Mycobacterial Secretion Systems ESX-1 and ESX-5 Play Distinct Roles in Host Cell Death and Inflammasome Activation

This information is current as Abdallah M. Abdallah, Jovanka Bestebroer, Nigel D. L. of September 26, 2021. Savage, Karin de Punder, Maaike van Zon, Louis Wilson, Cees J. Korbee, Astrid M. van der Sar, Tom H. M. Ottenhoff, Nicole N. van der Wel, Wilbert Bitter and Peter J. Peters

J Immunol 2011; 187:4744-4753; Prepublished online 28 Downloaded from September 2011; doi: 10.4049/jimmunol.1101457 http://www.jimmunol.org/content/187/9/4744 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2011/09/28/jimmunol.110145 Material 7.DC1 References This article cites 58 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/187/9/4744.full#ref-list-1

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Mycobacterial Secretion Systems ESX-1 and ESX-5 Play Distinct Roles in Host Cell Death and Inflammasome Activation

Abdallah M. Abdallah,*,†,1,2 Jovanka Bestebroer,†,2 Nigel D. L. Savage,‡ Karin de Punder,* Maaike van Zon,* Louis Wilson,‡ Cees J. Korbee,‡ Astrid M. van der Sar,† Tom H. M. Ottenhoff,‡ Nicole N. van der Wel,* Wilbert Bitter,† and Peter J. Peters*,x

During of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tu- berculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5—two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators—during the infection cycle. Using wild-type, Downloaded from ESX-1– and ESX-5–deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1b activation. The ESX-5 system also induces a -independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome http://www.jimmunol.org/ activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread. The Journal of Immunology, 2011, 187: 4744–4753.

athogenic secrete virulence proteins that can fa- this classification well. They have an uncommon and highly cilitate colonization and persistence in the host (1). There complex cell wall and contain multiple copies of a recently P are many different bacterial effector proteins, whereas the identified novel secretion system known as early secretory Ag

secretion systems used for protein delivery are more homoge- target 6 (ESAT-6) system (ESX), or type VII secretion system by guest on September 26, 2021 neous. Six different secretion systems have been described in (T7SS) (2). Gram-negative bacteria, whereas Gram-positive bacteria secrete A number of species of the Mycobacterium genus are important proteins mainly via the Sec or Tat system. Mycobacteria, although human and animal , and proteins secreted via T7SSs they have been considered as Gram-positive bacteria, do not fit contribute to their pathogenesis. The five known mycobacterial T7SSs are encoded by discrete gene clusters termed ESX-1 to *The Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital, 1066 CX ESX-5 (2, 3). ESX-3 is required for iron transport and as such is Amsterdam, The Netherlands; †Department of Medical Microbiology and Infection essential for the viability of Mycobacterium tuberculosis (4, 5), Control, Free University Medical Center, 1081 BT Amsterdam, The Netherlands; ‡Department of Infectious Diseases, Leiden University Medical Center, 2333 ZA whereas ESX-1 and ESX-5 are involved in the virulence of M. Leiden, The Netherlands; and xKavli Institute of Nanoscience, Technical University, tuberculosis and Mycobacterium marinum (6, 7). ESX-1 and ESX- 2628 CJ Delft, The Netherlands 5 have been found to secrete different effector proteins that 1Current address: King Abdullah University of Science and Technology, Thuwal, modulate host cell functions, although the molecular targets and Kingdom of Saudi Arabia. 2 mechanisms of action are yet to be identified (reviewed in Ref. 2). A.M.A. and J.B. contributed equally to this work. The substrates or structural components of ESX-1 include among Received for publication May 18, 2011. Accepted for publication August 30, 2011. others EsxA (ESAT-6) and EsxB (CFP-10). Within the phago- This work was supported by the European Union 7th Framework Program (Grant lysosome of , M. tuberculosis and M. marinum se- Agreement No. 201762), The Netherlands Leprosy Relief Foundation, the Turing Foundation, and the Aeras Global TB Vaccine Foundation via a grant from the crete ESX-1 substrates that interfere with the integrity of the Netherlands Directorate-General of Development Cooperation, Dutch Ministry of phagosomal membrane, leading to “leaky” membranes, phago- Foreign Affairs. somal rupture, and bacterial entry into the cytosol (8, 9). ESX-5 Address correspondence and reprint requests to Prof. Peter J. Peters, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX, Amsterdam, The Netherlands. E-mail is involved in the secretion of various proteins containing Pro-Pro- address: [email protected] Glu, Pro-Glu, and polymorphic GC-rich sequences, and ESX-5 The online version of this article contains supplemental material. mutants are unable to modulate macrophage responses

Abbreviations used in this article: CA-O74-Me, [L-3-trans-(propylcarbamoyl)oxir- (7, 10). ane-2-carbonyl]-L-isoleucyl-L-proline methyl ester; DPI, diphenyleneiodonium chlo- Macrophages are important both for bacterial control and for ride; ESAT-6, early secretory Ag target 6; ESX, early secretory Ag target 6 system; bacterial multiplication during the early stages of mycobacterial LDH, lactate dehydrogenase; NAC, N-acetyl-L-cysteine; ROS, reactive oxygen spe- cies; siRNA, small interfering RNA; STS, staurosporine; TBH, t-butyl hydroperox- infection. They respond to mycobacterial infection by production ide; T7SS, type VII secretion system; WT, wild-type; z-FY(t-Bu)-dmk, benzyloxy- of proinflammatory . IL-1b synthesis is strongly upreg- carbonyl-Phe-Tyr(t-Bu)-diazomethylketone. ulated upon infection with M. tuberculosis (11). IL-1b is first Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 synthesized as inactive pro–IL-1b and requires proteolytic matu- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101457 The Journal of Immunology 4745 ration by caspase-1 for cellular release (12). Caspase-1 itself needs like cells was induced with 10 ng/ml PMA (Sigma) for 24 h. RAW 264.7 activation by inflammasomes, protein complexes that sense cy- and A549 cells were trypsinized (0.5%; Life Technologies), washed with fresh medium without antibiotics, seeded in 24-well culture plates (Co- tosolic bacterial products and endogenous danger signals. Both b star), and allowed to grow to 70% confluency before infection. M. tuberculosis and M. marinum reportedly induce IL-1 secre- Monocytes were isolated from peripheral blood of healthy donors by tion through NALP3 inflammasomes in an ESX-1–dependent magnetic cell sorting using anti-CD14–coated beads. Isolated cells were manner (13–16). Recently, we observed that ESX-5 is also re- polarized with 50 U/ml GM-CSF (Novartis Pharma, Arnhem, The Neth- quired for IL-1b secretion upon infection with M. marinum (7). erlands) to obtain type 1 macrophages, 50 ng/ml M-CSF (R&D Systems) for type 2 macrophages, and 1000 U/ml GM-CSF and 500 U/ml IL-4 for Mechanisms regulating this event and the precise contribution of monocyte-derived dendritic cells (25). Cells were seeded 1 d before in- ESX-5 remain to be characterized. fection. Macrophages further respond to mycobacterial infection by induction of cell death. This is an effective innate immune mechanism in which , but not necrosis, is associated with For cryo-immunogold electron microscopy, THP-1 cells were seeded at 10 6 2 reduced bacillary viability. Sequestration and retention of myco- 3 10 per flask (75 cm ). For cytotoxicity and IL-1b secretion assays, 3 5 3 3 bacteria within apoptotic bodies allows for engulfment by newly THP-1 cells or type 1 macrophages were seeded at 5 10 and 3 10 cells/well in 24-well culture plates, respectively, in 1 ml RPMI/FCS. Cells recruited activated phagocytic cells (17, 18). Sly et al. (19) showed were infected with bacteria for 1 h at 33˚C for M. marinum and 37˚C for that virulent M. tuberculosis inhibits macrophage apoptosis by M. tuberculosis and 5% CO2. Supernatant was removed, and infected cells upregulation of the antiapoptotic Bcl-2 family member Mcl-1, were washed three times and incubated in fresh RPMI/FCS at appropriate supposedly by maintaining the integrity of the mitochondrial temperatures for the indicated times. For cell counting, undifferentiated THP-1 cells were used, and viability was determined by both trypan blue membrane and thereby preventing release of cytochrome c and exclusion and acridine orange staining. Cells were preincubated with in- Downloaded from activation of . Others have reported that virulent strains of hibitory compounds or proteins for 1–2 h before and during infection. M. tuberculosis prevent apoptosis and cause necrosis, thereby Electron microscopy promoting bacterial survival (20–22). Although the importance of macrophage cell death is evident, the underlying bacterial factors Cells were fixed with paraformaldehyde and glutaraldehyde for 2 h, sim- and the transport of these factors have not been identified. ilarly as described previously (9). After cutting 50-nm-thick sections at 2120˚C, sections were labeled with anti–LAMP-1 (H4A3; BD Bio- In this study, we examined the effects of ESX-1 and ESX-5 http://www.jimmunol.org/ sciences) or anti-CD63 (M1544; Sanquin) and protein A conjugated to 10 secretion systems on macrophage responses during infection nm gold (EM Laboratory, Utrecht University). Sections were examined with M. marinum and M. tuberculosis. Using wild-type and ESX- using an FEI CM10 transmission electron microscope. 1– and ESX-5–deficient strains, we demonstrate that these T7SSs Cytotoxicity assays differentially affect macrophage cell death and IL-1b secretion. Additionally, we identify cathepsin B as playing a major role in Cytotoxicity was monitored via measurements of cytosolic lactate de- both responses. hydrogenase (LDH) release (OD490nm) with CytoTox 96 assay (Promega). Maximum release was achieved by lysis of cells with 1% Triton-X, and supernatants of uninfected cells were used to assess spontaneous release. Materials and Methods Percentage cytotoxicity through LDH release (OD490nm) was calculated Reagents with the formula: [(test LDH release 2 spontaneous release)/(maximal by guest on September 26, 2021 release 2 spontaneous release)] 3 100. Where indicated, apoptosis was Z-VAD, Z-DEVD-FMK, staurosporine (STS), Z-IETD-FMK, Z-LEHD- induced with 1 mM STS and necrosis with 200 mM TBH. The neutralizing FMK, Z-VAD, Z-DEVD-FMK, t-butyl hydroperoxide (TBH), cyclo- ability of the TLR2 Ab was confirmed by challenging differentiated THP-1 sporin A, cytochalasin D, BAPTA-AM, N-acetyl-L-cysteine (NAC), glu- cells or TLR2-transfected HEK293 cells with Pam3CSK4 in the presence tathione, diphenyleneiodonium chloride (DPI), and nigericin were from or absence of the TLR2 Ab (not shown). Sigma-Aldrich. CA-O74-Me ([L-3-trans-(propylcarbamoyl)oxirane-2-car- bonyl]-L-isoleucyl-L-proline methyl ester), z-FY(t-Bu)-dmk (benzyloxy- IL-1b secretion carbonyl-Phe-Tyr(t-Bu)-diazomethylketone), and pepstatin A were from b Calbiochem. Anti-TLR2 (2392) was from Genentech. Anti-human TNF-a Cell supernatants were assayed for IL-1 by ELISA (Invitrogen) according was from R&D Systems. to the manufacturer’s instructions. Bacterial strains and growth conditions Small interfering RNA-mediated inhibition of cathepsin B expression Wild-type (WT) M. marinum E11 strain, ESX-5 mutant 7C1 with trans- poson in MMAR2676 (homolog of Rv1794) (7, 10), complemented ESX-5 Differentiated THP-1 cells were seeded overnight in 6-well plates. mutant with restored ESX-5 secretion (10), ESX-1 mutant fas3.1 or fas30 Transfection was carried out the next day using cathepsin B and control with transposons in eccB1 (23), complemented ESX-1 mutant with restored (scrambled) small interfering RNA (siRNA) according to the manu- ESX-1 secretion (23), and M. tuberculosis CDC1551 strain and the ESX-5 facturer’s protocol (Santa Cruz Biotechnology, Santa Cruz, CA). Control mutant [JHU1783-2086 (24); transposon mutant in Rv1783 with defective siRNA is an mRNA sequence that will not cause the specific degradation ESX-5 secretion (E.N.G. Houben, J. Bestebroer, J. Luirink, and W. Bitter, of any cellular message. After transfection, cells were infected with M. manuscript in preparation)] were grown in Middlebrook 7H9 liquid me- marinum as described earlier. Cell lysates were analyzed by Western blot dium or Middlebrook 7H10 agar supplemented with 10% Middlebrook analysis using anti-cathepsin B (CA10; Abcam). Equal loading was con- ADC or OADC, respectively (BD Biosciences) and 0.05% Tween 80. For firmed with anti–b-actin mAb (13E5; Cell Signaling). infection experiments, bacteria were grown to logarithmic phase, washed, and diluted in RPMI with glutamax-1 medium (Life Technologies) sup- IL-1b and caspase-1 processing plemented with 10% FCS (RPMI/FCS). To remove bacterial clumps, Type 1 macrophages or THP-1 cells (differentiated for 3 h with 300 ng/ml bacterial suspensions were subjected to low-speed centrifugation (300 3 g, 3 6 m PMA and extensively washed) were seeded at 1 10 cells/well in 12-well 10 min) after which the supernatant was passed through a 5- m filter and plates. After 24 h, cells were infected with M. marinum for 1 h at a mul- used at a multiplicity of infection of 10, unless indicated otherwise. tiplicity of infection of 10. Cells were washed three times, and serum-free Cells RPMI was added for 24 or 48 h. Where appropriate, inhibitory compounds were added during and after infection. Nigericin was added 2 h before Human acute promonocytic leukemia THP-1 cells (American Type Culture sample collection. Supernatants were collected, and proteins were pre- Collection), mouse macrophage cell line RAW 264.7 (American Type cipitated by methanol–chloroform extraction as described in Ref. 26. Cell Culture Collection), and human type II alveolar epithelial cells A549 lysates were made with RIPA lysis buffer, and protein concentration was (American Type Culture Collection) were cultured in or RPMI or DMEM determined (Pierce). Samples were separated by SDS-PAGE and were (Life Technologies) supplemented with 10% FCS, 100 U/ml penicillin, and transferred onto nitrocellulose membranes. Blots were probed with anti- 100 mg/ml streptomycin. Differentiation of THP-1 cells into macrophage- human caspase-1 p20 (D57A2; Cell Signaling) for detection of cleaved 4746 DISTINCT ROLES FOR ESX-1 AND ESX-5 caspase-1, anti-human caspase-1 p10 (sc-515; Santa Cruz Biotechnology) for electron microscopy, and quantification confirmed that both M. procaspase-1, anti-human IL-1b (MAB201; R&D Systems) for pro–IL-1b marinum and M. tuberculosis WT strains translocated from b b and IL-1 , and for equal loading with anti– -actin mAb (Cell Signaling). phagolysosomes into the cytosol (Fig. 1 and Supplemental Fig. Production and secretion of ESAT-6 1B). As we have previously described for M. tuberculosis (9), an ESX-1 mutant of M. marinum did not translocate and was retained Mycobacteria were grown to mid-logarithmic phase in Middlebrook 7H9 liquid medium supplemented with 0.2% dextrose and 0.05% Tween 80. in phagolysosomes (Fig. 1A). By contrast, ESX-5 deficiency did Proteins in the cell-free supernatants were precipitated with 10% TCA, and not affect translocation either in M. marinum, as shown before (7), cell pellets were sonicated. Proteins were separated by SDS-PAGE and or in M. tuberculosis (Fig. 1 and Supplemental Fig. 1B). Contrary visualized by immunoblot using mouse anti–ESAT-6 (Hyb76-8) and mouse to ESX-1, the ESX-5 mutant of M. marinum secretes ESAT-6 anti-GroEL2 (CS44; Colorado State University). (Supplemental Fig. 1A), confirming that ESX-1–dependent se- cretion is linked to translocation. Results When translocation of these mycobacterial species was quan- ESX-5 deficiency does not affect translocation from tified over time, the M. marinum ESX-5 secretion mutant showed phagolysosome to cytosol a delayed ability to translocate compared with the WT (Fig. 1B). To explore the role of T7SSs in pathogenicity of mycobacterial After the initial 24 h, the final percentage of cytosolic bacteria was infection, the subcellular localization of WT and mutant M. ma- similar to that of the WT. As previously demonstrated (9), M. rinum and M. tuberculosis was analyzed in human macrophage- tuberculosis only starts to translocate at 48 h postinfection. From like THP-1 cell line. It has been reported that the ESX-1 secretion this time point, the percentage of cytosolic M. tuberculosis was system is essential for mycobacterial translocation and virulence similar for WT and the ESX-5 mutant (Fig. 1C) during the entire Downloaded from (9). Therefore, we determined the subcellular localization of M. course of infection. This effect was thus not observed for the marinum E11 WT strain, an isogenic ESX-1 secretion mutant with M. tuberculosis ESX-5secretionmutant(Fig.1C). In conclu- transposon in eccB1 (23), and isogenic ESX-5 secretion mutant sion, whereas deficiencies in ESX-1 block translocation, deficient MMAR2676 (homolog Rv1794) (7, 10) in PMA-differentiated ESX-5 does not. THP-1 cells. Moreover, we analyzed M. tuberculosis CDC1551 ESX-1 and ESX-5 are involved in cell death

WT strain and an isogenic ESX-5 secretion mutant with trans- http://www.jimmunol.org/ poson in eccC5 (CDC1783; JHU1783-2086) (24). Cells infected While quantifying translocation of mycobacteria, a rapid induction with various mycobacteria were analyzed by cryo-immunogold of THP-1 cell lysis was observed for WT M. marinum, whereas the by guest on September 26, 2021

FIGURE 1. ESX-5 mutants translocate to the cytosol of infected macrophages. THP-1 cells were infected with M. marinum WT, ESX-1, or ESX-5 mutants (A, B) and M. tuberculosis WT and ESX-5 mutant (C). At different time points, cells were fixed and processed for electron microscopy and cryo- immunogold labeling with lysosomal marker CD63. A, Representative electron micrographs of cells infected with M. marinum ESX-1 mutant after 2 d (Mm1, top panels) or ESX-5 mutant (Mm5, bottom panels) after 5 d. Scale bars, 200 nm. B and C, Quantification of cells containing cytosolic M. marinum WT, ESX-1, and ESX-5 mutants (B)orM. tuberculosis WT and ESX-5 mutant (C). Depicted is the percentage of cytosolic bacteria at 4, 12, 24, and 72 h postinfection. Results depict representative electron micrographs and quantification of the percentage of cytosolic bacteria of three independent experi- ments. L, lysosome; M, . The Journal of Immunology 4747

T7SS mutants were less cytotoxic (7). To evaluate the contribution summary, viable mycobacteria need to be internalized by host cells of ESX-5 and ESX-1 to cell death, THP-1 cells were infected with to induce ESX-1 and ESX-5, T7SS-dependent cell death. WT M. marinum, or the ESX-5 or ESX-1 mutants, and cell death was monitored by measuring release of cytosolic LDH and by A nonapoptotic M. marinum-induced cell death trypan blue exclusion and acridine orange staining. Within 24 h, To delineate how T7SSs induce host cell death, the role of several WT M. marinum induced cell death in infected cells (Fig. 2A,2B). known cell death pathways was investigated. Caspases are involved By contrast, the ESX-5 secretion mutant and ESX-1 secretion in many apoptotic pathways (27, 28), and herein caspase-3 is mutant showed impaired cytotoxicity: at 24 h postinfection, the central (29). Caspase-1 and its conversion of IL-1b are involved in WT induced 40% cell death whereas the ESX-1 and ESX-5 mu- cell death induced through Type 3 secretion system substrates of tants induced 15 and 16%, respectively. A constant reduction in many Gram-negative bacteria (30), but caspase-1 is not required cell death of ∼50% was apparent during the whole 72-h culture for apoptosis (31) and can be involved in necrosis. Furthermore, for both mutants (Fig. 2A,2B). ESX-1 (23) and ESX-5 com- cell wall-associated and soluble mycobacterial factors have been plementations (10) resulted in restored induction of cell death reported to induce TLR2-dependent cell death (32, 33), and TNF- comparable with that of the WT strain (Fig. 2A). In addition to a is a classic cytotoxic host protein that induces cell death (34– cell death induction in THP-1 cells, ESX-5 cytotoxicity was also 36). Also, different cellular stresses such as the generation of re- examined in other phagocytic cell types, in primary cells as well active oxygen species (ROS) or increases in cytosolic Ca2+ lev- as in several cell lines (Supplemental Fig. 2). For all infections, els have been connected to Mycobacterium-induced death (37, compared with the WT, both the ESX-5 and ESX-1 mutants 38). We assessed the role of these pathways during M. marinum- showed significantly reduced induction of cell death of up to 70% induced cell death of macrophages by using specific inhibitory Downloaded from in human primary monocytes, monocyte-derived pro- and anti- agents or blocking Abs during infection. Even though some of inflammatory macrophage subsets, dendritic cells, as well as in these factors have been connected to M. tuberculosis-induced the murine macrophage cell line RAW264.7 and human type II death, they did not play a role in M. marinum-induced cytotoxicity alveolar epithelial cells A549. As we observed similar effects in of THP-1 cells as evident from the lack of any effect of the in- different cell types, we continued our experiments using THP-1 hibitors or blocking Abs used during infection, as shown in Sup-

cells, unless stated otherwise. Similarly to M. marinum, THP-1 plemental Fig. 3. http://www.jimmunol.org/ cell infections with WT M. tuberculosis also induced cell death, Necrotic cell death has also been reported for M. tuberculosis- which was reduced upon infection with the ESX-5 mutant (Fig. infected macrophages due to mitochondrial damage. We observed 2C,2D). At 24 h, the WT induced cell death in 30% of the cells, that cell treatment with cyclosporin A—which prevents mitochon- whereas the mutant was responsible for 16% cell death. Thus, drial pore opening (39)—prior to infection with M. marinum had mycobacterial cytotoxicity is dependent on ESX-5 and ESX-1. a marginally protective effect; the release of cytosolic LDH was Next, we examined whether active internalization of bacteria was reduced by 5–23% (Fig. 3A). M. marinum-induced cytotoxicity is required for induction of cell death. Treating THP-1 cells with cy- thus also partially dependent on mitochondrial depolarization and tochalasin D, which blocks actin polymerization and thus phago- therefore mostly resembles a nonapoptotic type of cell death. cytosis, made these cells resistant to cytotoxicity after infection by guest on September 26, 2021 with WT M. marinum or ESX-1 or ESX-5 mutants (Fig. 2E). As a Cathepsin B is involved in M. marinum- and M. tuberculosis- control, cytochalasin D did not inhibit STS-induced cytotoxicity. induced macrophage cytotoxicity Furthermore, heat inactivation of the bacilli also abrogated myco- Lysosomal proteases that translocate to the cytosol in response to bacterial-induced cytotoxicity in macrophages (data not shown). In various stimuli can mediate cell death. Previous infection studies

FIGURE 2. ESX-5 of M. marinum and M. tuberculosis is required for induction of cell death. A–D, THP-1 cells were infected with M. marinum WT, ESX-1, or ESX-5 mutants, ESX-5 complemented strain (ESX-5.C), or ESX-1 complemented strain (ESX-1.C) (A, B) and M. tuberculosis WT or ESX-5 mutant (C, D). At several time points, cell death was quantified by measuring cytotoxicity (A, C) through LDH release or cell viability through cell counts (B, D). The results are mean percentages of cytotoxicity 6 SD of three experiments, each performed in duplicate. E, Cytotoxicity postinfection with M. marinum or 1 mM apoptosis-inducing STS in the presence or absence of 5 mM cytochalasin D. 4748 DISTINCT ROLES FOR ESX-1 AND ESX-5 Downloaded from

FIGURE 3. Cell death is mediated by cathepsin B. A–C, THP-1 cells (A, B) or human primary type 1 macrophages (C) were infected with M. marinum WT, ESX-1, or ESX-5 mutants for 24 h in the presence or absence of 10 mM cyclosporin A (A)or40mM CA-O74-Me, pepstatin A, or z-FY(t-Bu)-dmk (B, C). D, Infection of THP-1 cells with M. tuberculosis or ESX-5 mutant in the presence of 40 mM CA-O74-Me. Where indicated, apoptosis was induced with 1 mM STS and necrosis with 200 mM TBH. Cell death was evaluated by cytotoxicity 24 h postinfection, and results are mean 6 SD of three experiments performed in duplicate. E, Cathepsin B was depleted in THP-1 cells by siRNA for 24 h. Subsequently, untreated, control-treated (siCon), and cathepsin B- http://www.jimmunol.org/ depleted (siCathB) cells were infected with WT M. marinum for 24 h, and cytotoxicity was determined. Results are mean 6 SD of a duplicate and representative of three experiments. F, Representative Western blot analysis of cathepsin B levels in cell lysates (50 mg) of siRNA-treated cells. b-Actin reflects loading control. with M. tuberculosis strain H37Rv showed that cathepsin B and L ESX-5 is required for inflammasome activation and IL-1b inhibitors reduced DNA fragmentation and macrophage cell death, release suggesting the involvement of lysosomal proteases in Mycobac- Upon infection, macrophage sensing of bacteria results in IL-1b terium-induced cell death (40, 41). In this study, we investigated secretion, and uncovering the bacterial substrates that induce IL- by guest on September 26, 2021 a possible role for lysosomal proteases cathepsin B, D, and L in M. 1b production is important to understand how host cells detect marinum-induced cell death. As shown in Fig. 3B, treatment of infection and possibly constrain disease. Inflammasome activation THP-1 cells with the cathepsin L inhibitor z-FY(t-Bu)-dmk or upon M. marinum infection can exacerbate disease (42) indicating cathepsin D inhibitor pepstatin A did not show any detectable a host-detrimental and bacterium-promoting role of the inflam- effect on cytotoxicity of THP-1 cells by M. marinum. By contrast, matory pathway during mycobacterial infection (15). Previous inhibition of cathepsin B by CA-074-Me reduced M. marinum- studies demonstrated that infection of macrophages with patho- infected THP-1 cell cytotoxicity by 55%. The cathepsin B in- genic mycobacteria results in the induction of IL-1b release, hibitor was also the only inhibitor used that displayed reduced which is dependent on both ESX-1 and ESX-5 (7, 13–15). Fig. 4A cytotoxicity upon infection of primary type 1 macrophages with shows that indeed, in contrast to WT M. marinum, both the iso- M. marinum (Fig. 3C). In addition, we observed the same effects genic ESX-1 and ESX-5 secretion mutants do not induce IL-1b during THP-1 infections with M. tuberculosis; inhibition of ca- secretion in human proinflammatory type 1 macrophages. The thepsin B also partially protected M. tuberculosis-infected THP-1 complemented ESX-1 and ESX-5 mutant showed restored in- cells (Fig. 3D). Finally, siRNA-mediated silencing of cathepsin B in duction of IL-1b secretion. As the ESX-1 effect is most likely due THP-1 cells was performed. Western blot analysis revealed that the to its inability to translocate from the phagolysosome to the cy- addition of cathepsin B-specific siRNA indeed resulted in a signif- tosol, we sought to identify how ESX-5 substrates drive IL-1b icant reduction of cathepsin B (Fig. 3F). This reduction of ca- production. thepsin B partially rescued macrophages from M. marinum-induced The secretion of IL-1b is dependent on two distinct signals: cell death (Fig. 3E) compared with the addition of control siRNA. induction of pro–IL-1b synthesis and caspase-1 activation. In our Notably, the contributions of ESX-5 and ESX-1 to cathepsin B- system, IL-1b secretion upon infection with M. marinum was mediated cytotoxicity are different. As observed for WT bacteria, indeed dependent on caspase-1 activation, as caspase-1 inhibitor cell death induced by the ESX-5 mutant was also affected by the z-VAD abrogated IL-1b secretion (Fig. 4B). Therefore, we inves- cathepsin B inhibitor; however, this difference was only marginal b for the ESX-1 mutant (Fig. 3B) in which case the bacterium tigated the effect of ESX-5 on pro–IL-1 synthesis and caspase-1 remains in the phagolysosome of THP-1 cells. The same result activation. Cell lysates from type 1 macrophage cells infected with was also obtained for M. tuberculosis WT and the ESX-5 mutant M. marinum ESX-5 secretion mutant showed reduced levels of b (Fig. 3C). Thus, M. marinum- and M. tuberculosis-induced cyto- pro–IL-1 expression compared with those of lysates from cells toxicity in macrophages is partially dependent on the release of infected with WT or complemented bacteria (Fig. 4C). This dif- lysosomal proteases via ESX-1–mediated translocation from the ference, however, does not account for the IL-1b discrepancy, as phagolysosome, and this type of cell death is predominantly due to induction of high and equal expression of pro–IL-1b by adding the proteolytic activity of cathepsin B. ESX-5 mediates cell death LPS during infection still resulted in strongly reduced secretion of through a yet unidentified mechanism. IL-1b in the ESX-5 mutant compared with that in the WT (Fig. The Journal of Immunology 4749 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. ESX-5 is required for inflammasome activation and IL-1b release. A, IL-1b secretion was quantified in supernatants of type 1 macrophages infected with M. marinum WT, ESX-1 mutant, ESX-5 mutant, ESX-1 complemented strain (ESX-1.C), or ESX-5 complemented strain (ESX-5.C) 24 h postinfection. Results are mean (6SEM) of two experiments performed in duplicate wells. B, IL-1b secretion upon infection with M. marinum WT in the presence of z-VAD. Results represent mean (6SEM) of three experiments. D, Macrophages were infected in the presence of LPS and analyzed for IL-1b release in the supernate and pro–IL-1b expression in the cell lysates (125 mg). b-Actin reflects loading control. C and E, Type 1 macrophages were infected with M. marinum WT, ESX-5 mutant, or ESX-5.C for 24 h. Levels of pro–IL-1b, IL-1b, or procaspase-1 were examined in cell lysates (125 mg) and caspase-1 p20 in precipitated supernatants. b-Actin reflects cell lysate loading control. F and G, Release of IL-1b (F) and caspase-1 p20 (G) was analyzed in supernatants after 24 and 48 h of infection with WT and ESX-5 mutant M. marinum. C–G, The results are representative of at least three experiments.

4D). Next, we examined the induction of procaspase-1 and its Inflammasome activation by M. marinum is dependent on ROS conversion to caspase-1. Although cell lysates from macrophages and potassium efflux infected with WT and ESX-5 mutant contained equal levels of Caspase-1 conversion is achieved upon assembly and activation procaspase-1 expression, active caspase-1 was absent for cells in- of inflammasomes, cytosolic multiprotein complexes converting fected with the ESX-5 mutant (Fig. 4E). The complemented strain procaspase-1 into active caspase-1. Inflammasomes can be acti- also induced caspase-1 activation, demonstrating ESX-5–specific vated by different signals, and we investigated which triggering effects. The differences in caspase-1 activation and IL-1b secre- tion were still apparent after a prolonged infection of 48 h (Fig. mechanisms play a role during M. marinum infections. As ROS are 4F,4G). In conclusion, optimal activation of caspase-1 and sub- known to induce inflammasome activation, we investigated IL-1b sequent release of IL-1b by M. marinum is dependent on ESX-5 secretion upon type 1 macrophage infection with M. marinum in substrates. the presence of the antioxidant NAC and the NADPH oxidase 4750 DISTINCT ROLES FOR ESX-1 AND ESX-5 inhibitor DPI. Both inhibitors affected bacterially induced IL-1b infection. For this purpose, THP-1 cell infections were first per- release (Fig. 5A). Additionally, generation of active caspase-1 was formed in the presence of the cathepsin B inhibitor CA-074-Me. affected in cells infected in the presence of DPI (Fig. 5B). Thus, Compared with untreated cells, cells treated with the inhibitor ROS play a role in inflammasome activation by M. marinum. failed to activate caspase-1 or secrete IL-1b upon infection with Potassium efflux was shown to be important for inflammasome M. marinum (Fig. 6A,6B). Inhibitors for other lysosomal pro- activation by M. tuberculosis (14). Involvement of this inflam- teases cathepsin D (pepstatin A) or cathepsin L [z-FY(t-Bu)-dmk] masome trigger during M. marinum infection of type 1 macro- showed no detectable effect, suggesting a specific requirement for phage was investigated by adding KCl to the infection medium. active cathepsin B. The importance of cathepsin B in IL-1b se- Both IL-1b secretion and caspase-1 activation were inhibited upon cretion in THP-1 cells was further confirmed through silencing increasing concentrations of KCl (Fig. 5C,5D). Subsequently, we experiments using siRNA (Fig. 6C). Successful but partial knock- investigated whether K+ efflux is sufficient to trigger inflamma- down of cathepsin B (Fig. 3F) completely inhibited IL-1b secre- some activation and IL-1b release in cells infected with ESX-5 tion upon infection. In addition to THP-1 cells, inhibition of ca- mutant M. marinum. Indeed, Fig. 5E shows that IL-1b was se- thepsin B using CA-074-Me prevented the activation of caspase-1 creted by cells infected by the ESX-5 mutant after addition of the and secretion of IL-1b in primary type 1 macrophages upon M. potassium ionophore nigericin. This effect was dependent on K+ marinum infection as well (Fig. 6D,6E). Thus, cathepsin B is in- efflux, as addition of extracellular KCl abolished IL-1b secretion. strumental in inflammasome activation (Fig. 7) during mycobac- Recovery of IL-1b secretion upon nigericin stimulation supports terial infection of macrophages. our finding that pro–IL-1b is sufficiently expressed in the ESX-5 mutant but not secreted effectively. Our results suggest that the Discussion Downloaded from loss of IL-1b secretion in the ESX-5 mutant is due to an inability Recently, it has become clear that pathogenic mycobacteria secrete to induce inflammasome activation through ROS generation and a variety of effector proteins into eukaryotic target cells that un- K+ efflux. dermine the host cellular immune response. For example, delivery of ESX-1 T7SS substrates into phagocytic cells can lead to trans- b Induction of IL-1 secretion is dependent on cathepsin B location of the bacteria into the host cytosol (8, 9) and can also

Inflammasome activation has recently been reported to be triggered modulate host cell responses (7). In this study, we demonstrate http://www.jimmunol.org/ by cathepsin B in the context of infection (43–45). Moreover, that T7SS substrates are linked to cell death and IL-1b conversion sterile stimuli induce inflammasome activation through release of in infected phagocytic cells and that ESX-5–secreted substrates activated cathepsins due to phagosomal rupture (26, 46), a process are involved in these processes. that we describe to be a determining factor for mycobacterial There have been reports of mycobacterial infection inducing pathogenicity. Hence, we examined a previously unexplored role either apoptosis (35, 47, 48) or necrosis (20, 49, 50). The dis- for cathepsins in inflammasome activation during mycobacterial crepancy could be caused by the use of different bacterial viru- by guest on September 26, 2021

FIGURE 5. Generation of ROS and potassium ef- flux are important for inflammasome activation by M. marinum. A and B, Release of IL-1b (A) and caspase- 1 p20 (B) was analyzed in supernatants of type 1 mac- rophages infected with M. marinum for 24 h in the presence or absence of 2 mM DPI and/or 10 mM NAC. C, IL-1b release was quantified for type 1 mac- rophages treated with 0–40 mM KCl during infec- tion with M. marinum WT for 24 h. D, Presence of pro–IL-1b, IL-1b, or procaspase-1 in cell lysates (125 mg) and caspase-1 p20 in precipitated supernatants was examined in type 1 macrophages infected with M. marinum in the presence of 40 mM KCl. b-Actin reflects cell lysate loading control. E, Type 1 macro- phages were infected with M. marinum ESX-5 mutant in the presence or absence of KCl (0 or 40 nM) for 24 h. Nigericin (Nig) was added for an additional 2 h. IL-1b release was determined in supernatants. The results are representative of at least three experiments. The Journal of Immunology 4751 Downloaded from http://www.jimmunol.org/

FIGURE 6. Cathepsin B is involved in inflammasome activation by M. marinum. A, B, D, E, THP-1 cells (A, B) or human primary type 1 macrophages (D, E) were infected with M. marinum WT or ESX-5 mutant in the presence of 40 mM cathepsin inhibitors. IL-1b secretion (A, D), caspase-1 p20 release (B, E), and procaspase-1 expression (E) were determined 24 h postinfection. b-Actin reflects cell lysate loading control. C, Cathepsin B was depleted in THP-1 by means of siRNA for 24 h. Subsequently, untreated, control-treated (siControl), and cathepsin B-depleted (siCathB) cells were infected for 24 h, and IL-1b secretion was determined. Representative Western blot analysis of cathepsin B levels in knockdown cell lysates (50 mg) is shown in Fig. 3F.

lence strains, cell types, types of infection, or host genetic sus- ables phagosomal escape, Koo et al. (13) have previously sug- by guest on September 26, 2021 ceptibility factors (51) or by the type of assay. In this study, we gested that ESX-1 allows entry of muramyl dipeptide or other show that in THP-1 cells, M. tuberculosis and M. marinum mainly bacterial products into cytosol to induce inflammasome activation. trigger a cathepsin B-dependent (nonprogrammed; without the The ESX-1 substrate ESAT-6 was subsequently reported to have intricate regulatory mechanisms that are characteristic of apo- a direct activation effect on inflammasomes (16). By contrast, in ptosis) necrotic type of cell death. Notably, the T7SSs ESX-1 and this study we have observed that the ESX-5 mutant also affects ESX-5 play different roles in this process. It has been shown that ESX-1 substrates facilitate the trans- location of pathogenic mycobacteria from phagolysosome into the host cytosol (9), most likely by disrupting the integrity of the phagolysosomal membrane. This is consistent with our data showing that the M. marinum ESX-1 mutant remains in the phagolysosome during prolonged infections. Similar to ESX-1, ESX-5 is also required for virulence (7), but its role in patho- genesis and the precise mechanisms of its involvement in myco- bacterial infection are yet to be defined fully. Our previous results and the results described in this study show that ESX-5 substrates do not play an important role in phagolysosome translocation, and ESX-5 deficiency seems to affect a later step in the host cell in- fection cycle. Despite this difference, both mutants affect mac- rophage cell death. We speculate that substrates of the ESX-1 system induce cell death indirectly by mediating bacterial trans- location into the host cell cytosol and concomitant cathepsin B FIGURE 7. Model of T7SS-induced translocation, necrosis, and IL-1b release from the phagosome, whereas ESX-5 substrates play a secretion. Pathogenic mycobacteria are phagocytosed and in time trans- different role in cell death induction, possibly through the action locate to the cytosol through ESX-1–secreted effector proteins. Phag- of secreted effectors as summarized in Fig. 7. osomal translocation results in cytosolic release of active cathepsin B and ESX-5 effector proteins. Together, these effectors result in inflammasome T7SSs are reported to mediate inflammasome activation and IL- activation, IL-1b secretion, and cell death. The effects on cell death and b 1 release during mycobacterial infections. A role for ESX-1 is inflammasome activation are probably independent of effector molecules described in NLRP3 inflammasome activation and IL-1b release secreted via ESX-1, although the extent of necrosis and IL-1b secretion are (13, 14). As mycobacterial peptidoglycan fragment muramyl di- (largely) dependent on the ESX-1–dependent bacterial translocation into peptide can activate NALP3 inflammasomes (52) and ESX-1 en- the cytosol. This illustration was produced using Servier Medical Art. 4752 DISTINCT ROLES FOR ESX-1 AND ESX-5 inflammasome activation, whereas secretion of ESAT-6 and trans- In conclusion, for a successful infection, two sequential mo- location into the cytosol are unaffected. As inflammasome acti- lecular mechanisms trigger two different cellular responses. First, vation upon infection with M. marinum is reported to exacerbate T7SS ESX-1 mediates translocation into the cytosol. This trans- disease without affecting bacterial growth (15), ESX-5 activation location process allows subsequent release of effectors secreted by of inflammasomes is most likely beneficial for the bacterium. the secretion system ESX-5 into the cytosol, which triggers cy- Therefore, we can conclude that ESX-1 and ESX-5 play distinct totoxicity and inflammasome activation. Together, the ESX-1 and roles in inflammasome activation. We hypothesize that ESX-1 ESX-5 determine different key events in the pathogenicity of affects both inflammasome activation and onset of cell death in- mycobacteria. Greater knowledge of the role of T7SSs in modu- directly through lysosomal rupture, translocation, and concomitant lating host cell responses will allow further understanding of tu- release of cathepsin B, whereas ESX-5 substrates influence these berculosis pathogenesis and may shape the design of future vac- processes probably more directly through a cytosolic mechanism. cines and better therapeutic drugs (58). We have observed that M. marinum further induced inflamma- some activation through the generation of ROS and KCl efflux, Acknowledgments suggesting involvement of NLRP3 inflammasome, which was We thank Ben Appelmelk for valuable discussions and Genentech (South recently also demonstrated by Carlsson et al. (15). San Francisco, CA) for providing human TLR2-specific mAbs. We are Lysosomal proteases of the cathepsin family have previously grateful to Ida Rosenkrand (Statens Seruminstitut, Copenhagen, Denmark) been implicated in a number of important cellular processes, such for anti–ESAT-6 (Hyb76-8) and Rogier Rooswinkel (The Netherlands Can- as protein degradation, Ag processing, and the execution of cell cer Institute, Amsterdam, The Netherlands) for providing different com- death (53, 54). M. tuberculosis and M. marinum both trigger the pounds. Downloaded from disruption of lysosomes, which leads to the release and subsequent activation of the lysosomal protease cathepsin B, triggering a Disclosures newly discovered cell death pathway as well as an inflammasome The authors have no financial conflicts of interest. activation pathway. Inhibition of cathepsin B protected against cell death and blocked caspase-1 activation, indicating that cathepsin References

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