; Omar Wagih (EBI) for the R script to 580 generate scatter plots; and Kyung-Min Noh (EMBL) for the H3.cs1 antibody. We are grateful 581 to all members of the Typas and Krijgsveld laboratories for their valuable discussions. We 582 acknowledge funding from EMBL for this research. JS, NL and HI were supported by 583 fellowships from the EMBL Interdisciplinary Postdoc (EIPOD) programme under Marie 584 Skłodowska-Curie Actions COFUND (grant number 291772). 585

586 AUTHOR CONTRIBUTIONS 587 Conceptualization: JS, JK and AT; Investigation: JS, NL, GS (proteomics), JS, JB, AS 588 (biochemistry and cell biology) and AS (bacterial growth curves); Resources: BIF, HSO 589 (cathepsin reactive probes) and AH, WDH (BMDM methodology); Software: JS, BED 590 (CellProfiler & R); Data Analysis: JS, NL, HI (proteomics), JS, BED (CellProfiler); Writing: JS 591 and AT with input from NL, JB, AH, AS, HI, GS, PB, WDH, PB, and JK; Figures: JS 592 (schematics, proteomic data, follow up cell biology), AS (bacterial growth curves); 593 Supervision: AT, JK and PB; Funding Acquisition: AT and JK. 594 595 DATA AVAILABILITY 596 Proteomic data has been deposited at ProteomeXchange 597 (http://www.proteomexchange.org/) under the identifier PXD010179. 598 599 CODE AVAILABILITY

600 The code and pipelines used for data analysis are available upon request. 601

602 DECLARATION OF INTEREST

603 The authors declare no competing interests.

604 FIGURE LEGENDS 605 Figure 1. Newly synthesised proteome enrichment detects diverse host responses 606 during STm infection. 607 (A) Experimental design of pSILAC-AHA labelling and subcellular fractionation. (1) 608 RAW264.7 cells were infected with STm 14028s grown to stationary phase at a MOI 100:1, 609 followed by centrifugation to facilitate host-pathogen contact. After a 25 min incubation, 610 extracellular STm were killed by moving cells first to media containing 100 µg/ml gentamicin 611 for 1 h, and then to media containing 16 µg/ml gentamicin for the remainder of the 612 experiment (Materials and Methods). Uninfected samples received a mock inoculum and 613 were otherwise treated identically to infected samples. (2) 3.5 hrs prior to harvest, cells were 614 washed and briefly starved for 30 minutes to remove residual amino acids, followed by 615 pSILAC-AHA labelling for 3 hours. (3) Conditioned media was harvested for the secretome 616 samples, whereas cells were washed and partially lysed with a mild detergent (Triton X-100) 617 to extract both the lysatome and nuclear samples. (4) Samples were mixed at 1:1 ratio 618 (infected: uninfected) and then fractionated by centrifugation to separate the secretome, 619 nucleome and lysatome. (5) Newly synthesised proteins containing AHA were covalently 620 linked to alkyne agarose beads via a Click-chemistry reaction. (6) After on-bead protease 621 digestion, peptides were quantified by LC-MS/MS. (I) and (H) refer to intermediate and 622 heavy isotopic amino acids respectively. 623 (B) GO term enrichment of differentially regulated host proteins (4, 8 and 20 h). Selected 624 enriched GO terms are depicted; all enriched GO terms can be found in Table S2. Node size

625 and colours depict the significance (p (-Log10) = right-sided hypergeometric test, Bonferroni 626 corrected) and number of proteins (blue shade), respectively. 627 628 Figure 2. STm infection induces distinct host proteome responses compared to LPS 629 across time and space. 630 (A) Upper panel, scatter plot of pSILAC-AHA lysatome data previously collected in response 631 to LPS stimulation (LPS was from Escherichia coli O111:B) 20 vs data collected after STm 632 infection from this study (Table S3). N.B. Due to differences in sample collection, the LPS 633 samples contain both the lysatome and nucleome fractions. Lower panel, histogram 634 containing protein expression from lysatome of RAW264.7 cells infected with STm for 4 hrs 635 after subtracting the corresponding LPS signal. Dotted blue lines in histogram indicate ± 2 636 S.D. from the mean and the dotted red line on inset GO term histograms indicates multiple- 637 test adjusted p value (Bonferroni corrected) cutoff of 0.05.

638 (B) Same as A but at later time point: 8 hrs after STm infection or LPS stimulation of 639 RAW264.7. Corresponding protein levels for both panels (A) and (B) in Table S4 and GO 640 terms in Table S6. 641 (C) Heatmap of secreted proteins from pSILAC-AHA labelled RAW264.7 cells after STm 642 infection or LPS stimulation, the latter data obtained previously 20. N.B. Only rows without 643 missing data points were used for analysis. To the right is a STRING network of vacuolar 644 proteins with increased secretion dynamics upon STm infection relative to LPS. Nodes 645 represent individual proteins and edges reflect experimentally determined interactions and 646 co-expression weighted data from STRING v10.5 74. 647 648 Figure 3. Lysosomal proteases are enriched in the nuclear fraction upon STm 649 infection. 650 (A) Histogram of nuclear proteins differentially regulated upon STm infection at 20 h, 651 lysosomal hydrolases are annotated in red. Blue and red dotted lines, as well as GO cellular 652 component enrichments (inset) as in Fig. 2a. 653 (B) Scatter plot of lysatome and nucleome data collected from RAW264.7 cells at 20 h post- 654 infection. Distributions of all proteins found in the lysatome (x-axis) and nuclear fraction (y- 655 axis) (blue), and lysosomal hydrolases (red). Black dotted line represents the line of best fit. 656 p = (unpaired Wilcoxon rank sum test). 657 (C) Boxplots displaying the relative fold change (infected/uninfected) of membrane bound 658 lysosomal vs soluble lysosomal luminal proteins from the lysatome and nucleome samples. 659 Box boundaries indicate the upper and lower IQR, the median is depicted by the middle 660 boundary and whiskers represent 1.5x IQR (see Table S7). 661 662 Figure 4. STm SPI-2 is required for nuclear cathepsin activity. 663 (A) Uninfected and wildtype STm-infected RAW264.7 cells (MOI = 100:1) were treated with 664 the cathepsin probe DCG04-Bodipy-FLike (5 µM), or DMSO vehicle for 3 h prior to 665 harvesting at 20 h post-infection. Tx-100 soluble (left) and insoluble (right) extracts were 666 separated by SDS-PAGE and then visualised using a fluorescence scanner (Ex 405 nm/Em 667 520 nm). Immunoblot with anti-GAPDH and coomassie staining of histones were used as 668 loading controls for lysatome and nucleome samples respectively. Data is representative of 669 two biological replicates (all blots shown in supplement). 670 (B) RAW264.7 cells infected with wildtype, ΔssaV (SPI-2) or ΔprgK (SPI-1) STm were 671 treated with the cathepsin probe DCG04-Bodipy-FLike (5 µM) and lysatome (left) and 672 nucleome (right) extracts were separated and visualised as described in A. Loading controls 673 as in A. Data is representative of two biological replicates (all blots shown in supplement).

674 (C) The cathepsin activity probe DCG04-Boclicky-TAMRA (5µM) was added to the media 2 675 h prior harvesting at 2 (upper) and 20 (lower) hours post-infection (MOI = 100:1) with STm 676 constitutively expressing GFP from pDiGc (see Methods). Cells were then fixed and stained 677 with Hoechst 33342 (DNA) and images acquired using a 20x objective. 678 (D) Violin plots of single cell nuclear cathepsin activity quantified in RAW264.7 cells infected 679 with wildtype or ΔssaV (SPI-2) STm constitutively expressing GFP from pDiGc (MOI = 680 100:1) for 20 h. Infected cells were treated with the cathepsin probe DCG04-Boclicky- 681 TAMRA 2 h prior to cell fixation. Nuclear cathepsin activity was measured by quantifying 682 DCG04-Boclicky-TAMRA signal that overlapped with Hoechst 33342 stained nuclei. Each 683 data point represents a measurement per cell nuclei, which was further classified as infected 684 or uninfected bystander based on the presence or absence of GFP expressing STm located 685 inside the host cell perimeter, respectively. Nuclear cathepsin activity was normalised to 686 nuclei area. Only infected cells containing similar bacterial loads per cell were compared. 687 Red bar = median. An unpaired Wilcoxon rank sum test was used to calculate p. A replicate 688 experiment yielded similar results (Fig S4). 689 (E) Cathepsin activity in fixed cells, prepared as in C), were visualised by confocal 690 microscopy. Depicted cells are representative of high and low nuclear cathepsin activity 691 distributions in D. Cells with high nuclear and perinuclear cathepsin activity are more readily 692 observed in cells infected with wildtype-STm compared to those infected with ΔssaV (SPI-2) 693 mutants, uninfected bystanders, and naïve cells from control wells not exposed to bacteria. 694 Scale bars represents 2 µm.

695 Figure 5. Nuclear cathepsin activity correlates with signatures of cell death. 696 (A) Nuclei were extracted from RAW264.7 cells treated with DCG04-Boclicky-TAMRA (5 µM) 697 for 2 h prior to harvest at 20 h post infection (MOI = 100:1), and were subsequently 698 counterstained with Hoechst 33342 and analysed by flow cytometry. Cells in G1, S or G2 699 phase of the cell cycle are separated by Hoechst 33342 staining on the x-axis (upper). 700 Cathepsin activity (DCG04-Boclicky-TAMRA) per nuclei is shown on the lower panel where 701 the value refers to the % of total DCG04-Boclicky-TAMRA positive nuclei in either sub-G1 or 702 G1/S/G2, red and black boxes, respectively. 703 (B) RAW264.7 cells were infected with STm 14028s (MOI 100:1) for 19 h. Pyroptotic cell 704 death was assessed by quantifying LDH release into culture supernatants of cells infected 705 with wildtype, SPI-2 (ΔssaV) and the effector deletion (ΔsifA) mutants in the presence or 706 absence of CA-074-Me (12.5 and 25 µM) or DMSO solvent control. Data is from two 707 independent experiments; each containing 8, 4 and 4 biological replicate wells infected with 708 wildtype, ΔssaV and ΔsifA mutants, respectively, per condition. Data represents the % LDH

709 release per condition relative maximum LDH release (see Methods). Box plots are depicted 710 as in Fig. 3C. An unpaired t-test was used to calculate p. 711 712 Figure 6. Cathepsin activity is required for STm induced Caspase-11 dependent cell 713 death. 714 (A) BMDMs were infected with wildtype STm (MOI 100:1), followed by incubation in the 715 presence of cathepsin inhibitor CA-074-Me at the indicated concentrations. At the indicated 716 times post-infection, cell death was measured as the % of LDH released into culture 717 supernatants. Data points represent the mean and error bars indicate the 95% CI. Time 718 points 0-14 are derived from three biological replicates per condition, whereas the 18 hour 719 time point contains combined data from 3 or more independent experiments, each 720 containing 3-4 biological replicates per condition. 721 (B) related to A), wildtype, caspase-11/1 -/-, caspase-11 -/-, NLRP3 -/- , NLRC4 -/- BMDMs 722 were infected with wildtype STm (MOI 100:1), followed by incubation in the presence of 723 cathepsin inhibitor CA-074-Me (12.5 µM) for 18 h. The % LDH released into culture 724 supernatants was measured 18 h post-infection. n denotes the combined data from 10 (wild 725 type) and 3 (mutant genotypes) independent experiments, each containing 3-4 biological 726 replicates per condition. 727 (C) BMDMs infected with wildtype STm (MOI = 100:1) were treated with CA-074-Me (12.5 or 728 25 µM) or DMSO control at 1 or 9 h post infection. Box plots are depicted as in Fig. 3C. An 729 unpaired t-test was used to calculate p. n denotes the combined data from 3 independent 730 experiments, each containing 3-4 biological replicates per condition.

731 Supplementary Figure S1. Increasing intracellular STm load over time co-occurs with 732 dynamic proteome changes. 733 (A) RAW264.7 cells infected with STm constitutively expressing GFP (pDiGc) at MOI 100:1 734 and incubated for the indicated times were analysed by CellProfiler. Images were captured 735 at 20x objective and analysis was conducted with CellProfiler to segment infected from 736 uninfected cells and quantify bacterial load per cell based on GFP fluorescence per infected 737 cell. CellProfiler quantification of bacterial count per infected cell (only infected cells) are 738 displayed as a histogram and shows increasing bacterial load with time. Data contains 739 combined counts from two biological replicate experiments, whereby each replicate received 740 reverse SILAC labels (see Methods). 2-5 fields of view (technical replicates) were acquired 741 per biological replicate and per time point. The combined total of host cells quantified is 742 indicated (n=). The dotted red line indicates distribution median. Representative images of 743 quantified cells are displayed to the right of histograms. Scale bars represent 50 µm. The % 744 of infected cells per fields of view (FOV) at the beginning of the experiment (i.e. immediately 22

745 post gentamicin 100 µg/ml treatment, t=0) is displayed as a boxplot on the far right. Box 746 plots are depicted as in Fig. 3C. 747 (B) Replicate correlation of infected vs. uninfected samples for the indicated time points and 748 different subcellular fractions - cytoplasmic and solubilised organelles within the Tx-100 749 soluble fraction (lysatome; upper), nuclear enriched fraction in the Tx-100 insoluble fraction 750 (nucleome-middle) and proteins secreted into the culture supernatant (secretome-lower). 751 Two biological replicates containing reversed SILAC labels were obtained for each cell 752 fraction and each time point (See Table S1).

753 Supplementary Figure S2. Nuclear cathepsin activity is observed within the first 8 754 hours of infection. Time course of nuclear cathepsin activity. Nuclei and STm were 755 enriched by sequential 50 x g and 8,000x g centrifugation steps, respectively. RAW264.7 756 cells were infected with wild type STm 14028s, and treated with DCG04-Bodipy-FLike (5µM) 757 for 4 hours prior to harvesting. Samples were separated by SDS-PAGE and visualised using 758 a fluorescent scanner (Ex 405 nm/Em 520 nm), followed by immunoblotting for the 759 lysosomal associated membrane protein LAMP1, the soluble cytoplasmic protein GAPDH 760 and Coomassie stained for histones as a loading control for nuclear extracts. L = lysatome, 761 N = nucleome, STm = STm enriched fraction.

762 Supplementary Figure S3. CtsL specific Histone 3 cleavage product is decreased by 763 STm infection. RAW264.7 cells were infected with STm at MOI 100:1 and harvested at 20 h 764 post infection. Whole cell lysates were immunoblotted with the CtsL specific cleavage 765 product of H3 (H3.cs1)42 and histone 3 (H3) as loading control. The experiment was 766 performed in biological duplicate per condition and were analysed in adjacent lanes.

767 Supplementary Figure S4. Nuclear cathepsin activity requires SPI-2. Replicate 768 experiment of Figure 4D. The cathepsin activity probe DCG04-Boclicky-TAMRA (5µM) was 769 added to the media 2 h prior to harvesting at 20 h hours post-infection (MOI = 100:1). Cells 770 were fixed and stained with Hoechst 33342 (DNA). Four fields of view per biological triplicate 771 well were acquired per condition and analysed using CellProfiler as per Fig 4D. Images were 772 acquired using a 10x objective. An unpaired Wilcoxon rank sum test was used to calculate p.

773 Supplementary Figure S5. STm growth is unaffected by cathepsin inhibitors in batch 774 culture conditions. 775 (A) STm 14028s growth was measured in the presence of the pan-cathepsin inhibitor E-64 776 in LB at 37 °C. STm was grown in the presence of E-64 5, 10 and 15 µM and DMSO solvent 777 controls. 23

778 (B) STm 14028s growth was measured in the presence of the selective cathepsin inhibitor 779 CA-074-Me in LB 6, 12.5 and 25 µM and DMSO solvent controls. Drug concentrations used 780 are indicated. Both A and B were performed in biological triplicate.

781 Supplemental information 782 Figure S1. Increasing intracellular STm load over time co-occurs with dynamic proteome 783 changes 784 Figure S2. Nuclear cathepsin activity time course of RAW264.7 infected cells. 785 Figure S3. CtsL specific Histone 3 cleavage product is not increased by STm infection 786 Figure S4. Nuclear cathepsin activity, repeat of Fig 4C. 787 Figure S5. STm growth is not inhibited by cathepsin inhibitors in batch culture conditions 788 Table S1. Replicate data of reversed pSILAC-AHA labelled RAW264.7 cells during STm 789 infection. 790 Table S2. GO term enrichment analysis of proteome changes during STm infection of 791 RAW264.7 cells. 792 Table S3. Merged pSILAC-AHA proteomic data from cells stimulated with LPS or STm 793 infection. 794 Table S4. STm infected Lysatome pSILAC-AHA labeled RAW264.7 cell data after 795 subtraction of LPS signal from Eichelbaum & Krijgsveld 2014b. 796 Table S5. STm infected Secretome pSILAC-AHA labeled RAW264.7 cell data after 797 subtraction of LPS signal from Eichelbaum & Krijgsveld 2014b. 798 Table S6. GO term enrichment of STm infected Lysatome, Nucleome and Secretome 799 pSILAC-AHA data data after subtraction of LPS signal from Eichelbaum & Krijgsveld 2014b. 800 Table S7. Luminal and membrane bound lysosomal proteins.

801 METHODS 802 No statistical methods were used to predetermine sample size.

803 Media, chemicals and reagents

804 The following chemical and reagents used were purchased from Sigma: E64 (cat. 3132), 805 DMSO (cat. D8418), L-methionine, L-cysteine, L-lysine, L-arginine L-glutamine. 806 Tris(carboxyethyl)phosphine (cat. C4706) and 40 mM 2-Chloroacetamide (cat. 22790), 807 Triton X-100 (x100), heat inactivated Fetal Bovine Serum (FBS) (F9665-500ML), Phalloidin- 808 ATTO 700 (79286-10NMOL), gentamicin (G1914), Gibco; DMEM 4.5 g/L glucose (41965), 809 DMEM 4.5 g/L glucose non GMP formulation ME 100073L1 (without L-lysine HCl and L- 810 arginine HCl), dialysed FBS (26400044), RPMI 1640 without (11835-030) & with (52400- 811 025) phenol red, respectively. DMEM containing high glucose, HEPES buffered and without 812 phenol was purchased from Thermo Fisher (21063029). Cathepsin inhibitor CA-074-Me was 813 purchased from EMD Millipore (205531), L-azidohomoalanine (AHA) from Click chemistry 814 tools (1066-100), cOmplete mini EDTA-free protease inhibitors from Roche (11873580001), 815 recombinant murine M-CSF from PeproTech (315-02), Hoechst 33342 from Life 816 Technologies (H3570), formaldehyde 16% (w/v) from Thermo Scientific PierceTM (28908) 817 and the CytoTox 96® Non-Radioactive Cytotoxicity kit from Promega (G1780). Antibodies 818 were mostly purchased from Cell Signalling; GAPDH(D16H11) cat. 8884, Histone H3(D1H2) 819 cat. 4499P, Sigma; anti-rabbit HRP (Sigma/GE - NA934-1ML) and anti-mouse HRP (Sigma, 820 HVZ-A4416-1ML).Anti-RNA polymerase Sigma 70 (RpoD) [2G10] cat. GTX12088 was 821 purchased from Acris antibodies. Mouse serum was purchased from Abcam (ab7486).

822 Bacterial culture conditions and strain construction 823 Salmonella enterica Typhimurium 14028s (STm) was cultured at 37°C in LB Broth (Lennox) 824 with agitation overnight in the presence of antibiotics for plasmid selection. Strains 825 expressing antibiotic resistance genes were selected and maintained on solid LB agar plates 826 containing citrate and 30µg/mL kanamycin (mutant selection) or 100µg/mL ampicillin (for 827 pDiGc plasmid). Mutant strains were retrieved from a single-gene mutant collection 75 , 828 followed by PCR confirmation and retransduction of the mutated loci into the wild type STm 829 14028s background using P22 phage. To visualise bacteria during infection via fluorescence 830 microscopy, a plasmid constitutively expressing GFP - pDiGc - 76 was introduced into 831 bacteria by electroporation followed by selection on LB agar containing ampicillin 100µg/mL 832 at 37°C.

833

834

835 Cell culture conditions 836 RAW264.7 cells (ATCCⓇTIB71TM) purchased from ATCC were routinely cultured in DMEM 837 containing 4.5 g/L glucose and passaged by detaching with accutase (StemCell; A1110501). 838 Only cells below passage number 15 were used for experiments. Bone marrow was isolated 839 from 8-12 week old mice from wild type C57Bl/6 mice, Casp1/11-/- 77, Casp11-/- 78, Nlrp3-/- 840 79 and Nlrc4-/- 80 (C57BL/6 genetic background). Femur and tibia were flushed with PBS. 841 The bone marrow cell suspension (from femur) was filtered through a 70 µm cell strainer 842 (Falcon), washed with 20 ml PBS, spun down at 1200 rpm (4 °C, 15 min) and resuspended 843 in 1 ml of 90 % heat-inactivated FBS (Life Technologies) + 10 % DMSO (Sigma) at a 844 concentration of 1x107, then transferred to liquid nitrogen for storage. For experiments, bone 845 marrow was thawed and washed in 10 mL of pre-warmed RPMI supplemented with 10% 846 FBS (Sigma) (RPMI+FBS). Cells were then resuspended in 20 mL RPMI+FBS without 847 phenol, supplemented with 50 µg/L gentamicin and 40 ng/mL M-CSF. N.B. M-CSF was 848 reconstituted in 0.1% bovine serum albumin (Carl Roth, cat. 8076.4), then aliquoted and 849 stored at -30°C. Cell suspensions were then split across two 10 cm petri dishes, then

850 incubated at 37°C, 5% CO2 for 6 days to allow bone marrow-derived macrophage (BMDMs) 851 differentiation. BMDMs were washed with 3 mL PBS, and detached by incubating cells in 3 852 mL cell dissociation buffer (5% FBS, 2.5 mM EDTA in PBS) and incubation on ice for 5 853 minutes. Resuspended BMDMs were pooled, then pelleted at 500 xg and resuspended in 20 854 mL RPMI+FBS(5%) without phenol and supplemented with 40 ng/mL M-CSF.

855 Proteomic sample preparation 856 RAW264.7 cell infections were performed as previously described 81. Approximately 18-20 h 857 prior to infection, RAW264.7 cells were seeded in DMEM containing 10% FBS 858 (DMEM+FBS) at a cell density of 0.9x105 per well in 6 well plates. Cell density from

859 overnight bacterial cultures grown in LB Broth (Lennox) at 37°C were measured (OD578) and

860 normalised to OD578. = 1. Cells were then washed in PBS and pelleted at 8,000 xg for 5 min. 861 To opsonize bacteria, pellets were resuspended in DMEM containing 10% mouse serum 862 and incubated at room temperature for 20 min. Opsonized bacteria and mock inoculum, 863 were added directly to wells containing RAW264.7 cells at an MOI 100:1 and centrifuged at 864 170 g for 5 min to promote bacterial uptake. Infected cells were incubated at 37°C at 5%

865 CO2 for 25 min. Infected cells were then washed once and media and replaced with 866 DMEM+FBS containing 100 µg/mL gentamicin and returned to the incubator for 1 hour. 867 Media was then replaced with DMEM+FBS containing 16 µg/mL gentamicin for the 868 remainder of the experiment; this step denotes t = 0 h. Therefore, for all experiments, t = 869 time since addition of DMEM+FBS containing 16 µg/mL gentamicin. 870 27

871 Cells were pulse-SILAC-AHA labeled as previously described 19 with the following 872 modifications. To deplete the cells of methionine, lysine and arginine, roughly 3.5 hours prior 873 to harvest, infected and corresponding control cells were washed thrice with prewarmed 874 PBS followed by a 30 min incubation in DMEM dropout media; DMEM containing 10% 875 dialysed FBS, 4.5 g/L glucose, 40 mM L-glutamine, 60 µg/mL L-cysteine and 16 µg/mL 876 gentamicin, but lacking L-methionine, L-lysine and L-arginine (GIBCO). This was then 877 replaced with DMEM dropout media media supplemented with 100 µM L-azidohomoalanine 13 15 13 15 878 and either 84 µg/ml [ C6, N4] L-arginine and 146 µg/ml [ C6, N2]L-lysine (heavy) or 84 13 879 µg/ml [ C6]L-arginine and 146 µg/ml [4,4,5,5-D4]L-lysine (intermediate) SILAC labels 880 (Cambridge Isotope Laboratories, Inc). Cells were then pulse labeled for 3 hours to allow 881 sufficient time for protein translation and subsequent trafficking throughout the cell. 882 883 For cell fractionation, conditioned media containing the “secretome” from pulse SILAC-AHA 884 labeled cells were collected as previously described 19 and stored at -80°C. RAW264.7 cells 885 were then washed three times with prewarmed PBS followed by partial lysis in 1 ml PBS 886 containing 0.1% Tx-100 and protease inhibitors (Roche: cOmplete, mini, EDTA-free) per well 887 for 10 minutes at room temperature. Intermediate and heavy isotopically labeled samples 888 corresponding to either infected or uninfected cells were combined in a 1:1 ratio. To isolate 889 Tx-100 resistant nuclei and bacteria, the lysate was centrifuged at 3,220 xg for 10 min at 890 4°C. Supernatant containing the “lysatome” was transferred to a separate tube and the 891 pellets containing the “nucleome” were washed with PBS containing 0.1% Tx-100 followed 892 by storage at -80°C. Thus, the lysatome is a nuclear-free cell lysate. 893 894 To enrich for the newly synthesised proteome, samples from two biological replicates that 895 were simultaneously pulse labeled with SILAC and AHA labels (biological replicates 896 contained reversed SILAC labels) for 3 h prior to harvest were harvested as previously 897 described 20, with the following modifications. Secretome and lysatome samples were 898 thawed and concentrated to a volume of ~250 ul using a 15 ml Amicon Ultra centrifugal 899 device with a 3 kDa cutoff at 4°C at 3,220 xg. Nucleome pellet was thawed and solubilised 900 in Lysis buffer (ThermoFischer Click-iT: C10416) followed by DNA shearing using a probe 901 sonicator. Newly synthesised proteins were then enriched using 100 ul of beads and 902 according to the manufacturer’s instructions with the following modifications. AHA-containing 903 newly synthesised proteins were reacted with the beads overnight (~16 hrs) at room 904 temperature with rotation. Samples were then centrifuged at 2,600 xg. Beads were then 905 washed three times with SDS buffer (1% SDS, 100mM Tris pH8, 5mM EDTA and 500mM 906 NaCl) followed by reduction and alkylation by resuspending the beads in 500 ul SDS buffer 907 containing 10 mM Tris(carboxyethyl)phosphine (Sigma: C4706) and 40 mM 2- 28

908 Chloroacetamide (Sigma: 22790). Samples were then incubated for 30 minutes at 37 °C with 909 constant agitation at 1,000 rpm. Beads were transferred to retention columns and washed in 910 the following sequence: 7x with 1 ml SDS wash buffer, 10x with 1 ml freshly prepared Urea 911 buffer (8M Urea, Tris-HCl pH 8.0), 10x with 20% 2-propanol and 10x with 20% acetonitrile. 912 Beads were then transferred to low protein binding tubes by resuspending in buffer

913 containing 100 mM Tris pH 8.0, 2 Mm CaCl2 and 4% acetonitrile. Beads were then 914 centrifuged at 2,600 xg for 1 minute and supernatant decanted. 915 916 For peptide preparation, on-bead digestion was carried out in 50 ul of digestion buffer (8M 917 Urea, Tris-HCl pH 8.0, 2.5% acetonitrile) by adding 2 µl of a 0.5 µg/µl LysC/Trypsin to each 918 tube and incubation at 37 °C for 4 hours with shaking at 1,000 rpm. Urea was then diluted by

919 adding 150 µl of buffer (100 mM Tris pH 8.0, 2 mM CaCl2 and 4% acetonitrile) and incubated 920 overnight at 37 °C. Supernatants were then transferred to fresh microfuge collection tubes.

921 Beads were then washed with 200 µl of H2O to collect residual peptides and collated. 922 Samples were acidified by adding 8 µl (2% of the sample volume) of a 10% formic acid

923 followed by acidification verification using a pH strip. 924 925 Peptides were desalted by binding to a Waters Oasis HLB 96-well µElution Plate (Waters: 926 186001828BA) using a vacuum manifold. Wells were preconditioned by passing 100 µl of 927 100% acetonitrile followed by 100 µl of Oasis buffer B (1% formic in 60% MeOH) then 100 µl

928 of Oasis buffer A (1% formic in H2O). Samples were then bound followed by sequential 300 929 µl, 200 µl and 100 µl washes with Oasis buffer A. To elute peptides, a collection tray was 930 loaded with glass vials over which the Oasis plate was carefully aligned. Peptides were 931 eluted in subsequent 50 µl and 25 µl of Oasis buffer B. Glass vials were then transferred to 2 932 ml centrifuge tubes, pulse spun then stored at -20°C. Secretome and nucleome fractions 933 were evaporated using a speedVac at 35°C for ~2 hrs, then resuspended in 20 µl of injection 934 buffer (96.9% water, 3% ACN and 0.1% Formic acid) for direct analysis by nano LC-MS/MS 935 on Velos orbitrap. 936 937 To reduce sample complexity, lysatome samples were subjected to high-pH reversed phase 938 fractionation. In brief, the fractionation was performed on an Agilent 1260 HPLC system 939 equipped with a variable wavelength detector (254nm). On the HPLC, fractionation was 940 performed on an XBridge BEH C18 column (1 x 100mm, 3.5 µm, 130Å, Waters). Elution was 941 performed at a flow rate of 0.1mL per minute using a gradient of mobile phase A (20mM 942 ammonium formate, pH 10) and B (acetonitrile), from 1% to 37.5% over 61 minutes. 943 Fractions were collected every 2 minutes across the entire gradient length and concatenated 944 into 8 final samples as discussed previously 82. Fractions were dried in a SpeedVac and 29

945 reconstituted in 0.1% formic acid prior to desalting on an Oasis uElution plate and analysis 946 by LC-MS/MS. Samples were then analysed on an Orbitrap Velos Pro (Thermo Fisher 947 Scientific) as previously described 20. 948 949 For proteomic data analysis, the raw data obtained by Orbitrap mass spectrometers were 950 processed using MaxQuant software (version 1.5.0.0). The MaxQuant implanted search 951 engine Andromeda was used to search the MS/MS spectra, against a mouse database 952 obtained from Uniprot. In the database, the sequences of frequently observed contaminant 953 proteins and the reversed sequences of all entries were included, to indicate the false- 954 positive search hints. Trypsin/P was chosen as the digestion enzyme, and only no more than 955 two miss cleavages were allowed for the peptide identification. Cysteine 956 carbamidomethylation was used as the only fixed modification, while methionine oxidation, 957 N-terminal acetylation and AHA replacement of methionine were chosen as the variable 958 modifications. The minimal peptide length was set to 7 amino acids. The mass tolerance for 959 peptides was 20 ppm in initial search and 6 ppm in main search. The maximal tolerance for 960 fragment ion identification was 0.5 Da. False discovery rates for the identification of proteins 961 and peptides were set to 1%. The minimal unique peptide number for protein identification 962 was set to 1%. At least two ratio counts (quantified peptides for each protein) were set for 963 the protein quantification. The "requantify" and "match between runs" were functionalized in 964 MaxQuant. In the pulse SILAC-AHA samples, only protein groups having a ratio of over 20% 965 identified peptides containing medium or heavy labels were kept for the further analysis. 966 Proteins identified as potential contaminants, with reverse sequences, and from only 967 peptides were removed. Average protein ratios were calculated, only if they were quantified 968 in both biological replicates. The data are available via ProteomeXchange 83 with the 969 identifier (PXD010179). Proteomic data was visualized using R (v 1.0.143) and Cytoscape 970 (v3.3.0). Heatmaps were visualised with Heatmapper 84.

971 Flow Cytometry of extracted nuclei 972 Tissue culture media from infected cells were replaced with fresh DMEM containing 5 µM

973 DCG04-Boclicky-TAMRA, followed by a 1 hour incubation at 37°C, 5% CO2. Cells were 974 washed thrice in PBS followed by solubilization in 0.1% Triton X-100. Nuclei were pelleted 975 (500 xg, 3 min), washed and resuspended in PBS containing 0.1% Tx-100. Nuclei were then 976 fixed by adding formaldehyde to 4% wt/vol (Thermo Scientific; 28908) and incubated for 15 977 minutes at room temperature, followed by washing in buffer containing 0.1% Triton X-100 978 containing 2 µg/ml Hoechst 33342. Nuclei were resuspended in 0.1% Triton X-100 979 containing 2 µg/ml Hoechst 33342 and subjected to flow cytometry. Nuclei samples were 980 acquired on the Attune NxT Flow Cytometer (Thermo Fisher Scientific) equipped with 405

981 nm, 488 nm, 561 nm, and 638 nm lasers. Hoechst was excited by the 405 nm laser line and 982 fluorescence signal was collected using a 440/50 bandpass filter and TAMRA excited by the 983 561 nm laser line and collected using a 585/15 bandpass filter. Post-acquisition analysis was 984 done in FlowJo software 10.0.08 (Tree Star, Inc.).

985 SDS-PAGE and Immunoblot 986 RAW264.7 cells were infected in 6 well plates tissue culture plates at an MOI 100:1 as 987 described above. To visualise cathepsin activity, infected and mock treated cells were 988 labeled with 5 µm DCG04-Bodipy-FLike (λex 488 λem520) 3-4 hours prior to harvest. Cells 989 were then washed three times with PBS, then partially lysed in 200 µL PBS lysis buffer (PBS 990 containing 0.1% Tx-100 and cOmplete mini EDTA-free protease inhibitors) on ice for 10 min. 991 Cell lysates were then collected and nuclei pelleted at 50 xg for 3 min, followed by a 992 subsequent spin at 500 xg to pellet bacterial cells. Nuclei and bacterial enriched pellets were 993 washed once and then resuspended in 200 µL PBS lysis buffer. Cell fractions were then 994 combined with 4x Laemmli buffer loading dye, heated to 95°C for 3 min. Samples were then 995 separated by SDS-PAGE and cathepsin activity measured using 488nm laser on Typhoon 996 scanner (FLA 9500). The bottom sections of SDS-PAGE gels were subjected to Coomassie 997 staining to visualise histones followed by western transfer to a 0.45µm PVDF membrane 998 (Millipore ImmobilinⓇ-P, IPVH00010). Infected whole cell lysates for analysis by SDS-PAGE 999 were generated by washing cells 3 times in PBS at the indicated time point and then directly 1000 lysing cells in 1x Laemmli buffer loading dye. Lysates were then heated to 95°C for 3 min 1001 and analysed by immunoblot. Prior to SDS-PAGE, nuclear DNA was mechanically sheared 1002 using a Hamilton syringe. 1003 1004 Membranes were blocked with 5% skim milk in TBS-T and probed with the following 1005 antibodies 5% skim milk in TBS-T overnight; RpoD 1:1000, GAPDH 1:10,000, LAMP1 1006 1:1,000, Lamin A 1:1,000, Histone H3 1:,1,000 and H3.cs1 1:1,000. Secondary antibodies 1007 were used at 1:5,000 and incubated with washed membranes for 1 h at room temperature. 1008 After washing, chemiluminescence substrate (GE Healthcare, RPN2106) was used for 1009 signal development and detected using x-ray film (Advantsta, L-07013-100) or a BioRad 1010 ChemoDoc Touch. X-ray film was converted to digital from by scanning at 300x300 dpi. 1011 Digital images were then cropped and contrast adjusted in Photoshop and incorporated into 1012 figures using Adobe Illustrator. Biological replicate samples were analysed on separate 1013 SDS-PAGE gels, unless otherwise stated.

1014

1015 31

1016 LDH release assay 1017 RAW264.7 cells were seeded in DMEM containing 10% FBS (DMEM+FBS) at a cell density 1018 of 2x104 per well in 96 well plates and infected as described above with the following 1019 deviations. Unless otherwise stated, inhibitors and DMSO solvent was added at t = 0 h (see 1020 definition above). BMDMs were seeded in RPMI+FBS(5%) without phenol and 1021 supplemented with 40 ng/mL M-CSF at 5x104 cells per well into a 96 well plate (Thermo, 1022 cat.167008) ~18-20 h prior to infection. The bacterial inoculum was prepared as described 1023 above. To opsonize bacteria, pellets were resuspended in RPMI containing 10% mouse 1024 serum and incubated at room temperature for 20 min. Opsonized bacteria were added 1025 directly to wells containing BMDMs or RAW264.7 cells at an MOI 100:1 and centrifuged at 1026 170 xg for 5 min to promote bacterial uptake. Infected cells were incubated at 37°C at 5%

1027 CO2 for 25 min. Cells were then washed once, media was replaced with RPMI + FBS(5%) 1028 without phenol, or DMEM+FBS(5%) without phenol for RAW264.7 cells, containing 100 1029 µg/mL gentamicin and returned to the incubator for 1 hour. Media was then replaced with 1030 RPMI+FBS(5%) or DMEM+FBS(5%) without phenol for RAW264.7 cells with 16 µg/mL 1031 gentamicin for the remainder of the experiment for BMDMs and RAW264.7 cells, 1032 respectively: this step denotes t = 0 h. LDH release as a measure of cell death was 1033 quantified according by measuring the % LDH release as previously described using the 1034 CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega:G1780) kit 85 according to 1035 manufacturer's instructions. Percentage cell death = 100 x (experimental LDH - spontaneous 1036 LDH) / (maximum LDH release - spontaneous LDH). Data was visualised in R and 1037 GraphPad Prism. Each experimental batch consisted of 3-8 biological replicate wells per 1038 condition, whereby single measurements (490nm) were acquired per well. This 1039 measurement was then normalised to the spontaneous LDH release from uninfected wells 1040 that were otherwise identically treated, using the aforementioned formula and treated as a 1041 separate data point. In our hands, the degree of overall LDH release upon STm infection 1042 displayed considerable variance across batches of BMDM cells. To reduce this variance, 1043 batches whereby wildtype STm induced more than >50% LDH release were removed. All 1044 conditions/mutants for a single experiment (batch) were contained within a single 96 well 1045 plate.

1046 Microscopy 1047 RAW264.7 cells were seeded in DMEM containing 10% FBS (DMEM+FBS) at a density of 1048 4x105 cells per well in 24 well glass bottom tissue culture plates (Greiner, 662892). Cells 1049 were infected with pDiGc expressing bacteria as described above (MOI 100:1). At the 1050 indicated times, cells were then treated with the cell permeable cathepsin probe DCG04- 1051 Boclicky-TAMRA for 2 hours prior to harvest. Cells were washed three times with PBS and

1052 then fixed with in PBS solution containing 4% formaldehyde. Cells were then stained 1053 overnight with 2 µg/mL Hoechst 33342 and 70 ng/mL Phalloidin in PBS containing 0.05% 1054 Tx-100 to visualize host cell nuclei and host cytoskeleton, respectively. Images of stained 1055 cells were acquired on a Zeiss Cell Observer microscope using a 20x objective. Images 1056 were processed using CellProfiler software 2.1.1 as previously described 86. Nuclear 1057 cathepsin activity was quantified by measuring the integrated DCG04-Boclicky-TAMRA 1058 signal intensity that overlapped with the host nucleus defined by the Hoechst 33342 channel. 1059 Cathepsin activity of individual nuclei was normalised to the nuclear size (area), and only 1060 infected cells containing similar bacterial loads were compared between strains. A replicate 1061 experiment was performed by seeding 2x104 cells per well of a 96 well glass bottomed plate, 1062 infected with an MOI 100:1 and processed as described above. Images for this experiment 1063 were acquired on a Nikon Ti-E with a pate loader for 96 well plates using a 10x objective. 1064 1065 Confocal images were acquired on an Olympus confocal laser scanning microscope 1066 (FV3000) using an 60x oil immersion objective. Samples were prepared as described above 1067 in a glass bottomed 96 well plate. After three washes in PBS, cells were fixed with 4% 1068 formaldehyde and 0.4% Tx-100 in PBS for 1 hour at room temperature. Pictures were 1069 captured using the 405 nm laser for excitation for Hoechst 33342, 488 nm for GFP 1070 expressing (pDiGc) STm and 561 nm for DCG04-Boclicky-TAMRA. Image overlays and grey 1071 scale conversions were done in FIJI, then images were cropped in Photoshop before figure 1072 construction with Adobe Illustrator. 1073 1074 Bacterial growth curves 1075 Overnight bacterial cultures grown at 37°C in LB (Lennox) were pelleted at 8000 xg and

1076 washed 3 times in fresh media, then back-diluted to OD578 = 0.005 in the presence of 1077 inhibitors and solvent at the indicated concentrations. Cells were dispensed, 100 µL per well, 1078 into a round bottom 96 well plate in triplicate (Thermo, Nunclon Delta Surface, cat. 168136), 1079 then covered with a sealable transparent breathable membrane (Breathe-Easy by Divbio, 1080 cat. BEM-1). Cell growth was quantified by measuring the absorbance at 578 nm at 20 min 1081 intervals with constant shaking at 37°C. 1082 1083 Statistical analyses 1084 For cell death (LDH release) assays, significance testing was performed in R using the 1085 unpaired t-test (Figure 5B, 6B & C). To avoid assuming any shape of the distribution for 1086 single cell nuclei analysis of cathepsin activity, statistical testing was performed in R using 1087 the unpaired Wilcoxon rank sum test (non-parametric) (Figure 4D & S4). Gene Ontology 1088 (GO) enrichment was calculated with the ClueGO plugin v2.1.7 of Cytoscape v3.3.0. For 33

1089 global analyses described (Figure 1B), GO enrichment was performed on proteins displaying

1090 ±1.5 Log2 fold change (infected/uninfected). For comparison between STm and LPS 1091 stimulated cells in (Figure 2A & B), GO enrichment was performed on proteins ±2 s.d. of the

1092 Log2 fold change (infected/uninfected). A custom reference set was used for each test and 1093 contained all quantified proteins within the same fraction. GO enrichment was performed 1094 separately on up- and down-regulated proteins using the right-sided hypergeometric test, 1095 and corrected for multiple testing using the Bonferroni step-down (Holm) method. This test 1096 was chosen for stringent error control. 1097

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38 bioRxiv preprint doi: https://doi.org/10.1101/455048; this version posted November 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. A B pulse SILAC + AHA Time 1 Uninfected (I)+AHA Infection (Hrs) 4 8 20

Infection response to virus Infected (H)+AHA

GO:0009615 2 Starve (30 min) response to bacterium 4h pulse SILAC + AHA (3 hr) Labelling 8h GO:0009617 20h defense response to other organism

0 4 8 15 20 GO:0098542 Infection Hrs cellular response to cytokine 3 stimulus STm Harvest GO:0071345 Secreted response to interferon-beta proteins Lysatome Nucleus GO:0035456 cellular response to Wash lipopolysaccharide

GO:0071222 extrinsic apoptotic signaling Conditioned media Partial lysis (+mild detergent) pathway 4 nucleic acid metabolic process Pooling Uninfected Infected Uninfected Infected GO:0097191

I-kappaB kinase/NF-kappaB GO:0090304 signaling

DNA replication initiation GO:0007249

cellular response to GO:0006270 Fractionation interferon-gamma

GO:0071346 defense response to protozoan

Collection PS PS GO:0042832 symbiont-containing vacuole membrane

Discard lysosome GO:0020005

nuclear nucleosome Nucleome GO:0005764 Nucleome Secretome DNA replication-dependent Lysatome GO:0000788 nucleosome assembly

lysosome GO:0006335 Old positive regulation of intrinsic Newly apoptotic signaling pathway by 5 Secretome GO:0005764 synthesized p53 class mediator protein GO:1902255 mitochondrion transport along New microtubule enrichment Cu(I), 18 h GO:0047497 Room temperature

Relative Quanti cation N.D. 1 10 20 30 6 not regulated

regulated (-Log10) Node size Mass adjusted .P value adjusted .P spectrometry Regulation Intensity (infected/uninfected Log2) 0 76 149 m/z > 1.5 fold (Up)

< 1.5 fold (down) Node fill Number of proteins Node border

Figure 1 bioRxiv preprint doi: https://doi.org/10.1101/455048; this version posted November 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. A B 6 6 2 2 4 h 8 h

3 3

0 0

−3 −3 r = 0.65 r = 0.635 p < 2.2e-16 p < 2.2e-16 n = 3014 LPS 3h (treated/control Log ) n = 3016 LPS 8h (treated/control Log ) −6 −6

Rgl1LplAnxa5 −6 −3 0 3 6 −6 −3 0 3 6 STm 8h (infected/uninfected Log ) STm 4h (infected/uninfected Log 2 ) 2

500 regulation of endothelial 500 response to virus DNA duplex unwinding regulation of inflammatory response cell apoptotic process cellular response to Cell cycle cyclin−dependent protein serine/ regulation of cell migration 400 interferon−beta 400 threonine kinase regulator activity Tlr7 IMP metabolic process defense response to GO Term regulation of apoptotic process Plau regulation of vascular smooth other organism GO Term 1e−02 1e−01 1e+00 GO Term muscle cell proliferation Pak1

response to bacterium GO Term 300 cellular response to 300 adjusted P Value negative regulation of cytokine stimulus Tjp2 response to virus Cd40 Serpine1 apoptotic process Cd72

positive regulation of tyrosine Tlr13 1e−01 1e+00 phosphorylation of STAT protein 1e−03 1e−01 200 Pak1 200 adjusted P

nr. proteins nr. proteins adjusted P Value 1e−01 Value

1e−05 1e−03 Serpine1 Tlr7 Lyz2 adjusted P Value C3 Olr1 Calcrl Ldlr Lpl

100 100 Cd40 Plau Olr1 Calcrl Fcgr1 Lyz2

0 0 −4 −2 0 2 4 −4 −2 0 2 4 STm Lysatome (4h) - LPS Lysatome (3h) STm Lysatome (8h) - LPS Lysatome (8h)

C LPS STm Time Rnase4 GusB Igfbp4 Plau Elimin2 Spink5 Lman2 Grn Pld4 Igsf8 Pam Psap Cfp Mfge8 CtsA CtsD Cst3 Clstn1 C3 Dag1 CtsB Lpl Csf1r Lyz2 CtsL1 Adam15 CtsZ Lfng Lcp1 Ppt1 Capg Eno1 CtsC Lgals3 Gsn Lgmn CtsS CtsL1 CtsC Cd14 Ccl9 Spp1 Aplp2 H2-K1 Lamc1 App H2-D1 H2-L Ldlr Grn CtsS

Secreted proteins C3 CtsB Gusb Spink5 CtsA Ppt1 Lgmn Psap CtsD CtsZ Rnaset2 Sdf4 Plod1 Napsa Qsox1 Nucb1 Ecm1 Nucb2 Atp6ap1 B2m Lgals3bp Gpnmb Cxcl10 Ccl4 Serpine1 Ccl2 Il1rn −2 0 2 Figure 2 3 8 19 4 8 20 Hrs Z−Score bioRxiv preprint doi: https://doi.org/10.1101/455048; this version posted November 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

A C response to bacterium 100 6 lysosome 2

proton−transporting two−sector

80 ATPase complex, catalytic domain GO Term 4 Nucleome TAP complex n = 1084 Atp6v1e1 60 1e-02 1e+00 Atp6v1a ected 20 h (Log ) Lgmn adjusted P value 2 nr. proteins 40 Psap Ctsl1 Atp6v1b2 CtsZ CtsA CtsB CtsS CtsD Mki67 Hist1h4a H3f3a Lysosomal proteins Lysosomal 0 20 infected/uninf

0 −2 13 10 8 8 = n −6 −4 −2 0 2 4 6 infected/uninfected 20 h Lysosome component lumen lumen (Log ) 2 membrane membrane

p = 0.026 Cell fraction B All (n = 740) Lysosomal hydrolases (n = 8)

Ctsl1 Sqstm1 p = 2.403e-06 CtsbPrdx1 Serpine1 Ptgs2 Ctsz Prdx4 5 Lgmn Nos2 Ctsd Lgals9 Il33 Ctsa Psap Ifi202 Il1rn Irg1 Noc4l Ctss Oasl1 Isg15 Sf3b3

2 Rnf213 Fosl2 Phf11 Impdh2 0 Lsm5 (Log ) Sun2 Uhrf1 H3f3a;Hist1h3b;Hist1h3a;H3f3c Hist1h4a Mki67 r = 0.586 −5 p < 2.2e-16 n = 740

infected/uninfected Nucleome 20 h −5 0 5 infected/uninfected Lysatome 20 h (Log ) 2

Figure 3. bioRxiv preprint doi: https://doi.org/10.1101/455048; this version posted November 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

A -- Wt Wt STm -- Wt Wt STm kDa - + - + Cath. Probe 488 kDa - + - + Cath. Probe 488 50 50 pro 37 CtsX 37 CtsB activity

25 activity CtsS 25 Cathepsin CtsL Cathepsin

GAPDH Histones (Coomasssie)

B Lysate Nuclear

kDa Wt SPI-2 SPI-1 kDa Wt SPI-2 SPI-1

50 50 pro 37 CtsX 37

CtsB activity activity Cathepsin 25 CtsS Cathepsin 25

GAPDH Histones (Coomasssie)

p = 1.86e-06 C D p = 7.86e-11 Hoechst 33342 DCG04-Boclicky-TAMRA Hoechst 33342/ p = 0.0072 (DNA) (Cathepsin activity) Cathepsin/STm p = 0.03 0.04

2h 0.03 STm Wt STm pDiGc

0.02 20h

STm Wt STm pDiGc 0.01 nuclear cathepsin activity (20h)

337 337 192 192 = n

Wt SPI-2 Wt SPI-2 bystanders Infected E uninfected Wild type SPI-2 naïve bystander infected (low) infected (high) infected (high) infected i ii iii iv v vi Merge DNA activity Cathepsin Cathepsin S Tm

Figure 4. bioRxiv preprint doi: https://doi.org/10.1101/455048; this version posted November 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. A Uninfected Wildtype SPI-2

1.5K sub-G1 G1/S/G2 sub-G1 G1/S/G2 1.5K sub-G1 G1/S/G2 1.2K G1 G1 G1

900 1.0K 1.0K Count Count

Count 600 Count Count Count 500 500

300 G2 G2 G2 0 0 0 6 6 6 10 0.16 % 0.31 % Total Total 10 3.12 % 2.48 % Total 10 0.97 % 2.63 % Total TAMRA+ = TAMRA+ = TAMRA+ = 5 5 5 10 0.47 % 10 5.6 % 10 3.6 %

4 4 4 10 10 10

3 3 3 10 10 10 YL1-A :: TAMRA-A YL1-A YL1-A :: TAMRA-A YL1-A YL1-A :: TAMRA-A YL1-A Cathepsin activityCathepsin Cathepsin activityCathepsin Cathepsin activityCathepsin (DCG04-Boclicky-TAMRA) (DCG04-Boclicky-TAMRA) (DCG04-Boclicky-TAMRA) 0 0 0

0 200K 400K 600K 800K 1.0M 0 200K 400K 600K 800K 1.0M 0 200K 400K 600K 800K 1.0M DNA DNA DNA VL1-A :: DNA-Pacific Blue -A VL1-A :: DNA-Pacific Blue -A VL1-A :: DNA-Pacific Blue -A (Hoechst 33342) (Hoechst 33342) (Hoechst 33342)

B RAW264.7 NS 40 NS

p = 2.3e-03 p = 2.6e-02 30

p = 1.8e-02 10 NS % cell death - LDH release (19 h)

16 16 16 888888 = n - 12.5 25 - 12.5 25 - 12.5 25 CA-074-Me (μM) Wt SPI-2 sifA

Figure 5. bioRxiv preprint doi: https://doi.org/10.1101/455048; this version posted November 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

A BMDMs C BMDMs 40 p = 2e-05 DMSO 25 p = 3.1e-05 30 CA-074Me (12.5μM)

CA-074Me (25μM) 20 p = 9.3e-04 20 p = 2.0e-04

15 10

0 10 % cell death - LDH release 4 8 12 16 20 Time (h) 5 % cell death − LDH release 18h B BMDMs 0 p = 5.3e-05 p = 5.1e-03 9 10 10 9 10 10 = n 50 p = 8.1e-04 CA-074-Me (μM) --12.5 25 12.5 25 Drug addition poi (h) 1 9 40

30 NS 20

0 % cell death − LDH release 18h −10 24 25 9 9 9 9 9 9 = n - + - + - + - + CA-074-Me (12.5μM)

Wild type Casp11 -/- NLRP3 -/- NLRC4 -/-

Figure 6. bioRxiv preprint doi: https://doi.org/10.1101/455048; this version posted November 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

A DNA - Actin - STm 300 t = 0 100 200 n = 697 0 100

0 80 200

150

100 4 n = 552 60 50

0 120 count 90 40

60 n = 343 8

0 Hours post infection 20 150 % infected cells per vield of view 100 n = 861 20 50 0

0 2 biological replicates 0 10 20 30 n = 9 replicate FOV (total) 2224 cells (total) Salmonella per cell

B 4h 8h 20h Lysatome 2 Nucleome infected/uninfected (Log ) - Replicate 2 Secretome

infected/uninfected (Log ) - Replicate 1 2

Figure S1 bioRxiv preprint doi: https://doi.org/10.1101/455048; this version posted November 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

B) 0 - 4 hrs 4 - 8 hrs 16 - 20 hrs

L N STm LN StmL N STm

75 50 * 37 CtsX CtsB (488 nm) 25 CtsS CtsL probe Cathepsin

GAPDH

Histones (Coomassie)

RpoD (STm)

Fig S2. bioRxiv preprint doi: https://doi.org/10.1101/455048; this version posted November 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Uninfected STm

H3.cs1

Fig S3. bioRxiv preprint doi: https://doi.org/10.1101/455048; this version posted November 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

p = 0.00173 p = 2.2e-16 p = 2.2e-16 p = 2.2e-16

0.04

0.03

0.02

0.01

nuclear cathepsin activity (20h) 0.00 6248 5684 3136 875 = n Wt SPI-2 Wt SPI-2 bystanders Infected

Fig S4. bioRxiv preprint doi: https://doi.org/10.1101/455048; this version posted November 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

A) B)

1.0 1.0 0.9 E-64 0.9 CA-074-Me 0.8 0.8 0.7 0.7

0.6 Drug 0.6 A) Drug DMSO 0.5 0.5 DMSO 0.4 E-64 0.4 CA-074-Me

0.3 0.3 OD578 OD578 Concentration μM Concentration μM 0.2 5 0.2 6.25 10 12.5 20 25 0.1 0.1

0 5 10 15 20 25 0 5 10 15 20 25 Time (hrs) Time (hrs)

Fig S5. bioRxiv preprint doi: https://doi.org/10.1101/455048; this version posted November 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Supplementary Figure - Uncropped scans

Relates to Figure S3 Relates to Figure S2

kDa 75 * CatX CatB 25 CatS CatL Cathepsin activityCathepsin Alexa Fluor 488 nm Fluor Alexa Short exposure Chemiluminescence Chemiluminescence Histones GAPDH RpoD Longer exposure Longer Coomassie

Relates to Figure 4A Biological replicate of Figure 4A

-- Wt Wt -- Wt Wt STm 75 - + - + - + - + Cath. Probe 488 * CatX 75 CatB 25 CatS CatL * CatX CatB CatS

Cathepsin activityCathepsin 25 Alexa Fluor 488 nm Fluor Alexa CatL Cathepsin activityCathepsin Alexa Fluor 488 nm Fluor Alexa Histones Coomassie GAPDH Chemiluminescence Histones Coomassie bioRxiv preprint doi: https://doi.org/10.1101/455048; this version posted November 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Supplementary Figure - Uncropped scans

Relates to Figure 4B.

Uncropped from Figure 4B (TR2 of BR1)

75 GAPDH

25 Chemiluminescence Alexa Fluor 488 Fluor Alexa Histones Laser scan (Typhoon) Coomassie

Cathepsin activity DCG04-FL BR = Bioligical replicate TR = Technical replicate Alexa Fluor 488 nm Coomassie

BR 1 12 1 1 2 1 1 2 1 1 2 BR 1 12 1 1 2 1 1 2 1 1 2 TR 1 1 1 1 1 1 1 1 1 1 1 1 TR 1 1 1 1 1 1 1 1 1 1 1 1 STm --- Wt Wt Wt ssaV ssaV ssaV prgK prgK prgK STm --- Wt Wt Wt ssaV ssaV ssaV prgK prgK prgK Cath. Probe 488 - + +- + + - + -+ ++ Cath. Probe 488 - + +- + + - + -+ ++

250 250 150 150 Lysatome 100 100 75 75

50 50

37 CatX 37 CatB CatS 25 CatL 25

20 20

Nucleome (20x g pellet) Nucleome (20x g pellet)

BR 1 1 1 1 1 1 BR 1 1 1 1 1 1 TR 1 1 1 1 1 1 TR 1 1 1 1 1 1 STm -- Wt Wt ssaV prgK STm -- Wt Wt ssaV prgK Cath. Probe 488 - + - + ++ Cath. Probe 488 - + - + ++ kDa 250 kDa 250 150 150 100 100 75 75 Nucleome 50 50

37 * 37

25 25

20 20

Nucleome (20x g pellet) Nucleome (20x g pellet) BR 2 2 2 2 2 2 TR 1 1 1 1 1 1 BR 2 2 2 2 2 2 STm -- Wt Wt ssaV prgK TR 1 1 1 1 1 1 Cath. Probe 488 - + - + ++ STm -- Wt Wt ssaV prgK Cath. Probe 488 - + - + ++ kDa 250 150 kDa250 150 100 100 75 Nucleome 75 50 50 * 37 37

25 25 20 20