Stem cell-derived synthetic embryos self-assemble by exploiting cadherin codes and cortical tension

Mammalian embryos sequentially differentiate into trophectoderm and an inner cell mass, the latter of which differentiates into primitive endoderm and epiblast. Trophoblast stem (TS), extraembryonic endoderm (XEN) and embryonic stem (ES) cells derived from these three lineages can self-assemble into synthetic embryos, but the mechanisms remain unknown. Here, we show that a stem cell-specific cadherin code drives synthetic embryogenesis. The XEN cell cadherin code enables XEN cell sorting into a layer below ES cells, recapitulating the sorting of epiblast and primitive endoderm before implantation. The TS cell cadherin code enables TS cell sorting above ES cells, resembling extraembryonic ectoderm clustering above epiblast following implantation. Whereas differential cadherin expression drives initial cell sorting, cortical tension consolidates tissue organization. By optimizing cadherin code expression in different stem cell lines, we tripled the frequency of correctly formed synthetic embryos. Thus, by exploiting cadherin codes from different stages of development, lineage-specific stem cells bypass the preimplantation structure to directly assemble a postimplantation embryo.

endoderm lineages were defined by the expression of their respective markers and showed similar dynamics of cadherin expression in ETX and natural embryos ( Fig. 1d and Extended Data Fig. 1a). In natural embryos, E-cadherin was expressed in all lineages from E4.5 to E6.5; P-cadherin expression was elevated only in trophectoderm after implantation (E5.5 and E6.5); and K-cadherin expression was elevated only in primitive endoderm before implantation (E4.5) when it sorts below the epiblast ( Fig. 1d and Extended Data Fig. 1b). We verified that the corresponding proteins, similar to mRNA, were differentially expressed in ES or TS colonies (Extended Data Fig. 1c), day 4 ETX embryos and E5.5 natural embryos (Extended Data Fig. 1d). Therefore, XEN cells most resemble E4.5 primitive endoderm cells of the preimplantation embryo, whereas TS cells resemble extraembryonic ectoderm cells of the postimplantation embryo.
ES cells readily form chimeras with eight-cell-stage embryos and sort to the epiblast lineage. Given that E-and K-cadherin were differentially expressed in the epiblast and primitive endoderm of natural preimplantation embryos, we examined whether overexpression (OE) of these cadherins in ES cells would affect their subsequent sorting in the blastocysts of chimeras (Fig. 1e). We found that wild-type ES cells (n = 32 embryos) and ES cells overexpressing E-cadherin (Cdh1 OE) contributed exclusively to the epiblast of the chimeras (n = 16 embryos) (Fig. 1f,g). In contrast, ES cells overexpressing K-cadherin (Cdh6 OE) frequently contributed to the primitive endoderm (n = 16 embryos) (Fig. 1f,g and Extended Data Fig. 1e). These data are consistent with K-cadherin promoting primitive endoderm localization and E-cadherin promoting epiblast localization.
We noted that ES cells overexpressing P-cadherin (Cdh3 OE) were excluded from the preimplantation embryo (n = 13 embryos) and sorted outside the trophectoderm (Fig. 1f,g and Extended Data Fig. 1f), consistent with low P-cadherin expression in all lineages of the blastocyst and elevated P-cadherin expression in the trophectoderm only in the postimplantation natural embryo.
To evaluate whether differential adhesion plays a role in ETX embryo self-assembly, we used atomic force microscopy (AFM) to determine the cell-cell adhesion of ES, TS and XEN cells in vitro (Fig. 2a,b). We found that the mean adhesion forces between ES-ES cell couples (1.94 ± 0.54 nN) and TS-TS cell couples (2.20 ± 0.85 nN) were significantly higher than those between Stem cell-derived synthetic embryos self-assemble by exploiting cadherin codes and cortical tension Min Bao 1,2 , Jake Cornwall-Scoones 1,3 , Estefania Sanchez-Vasquez 1 , Dong-Yuan Chen 1 (Fig. 2c). We also inferred adhesion forces from the contact angles between cells 12 (Fig. 2d). The contact angles at ES-ES, TS-TS and XEN-ES junctions were greater than the contact angles between ES-TS, XEN-XEN and TS-XEN cells ( Fig. 2e and Extended Data Fig. 2a), in agreement with the AFM measurements.
To compare cell-cell contact angles in ETX and natural embryos, we employed the imaging surface analysis environment (ImSAnE) algorithm 13,14 to extract focal planes from three-dimensional (3D) stacks of E-cadherin-stained day 4 ETX and E5.5 natural embryos and unrolled these into 2D projections (Extended Data Fig. 2b). In agreement with cell contact angle measurements in stem cell doublets, we found that the homotypic contact angles were larger than the heterotypic contact angles in ETX and natural embryos (Extended Data Fig. 2c). We also noted that the contact angle between XEN-XEN and VE-VE cells at the surface of ETX embryos and natural embryos was close to 180° (Extended Data Fig. 2c,d), indicating a smooth boundary interface and reflecting the high relative tension along the interface after self-organization. Together, these measurements indicate differential cadherin expression and differential adhesion between stem cells that build-and lineages that comprise-ETX embryos.
To examine the potential relationship between differential cadherin expression and differential adhesion, we measured adhesion forces between ES and TS cells and immobilized E-cadherin (Cdh1) or P-cadherin (Cdh3) substrate (Fig. 2f). ES cells exhibited higher adhesion with immobilized E-cadherin (2.13 ± 0.83 nN) than with P-cadherin (1.07 ± 0.54 nN), whereas TS cells displayed comparable adhesion forces with both E-cadherin (2.02 ± 0.89 nN) and P-cadherin (2.41 ± 0.86 nN). We then used RNA interference to knockdown (KD) cadherins in stem cells. E-cadherin KD reduced ES-ES adhesion fourfold. P-or E-cadherin KD similarly reduced TS-TS adhesion, suggesting that downregulation of one cadherin was sufficient to decrease the TS-TS adhesion force below a critical threshold ( Fig. 2g and Extended Data Fig. 2e). Depletion of either E-or P-cadherin from XEN cells did not affect their homotypic adhesion (Fig. 2g). Thus, E-cadherin is required for the homotypic adhesion of ES cells, whereas both E-and P-cadherin are required for the homotypic adhesion of TS cells.
To assess whether the measured adhesion forces are sufficient to generate ETX embryos, we simulated assembly using the cellular Potts model (CPM) 15 , re-sampling our AFM adhesion force measurements to provide parameters (Fig. 2h). This analysis showed that, among the many sorted configurations possible with three cell types, ETX-like structures were the most favoured ( Fig. 2i)  In b and c, cadherin-and protocadherin-related transcripts are highlighted in orange. d, Violin plots showing Cdh1 (top), Cdh3 (middle) and Cdh6 gene expression (bottom) from scrNA-seq in natural and ETX embryos at different stages. NE45, NE55 and NE65 represent natural embryos collected at day 4.5, 5.5 and 6.5. ETX4, ETX5 and ETX6 represent ETX embryos collected at day 4, 5 and 6. e, Schematic of chimera aggregation. Cadherin OE ES cells expressing H2B-rFP were aggregated with eight-cell-stage wild-type embryos. Their contribution to either EPI (red), PE (green), TE (blue) or excluded cells was assessed at E4.5. Orange, chimeric contribution. f, Chimeras stained for rFP (magenta), Sox17 (green) and DNA (DAPI; grey). Scale bars, 50 μm. The magnified images show the regions indicated by dashed boxes to the left (scale bars, 10 μm). The experiments were repeated three times. WT, wild type. g, Percentage of cells contributing to EPI, PE, TE or excluded cells in chimeras, as in e. The data are presented as violin plots. Each dot corresponds to an embryo. n = 32 embryos for wild-type ES chimeras (3365 cells in total), n = 16 embryos for Cdh1 OE ES chimeras (1787 cells in total), n = 13 embryos for Cdh3 OE ES chimeras (1574 cells in total) and n = 16 embryos for Cdh6 OE ES chimeras (1894 cells in total). Statistical significance was determined by one-way ANOVA with a multiple comparison test. Numerical data are available as source data.

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Next, we determined how the observed cadherin code affects the efficiency of ETX embryogenesis. Single-cell suspensions of ES, TS and XEN cells seeded into microwell plates assembled into multiple structures, of which 15.4% formed ETX structures recapitulating postimplantation embryo morphogenesis ( Fig. 3a and Supplementary Video 1). In contrast, 38.2% of structures had more than one ES compartment, 30.8% had more than one TS compartment and 12.8% had mislocalized XEN cells or lacked an outside XEN layer; we termed these missorted ETX structures ( Fig. 3b-d). The proportion of correctly sorted ETX embryos  Letters NaTurE CEll Biology plateaued at 15% after the first day of culture (Extended Data Fig. 3a). Thus, the three cell types undergo a sorting phase within the first 24 h of seeding before becoming consolidated into compartments. We hypothesized that cells can no longer sort during the consolidation phase due to their low mobility. To test this, we filmed ETX embryo formation by time-lapse microscopy and tracked cell mobility (Extended Data Fig. 3b). This revealed all cell types to be mobile during the cell sorting stage, becoming relatively immobile during the tissue consolidation stage (Extended Data Fig. 3c,d). XEN cell sorting fell into a particular two-phase pattern. XEN cells sorted efficiently; over 90% of XEN cells formed a monolayer, first enveloping ES cells and then spreading to cover TS cells. KD of E-cadherin (Cdh1) or K-cadherin (Cdh6), which are co-expressed in XEN cells, reduced the frequency of ETX embryos having a continuous XEN layer (Fig. 3e,f). However, XEN cells with OE of Cdh1 or Cdh6 often missorted within these compartments (Fig. 3e,f). Thus, an optimal balance of E-cadherin and K-cadherin contributes to the proper sorting of XEN cells in ETX embryos.
Differential adhesion cannot fully account for the ability of XEN cells to envelop the TS layer because the adhesion force between ES and XEN cells is larger than that between ES and TS cells, and XEN cells were found between the ES and TS compartments in approximately 10% of CPM simulations that sampled these data (Fig. 2i). The discrepancy between the predicted interfacial hierarchy for the sorted configuration in the ETX embryo and the measured differential adhesion force led us to hypothesize that the low number of XEN cells we used for making ETX embryos was insufficient to cover all ES cells during the sorting stage. To test this, we seeded ES and TS cells with between five and ten XEN cells and fixed the nascent structures at days 1 and 3. We found that low numbers of XEN cells first covered the ES cells only; subsequently, the TS cells enveloped the entire structure (Extended Data Fig. 3e,f). When we seeded approximately ten ES cells per structure, the XEN cells completely covered the ES cells, thereby excluding TS cells (Extended Data Fig. 3e,f), consistent with our measurements of differential adhesion.
Previous studies have reported a role for cortical stiffness in cell sorting, particularly in cell externalization [16][17][18] , prompting us to consider whether cortical tension may influence the capacity of XEN cells to form their external monolayer. Indeed, our AFM 17 measurements indicated that cortical stiffness is lower in XEN cells than in either TS or ES cells (Extended Data Fig. 3g). To determine whether differences in cortical stiffness between the different stem cell types of ETX embryos were due to differential actomyosin activity, as in other systems 17,19,20 , we measured cortical stiffness in the presence of blebbistatin (a myosin inhibitor 21 ). Blebbistatin reduced the cortical stiffness of both ES and TS cells to the same level as in XEN cells (Extended Data Fig. 3h). We also found that well-sorted ETX embryos treated with either blebbistatin or cytochalasin D (an actin depolymerizer 22 ) at day 3 for 24 h-once the primary sorting phase was completed-failed to maintain efficient sorting compared with control ETX embryos (Extended Data Fig. 3h). Moreover, when we treated well-sorted ETX embryos with either blebbistatin or cytochalasin D for 24 h at day 3, once the primary cell sorting phase was complete, more than 80 and 85% of blebbistatin-and cytochalasin D-treated structures, respectively, failed to maintain sorting compared with 18% of control ETX embryos (Extended Data Fig. 3h).
To further test the role of cortical stiffness on XEN cell externalization, we used a CPM in which cortical stiffness can be tuned independently. We found that lower stiffness increased both the sorting efficiency and speed of XEN cell externalization (Extended Data Fig. 3i), suggesting that the softness of XEN cells is important for this event. Together, these data suggest that, in addition to the differential expression of distinct cadherins, cortical stiffness plays a role in the self-assembly of stem cells into ETX embryos.
Next, we examined the function of the cadherin code in ES and TS cells during ETX embryo assembly. KD of P-cadherin (Cdh3) in TS cells, but not in ES cells, resulted in TS mislocalization and disrupted ETX embryogenesis. Similarly, KD of E-cadherin (Cdh1) in ES cells, but not TS cells, disrupted ETX embryogenesis. ETX embryo formation still occurred following E-cadherin depletion from TS cells ( Fig. 3g and Extended Data Fig. 4a), suggesting that differential expression of P-cadherin between ES cells and TS cells is sufficient to drive their sorting. We noticed that E-cadherin and P-cadherin showed different levels of expression in individual wild-type ES and TS cells, respectively (Extended Data Fig. 4b-d).
We considered that subsets of wild-type stem cells with low cadherin expression compromise ETX embryo formation. Indeed, when we combined wild-type ES and XEN cells with either a P-cadherin OE subset or a P-cadherin KD subset of TS cells, P-cadherin KD TS cells mislocalized to the ES compartment. Similarly, when combining wild-type XEN and TS cells with either an E-cadherin OE subset or an E-cadherin KD subset of ES cells, we observed mislocalization of E-cadherin KD ES cells in the TS compartment (Extended Data Fig. 5a,b). Thus, populations of ES and TS cells with low E-cadherin and low P-cadherin expression, respectively, compromise sorting in ETX embryos. Strikingly, mixing E-cadherin OE ES cells and P-cadherin OE TS cells with wild-type XEN cells increased ETX embryogenesis efficiency by almost threefold from approximately 15% with wild-type stem cells to approximately 42% with the OE cells (Fig. 3h). The time course of the sorting of E-cadherin OE ES cells, P-cadherin OE TS cells and XEN cells revealed that around 30% of these structures were well-sorted 12 h after cell seeding compared with 6.8% of wild-type structures (Extended Data Fig. 5c,d).   Thus, the sorting rate is increased following cadherin OE, as suggested in simulations 23 . Together, these results indicate that variable E-cadherin expression in ES cells and P-cadherin expression in TS cells limits the efficiency of ETX embryo formation. As implantation-stage embryo morphogenesis requires both lumenogenesis and basement membrane formation, we wanted to determine whether cadherin-enhanced ETX embryo self-organization also improved these events. ETX embryos generated from wild-type stem cells formed a central lumen-corresponding to the lumen of the epiblast rosette at implantation 24,25 -within the ES compartment by day 2. By day 3, multiple lumens developed in the TS compartment, corresponding to the multiple lumens of the E5.5 extraembryonic ectoderm, and these unified into a single cavity between days 4 and 5, as in natural development by E6.0 (Fig. 4a). Such a single unified cavity formed in over 90% of properly sorted ETX embryos but in fewer than 5% of ETX structures with missorted ES and TS cells (Extended Data Fig. 6a). Moreover, ETX structures with missorted XEN cells lacked cavities entirely (Extended Data Fig. 6b). Importantly, proper sorting and amniotic cavity-like formation were observed in only 9% of structures built from wild-type cells but in 40% of structures built from E-cadherin OE ES cells, P-cadherin OE TS cells and wild-type XEN cells (Fig. 4b,c). Moreover, the structures formed from cadherin OE cells, and the cavities within them, were longer than in ETX embryos built from wild-type cells (Extended Data Fig. 6c,d) after 3 d in culture. Together, this indicates that E-and P-cadherin OE in ES and TS cells, respectively, promotes cavity formation in ETX embryos.
Lumenogenesis requires signalling from the basement membrane, produced by the visceral endoderm 24,26 . Accordingly, we found that ETX structures with missorted XEN cells, which lacked a cavity, also failed to establish a basement membrane (Extended Data Fig. 6e). A continuous laminin-containing basement membrane was detected in 78% of structures built from E-cadherin OE ES cells, P-cadherin OE TS cells and XEN cells (Fig. 4d) but in only 45% of structures made from wild-type ES, TS and XEN cells (Fig. 4e). Thus, elevated expression of E-and P-cadherin in ES and TS cells, respectively, increases the successful formation of basement membrane, lumen and correctly sorted ETX embryos (Fig. 4f).
Our findings shed light on the remarkable self-assembly of stem cells into synthetic embryos 10,11,[27][28][29][30][31][32] . We show that this requires a cadherin code that, through strong homotypic interactions, sorts ES and TS cells into distinct compartments. In contrast, heterotypic interactions enable XEN cells to first surround ES and then TS cells. Although XEN cells have a cadherin code resembling preimplantation primitive endoderm, they nevertheless attain the ability to support synthetic postimplantation morphogenesis (Extended Data Fig. 6f). These differences between natural and ETX embryos highlight the distinct use of common rules between biological development and bioengineering. Synthetic embryo assembly utilizes these codes in a distinct way: XEN cells use the preimplantation code of primitive endoderm to sort in a layer below ES cells, whereas TS cells use the postimplantation code of extraembryonic ectoderm to sort as a cluster above ES cells.
The outcome of cell sorting has been modelled previously by considering cell-specific differences in interfacial energies maximizing the most energetically favourable cell interfaces 15,[33][34][35][36] . Disparity in interfacial energy was considered to reflect adhesion differences, with cadherins being the best-characterized effectors 1,37 , as espoused in the differential adhesion hypothesis (DAH) 33,38 . In accord, we now show that cell sorting is driven in ETX embryos by increased cadherin-mediated homotypic interactions in relation to heterotypic interactions. The later development of the differential interfacial tension hypothesis (DITH) 16,17,[39][40][41] invoking the role of differential cortical tension in sorting resonates with our findings on XEN cell externalization in self-assembly. Together, our observations support the balance between adhesion and tension (DAH versus DITH) as in biophysical models of cell sorting. However, incomplete ES-TS sorting still results in local order, emphasizing a need for global-scale sorting to fully recapitulate natural morphogenesis. DAH and DITH only account for local sorting to form homotypic clusters of ES and TS cells, as seen even in missorted structures. For complete sorting, ETX embryos must escape from locally correct neighbourhoods within globally incorrect patterns to explore alternative conformations. If cells remain in local minima before cell sorting is complete, structures will remain missorted.
The importance of the cell type-specific cadherin code is illustrated by our finding that established wild-type stem cell lines show heterogeneous cadherin expression, with some subsets below the threshold required to support proper sorting. Elevating E-cadherin in ES cells and P-cadherin in TS cells substantially improves ETX embryogenesis efficiency (Fig. 4f). This identifies a broader challenge in synthetic biology; namely, characterizing and ameliorating the impacts of heterogeneity in stem cell lines-factors that remain for the most part undefined but reported 35,[42][43][44] . Such heterogeneity might affect the distribution of cell-cell cohesive properties within the same cell population 45 , confounding the hierarchy of interactions necessary to drive self-organization of other organoid structures 46,47 . Thus, the principles of self-organization that we now describe can provide a means for increasing the efficiency of formation of different types of organoids by modulating interactions and the physical properties of cells.

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green), E-cadherin (monochrome) and DAPI (grey) staining in day 4 cadherin OE ETX structures formed by combining E-cadherin OE ES cells with P-cadherin OE TS cells and wild-type XEN cells. ETX structures formed by combining
wild-type cells were used as a control. Scale bars, 100 μm. c, Comparison and quantification of joined cavity formation in cadherin OE and control ETX structures. n = 361 (control group) and n = 253 (cadherin OE group). N = 5 for each condition. The data are presented as means ± s.d. Statistical significance was determined by unpaired two-tailed Student's t-test. d, representative image showing Oct4 (red), Gata4 (green), laminin (monochrome) and DAPI (blue) staining in day 4 cadherin OE ETX structures formed by combining E-cadherin OE ES cells with P-cadherin OE TS cells and wild-type XEN cells. ETX structures formed by combining wild-type cells were used as a control. Scale bars, 100 μm. e, Quantification of the structures that contained continuous or discontinuous laminin. n = 40 ETX structures per condition. N = 3. The data are presented as means ± s.d. Statistical significance was determined by unpaired two-tailed Student's t-test. f, Self-organization principles in stem cell-derived ETX embryos. Differential expression of E-, K-and P-cadherins enables the sorting of ES (epiblast-like), XEN (VE-like) and TS (TE-like) stem cells. Wild-type ES cells with low E-cadherin expression and wild-type TS cells with low P-cadherin expression exhibited detrimental global sorting efficiency. This could be overcome by overexpressing E-cadherin in ES cells and P-cadherin in TS cells to increase the efficiency of ETX embryo formation. Proper morphogenesis, including cavity formation, basement membrane formation (purple) and symmetry breaking can only be observed in well-sorted structures. Numerical data are available as source data.
TS cells (wild type) were cultured on mitomycin C (M4287; Sigma-Aldrich)-treated CF1 mouse embryonic fibroblasts (MEFs) in TSF4H medium with RPMI 1640 (M3817; Sigma-Aldrich) containing 20% foetal bovine serum Mouse embryos. Mice were maintained according to national and international guidelines. All experiments were regulated by the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body. Experiments were approved by the Home Office. Animals were inspected daily and those that showed health concerns were culled by cervical dislocation. Six-week-old female CD-1 mice were used in all of the animal experiments. All experimental mice were free of pathogens and were on a 12 h light/12 h dark cycle, with unlimited access to water and food. The temperature in the facility was controlled and maintained at 21 °C.
Generation of cell lines. The experiments were performed using mouse E14 wild-type ES cells 48 (derived in the laboratory of M.Z.-G.). The H2B-CFP ES cells were a gift from M. Elowitz, the wild-type TS cells were a gift from J. Nichols, the EGFP TS cells were a gift from J. Rossant, the wild-type XEN cells were a gift from E. Na and the H2B-RFP XEN cells were derived from wild-type XEN cells. Cdh1 and Cdh6 OE ES cells were generated from E14 wild-type ES cells, the Cdh3 OE TS cells were generated from wild-type TS cells and the Cdh1 and Cdh6 OE XEN cells were generated from wild-type XEN cells (see below).
To generate cadherin OE ES, TS or XEN cell lines, 0.5 μg of a super piggyBac transposase expression vector (PB210PA-1; System Biosciences) and 2 μg Cdh1-pHygro, Cdh3-pHygro or Cdh6-pHygro plasmid were co-transfected into cells using Lipofectamine. Cells were passaged for 24 h after transfection and subjected to selection in medium containing 50 μg ml −1 hygromycin (10687010; Thermo Fisher Scientific) for 1 week. A similar approach was used to generate the stable nuclear reporter H2B-RFP XEN cell line.
Cloning. Cloning procedures were performed using Gateway technology (Thermo Fisher Scientific). The fragment of interest (Cdh1, Cdh3 or Cdh6) was amplified by PCR to introduce attB sites. These fragments were cloned into the pDONR221 vector (a gift from J. Silva) using BP clonase II (11789020; Thermo Fisher Scientific). The fragment of interest (Cdh1, Cdh3 or Cdh6) was subcloned into a pHygro vector containing a hygromycin resistance cassette for expression in stem cells. The recombination reaction was carried out using LR Clonase II (11791100; Thermo Fisher Scientific).

Cell doublets experiment.
To measure cell-cell contact angles, 1200 dissociated ES, TS or XEN cells were mixed in pairs and seeded onto AggreWell plates (34411; STEMCELL Technologies) pretreated with rinsing solution (07010; STEMCELL Technologies). Cells were centrifuged at 100g for 3 min. After 1 h incubation at 37 °C, cells were collected and fixed for immunostaining.

Cadherin-coated surface preparation.
To measure the adhesion forces between cells and cadherin-coated surfaces, we followed a previous study 49 . Briefly, gold-coated glass cover slips (AU.0100; Platypus) were cleaned in argon plasma for 30 s and subsequently functionalized by immersion in thiol solution (P50757; Sigma-Aldrich) for 16 h, then rinsed with EDTA-buffer (15575-020; Thermo Fisher Scientific) to remove excess thiol. The cover slips were subsequently incubated with 10 μg ml −1 recombinant E-cadherin (8875-EC-050; R&D Systems) or P-cadherin (761-MP-050; R&D Systems) for 12 h at 4 °C. Before making force measurements, the cadherin-coated surfaces were washed with HEPES (15630106; Thermo Fisher Scientific) and activated by incubation in the same buffer for 30 min.
Adhesion force measurement. Cell-cell or cell-cadherin adhesion forces were measured using an atomic force microscope (Bruker NanoScope) coupled to a confocal microscope (TCS SP5II; Leica). Tipless silicon nitride cantilevers were V shaped, with nominal spring constants (60 pN nm −1 ; NP-0; Veeco Instruments). The atomic force microscope cantilevers were plasma cleaned before functionalization with concanavalin A, as described previously 17,49 . The system was calibrated in cell-free medium at 37 °C before each experiment by measuring the deflection sensitivity on a glass surface, allowing the cantilever spring constant to be determined in situ. Before loading the sample, the sample stage movement was calibrated using NanoScope software (version 6.13). Before measurements, cells were dissociated with TrypLE (12604013; Thermo Fisher Scientific) and resuspended in HEPES-buffered cell culture medium (15630056; Thermo Fisher Scientific). Cell suspensions were loaded into the atomic force microscope sample chamber and a single cell was captured by pressing the cantilever onto the cell with a contact force of 500 pN for 1 min. The cell was lifted from the surface and allowed to establish firm adhesion on the cantilever for 5 min. To measure the cell-cell adhesion force, the captured cell was lowered to contact with another single cell cultured on a gelatin-coated glass-bottom Petri dish (FD35; WPI). To measure the cell-cadherin adhesion force, the captured cell was lowered to contact cadherin-coated cover slips. The approach and retraction speeds were kept constant at 10 μm s −1 with a contact force of 2 nN. Three force curves were acquired for each cell. The captured cell was left to recover for 3 min between different adhesion force measurement cycles before it was adhered to the surface in a different position. Before and after every single measurement, we checked that our probing cell remained on the cantilever by direct observation. Maximal cell adhesion forces as well as the single rupture force step height were extracted from retrace curves using JPK IP software.
Cell cortical stiffness measurements. The stiffness of cells was measured using an atomic force microscope (Bruker NanoScope) coupled to a confocal microscope (TCS SP5II; Leica), as described previously 50,51 . The point-and-shoot procedure (NanoScope software; Bruker) was used to measure cell stiffness. All cells were kept in CO 2 -independent cell culture medium during the measurement.
A fluorescent 10 μm polystyrene bead (Invitrogen) was glued to silicon nitride cantilevers with nominal spring constants of 0.06 N m −1 (NP-S type D; Bruker). Indentations were performed using the single force option with a total indentation depth of 50-100 nm. To obtain cell stiffness values from force curves, PUNIAS software was used as described previously 50,51 . Multiple force displacement curves (at five different locations) were fitted to the Hertz model to calculate cell cortical stiffness (Young's modulus).
Stem cell-derived ETX embryo generation. ETX embryos were generated as described previously 10 . Approximately 6000-7000 ES cells, 15,000-19,000 TS cells and 5000-6000 XEN cells were added dropwise into AggreWell plates having 1200 microwells in one well (34411; STEMCELL Technologies). The microwells were treated with rinsing solution (07010; STEMCELL Technologies). Cells were centrifuged at 100g for 3 min. 1.5 ml c-IDG medium containing 7.5 nM ROCK inhibitor (72304; STEMCELL Technologies) was added dropwise to each well. On the following day (day 1), 1 ml medium was removed gently from each well and replaced with 1 ml fresh c-IDG medium without ROCK inhibitor. This step was repeated once to fully remove the ROCK inhibitor. On day 2, 1 ml c-IDG medium was replaced with 1 ml fresh medium. On day 3, the media was replaced with IVC1 medium 10,24,52 Table 1.

RNA extraction and real-time qPCR.
Total RNA was extracted from cells using TRIzol Reagent (15596-026; Invitrogen). Real-time qPCR was performed with SYBR Green PCR Master Mix (4368708; Applied Biosystems) and StepOnePlus Real-Time PCR System (Applied Biosystems). The fold change in mRNA expression was determined using the ΔΔCt method with Gapdh as an endogenous control. For the qPCR primers used, see Supplementary Table 1.
scRNA-seq sample preparation and dissociation. Natural and ETX embryos were transferred to Falcon tubes, washed with PBS and incubated in TrypLE Express (12604013; Gibco) for 15 min at 37 °C to dissociate them into single cells. If clumps remained, the incubation was extended for an additional 5 min at 37 °C and the sample pipetted further. Samples were filtered to remove large clumps, centrifuged at 200g for 5 min and resuspended in PBST (containing 0.02% Tween 20) and then processed for encapsulation, as previously reported 29,53 . For E5.5 embryos, one litter of 12 embryos was dissociated together. A total of 15 ETX embryos were dissociated for sequencing. Cells in culture were dissociated into a single-cell suspension using TrypLE Express (12604013; Gibco) and multiplexed using MULTI-seq lipid-modified oligos before running on two 10X Genomics lanes using single-cell 3′ version 3 reagents as reported 54 .
scRNA-seq analysis. A previously submitted and filtered scRNA-seq dataset comprising the ETX and natural embryos was downloaded from the Gene Expression Omnibus repository (GSE161947) 29 . The count matrix was loaded into Seurat version 3 (ref. 55 ), the fraction of counts mapping to mitochondrial genes was computed and the object was then log-normalized to a scale factor of 10,000. The 2,000 most variable genes were computed, the object was scaled and the percentage of mitochondrial counts was regressed out. Dimensional reduction was performed with principal component analysis and the data were projected on a uniform manifold approximation and projection low-dimensional space using 20 principal components. The embryonic, endoderm and trophectoderm lineage identity was pooled from previous annotations 29 , corresponding to clusters with high Dnmt3b, Gata4 and Lamb1 and Cdx2 and Gata2 expression levels, respectively. The average expression was computed using the average expression function and the latter was log 2 normalized. The expression levels of cadherins and protocadherins were subsequently plotted on a heatmap. The uniform manifold approximation and projection plots were directly plotted using the methodology described recently 29 after keeping the ETX and natural embryo samples only and re-computing the neighbourhood graph (five neighbours and 30 principal components; code at https://github.com/fhlab/ scRNAseq_inducedETX).

Time-lapse imaging.
To perform time-lapse imaging, cells were seeded on Gri3D PEG-hydrogel dishes with glass bottoms (SUN Bioscience) and imaged under a spinning-disc microscope (3i) with a Zeiss EC Plan-NEOFLUAR 20×/0.5 objective in a humidified chamber at 37 °C with 5% CO 2 . The structures were imaged every 5-10 min by collecting image stacks of 10 μm z-planes. Images were processed using SlideBook 5.0 (3i). Raw data were processed using the open-source image analysis software Fiji. For single-cell tracking, Imaris image analysis software (Bitplane) was used.
Quantification and statistical analysis. Criteria for selecting ETX embryos. Egg cylinder structures with one TS-derived compartment and one ES-derived compartment, covered by an outside XEN-derived visceral endoderm-like monolayer were considered to be well-sorted ETX embryos for analysis. Structures that did not fulfil these criteria were considered to be missorted ETX structures. Structures containing all three types of cells were collected and counted for quantification.
Image data acquisition, processing and quantification. Fluorescence images were acquired using an inverted Leica SP8 confocal microscope (LEICA software LAS X; Leica Microsystems) with a Leica FLUOTAR VISIR 25× or 40× objective. Images were acquired with 0.5-3.0 μm z-separation. To screen entire structures, the tile-scan imaging mode with automatic image stitching of the SP8 confocal microscope was used. All images were analysed and processed using Fiji software (http://fiji.sc). For digital quantifications and immunofluorescence signal intensity graphs, laser power and detector gain were maintained constant to permit quantitative comparisons of different experimental conditions within a single experiment.
To evaluate cell-cell contact angles in 3D ETX and natural embryos, ImSAnE 13,14 was employed to extract planes of the embryos from 3D stacks of E-cadherin and unroll them into a two-dimensional projection. Different lineages were indicated by different nuclear markers during analysis. Geometric observables as well as general distortions in projections can be correctly quantified using built-in correction methods in MATLAB 14 .
Numerical simulations. CPM. A CPM 15 was used to infer the predicted distributions of conformations given measurements of cell adhesion from AFM, as well as to determine the roles of cortical stiffness on the self-organization of ETX embryos. We parameterized adhesion strengths using cohesion forces between pairs of cell types, which were directly measured by AFM (Supplementary Table 2). For each simulation, we sampled this distribution to build the adhesion (J) matrix. Specifically, for a given element in this matrix, we sampled (with replacement) the set of AFM cohesion forces measured between pairs of cell types (for example, ES-ES, ES-TS and so on), performed around 500 times to establish an ensemble of J matrix samples. Each J matrix sample was used to perform a CPM simulation, generating an ensemble distribution of conformations over time. Simulations evolve via a stochastic minimization of an energy function (see equation (1) in the Supplementary Information) that accounts for both differential affinity and other physical properties of cells. Simulations were scored at each time point for being one of the 16 possible sorted configurations (Fig. 2i) by determining whether each cell type was enveloping and/or contiguous (see Supplementary Information for details). To test for the importance of softness in XEN cell externalization, we varied the cortical stiffness parameter for XEN cells ( λ XEN P ) and repeated the above simulation procedure.
Statistics and reproducibility. Statistical tests were performed using GraphPad Prism (versions 8.0 and 7.0a) software (with the exception of the analysis of sequencing data). Data with a Gaussian distribution were analysed using a two-tailed unpaired Student's t-test (two groups) or one-way analysis of variance (ANOVA) (multiple groups) with Tukey's multiple comparison test. Significant differences in the variance were taken into account using Welch's correction. Data that did not have a Gaussian distribution were analysed using a Mann-Whitney U-test (two groups) or Kruskal-Wallis test (multiple groups) with Dunn's multiple comparison test. For all quantifications, a minimum of three independent experiments were performed. The in vitro cell experiments were not randomized as it was not necessary. For experiments with chemical inhibitors, samples were randomly allocated to control and experimental groups. Embryos were randomly allocated to control and experimental groups for the in vivo experiments. Data collection and analysis were not performed blind to the conditions of the experiments. No statistical method was used to predetermine sample sizes. Sample sizes were determined based on previous experimental experience. The sample sizes used to derive statistics are provided in each figure caption. No data Extended Data Fig. 2 | Differential adhesion force in ETX embryos. (a) representative images of cell doublets for homotypic and heterotypic cell pairs (green, Gata4; cyan, Tfap2c; gray, Oct4; red, F-actin). Experiments were repeated 3 times. Scale bar is 10 μm. (b) Use of ImSAnE 'Unrolling' algorithm to project 3D E-cadherin staining stacks onto a 2D plane. Cell contact angles were quantified using built-in correction methods. Geometric observables as well as generally distortions in projections can be correctly quantified. Scale bar is 100 μm. (c) Cell-cell contact angle measurements based on ImSAnE method in day 4 ETX and E5.5 natural embryos. Total measured cell pairs in ETX embryos: ES-ES: n = 24; TS-TS: n = 15; XEN-XEN: n = 16; ES-TS: n = 24; XEN-TS: n = 19; XEN-ES: n = 16; XENi-XENi: n = 16. Total measured cell pairs in natural embryos: EPI-EPI: n = 20; TE-TE: n = 17; VE-VE: n = 22; EPI-TE: n = 24; EPI-VE: n = 16; TE-VE: n = 19; VEi-VEi: n = 24. Data are presented as box-whisker plots, black line inside the box indicates the median value and the error bar shows min to max value. Statistics calculated by one-way ANOVA with a multiple comparison test. (d) Enlargement of the boundary area in a day4 ETX embryo stained with E-cadherin, with homotypic contacts highlighted in blue (TS-TS), red (ES-ES) and purple (XEN-XEN), and the heterophilic boundary interface in yellow. Angles formed at tricellular junctions between different types are indicated: EX, TX and ET, angles between heterotypic contacts (ES-XEN, TS-XEN and ES-TS); EE, TT and XX, angles between homotypic contacts (ES-ES, TS-TS and XEN-XEN). XXi indicates contact angles of XEN cells at cell-medium interface. Experiments were repeated 6 times. Scale bar represents 20 μm. (e) E-cadherin and P-cadherin mrNA expression in cells after downregulation of E-or P-cadherin by rNAi, scrambled sirNA was used as a control. P-cadherin mrNA expression in ES cells after overexpression of P-cadherin. N = 4 for all conditions. Data are presented as mean ± SD. Statistics calculated by unpaired two-tailed Student's t test. Source numerical data are available in source data.

NaTurE CEll Biology
Extended Data Fig. 3 | Differential cadherin code and cortical tension regulate self-organization in ETX embryos. (a) Time course of formation of correctly-sorted ETX embryos following seeding. 0.5-h: 0/515 structures; 12-h: 79/1292 structures; 24-h: 160/1074 structures; 48-h: 134/888; 72-h: 93/702 structures. N = 3 for each condition. Data are presented as Mean ± SD. Statistics calculated by unpaired two-tailed Student's t test. (b) Live cell imaging and tracking. H2B-rFP-XEN (green), H2B-CFP-ES (red) and EGFP-TS (blue) were overlaid with Imaris cell-tracking spheres. (c) Quantification of mobility for different types of cells during self-organization. Data are presented as Mean ± SD at different time points. (d) The bar graph shows the average mobility for different cell types during self-organization at different time ranges after cell seeding. Data are presented as Mean ± SEM. 12 structures from 3 independent experiments were imaged for quantification. Statistics calculated by unpaired two-tailed Student's t test. (e) Examples of structures made from low (control) and high number of XEN cells, stained at day-1 and day-3. Experiments were repeated 3 times. Scale bar, 10 μm. (f) Schematic of morphological transitions when using low and high number of XEN cells. (g) Cortical stiffness measurements for indicated cell types before and after treatment with Blebbistatin (Bleb). Total measured cell numbers for each condition: ES: n = 58; ES + Bleb: n = 34; TS: n = 68; TS + Bleb: n = 31; XEN: n = 68; XEN + Bleb: n = 35. Data are presented as Mean ± SD. Statistics calculated by ANOVA with a multiple comparison test. (h) Day 3 wellsorted ETX embryos were cultured with either blebbistatin, cytochalasin D or DMSO (control) during consolidation stage for 24 hrs and immuno-stained to reveal the indicated markers. Quantification shows the percentage of disorganized ETX structures. n = 84 (Bleb treated), n = 83 (Cyto D treated) and n = 75 (control), N = 3 for each condition. Data are presented as Mean ± SD. Statistics calculated by unpaired two-tailed Student's t test. Scale bar, 100 μm.
(i) CPM modelling shows the effect of XEN cell stiffness (λ P ) on externalization efficiency (N = 474). The sorting efficiency calculated for each time-point is plotted as a heat-map, overlayed with contours (white dotted lines). Source numerical data are available in source data.


A newly identified flavoprotein disulfide reductase Har protects Streptococcus pneumoniae against hypothiocyanous acid

Hypothiocyanous acid (HOSCN) is an antimicrobial oxidant produced from hydrogen peroxide and thiocyanate anions by heme peroxidases in secretory fluids such as in the human respiratory tract. Some respiratory tract pathogens display tolerance to this oxidant, which suggests that there might be therapeutic value in targeting HOSCN defense mechanisms. However, surprisingly little is known about how bacteria protect themselves from HOSCN. We hypothesized that tolerant pathogens have a flavoprotein disulfide reductase that uses NAD(P)H to directly reduce HOSCN, similar to thioredoxin reductase in mammalian cells. Here, we report the discovery of a previously uncharacterized flavoprotein disulfide reductase with HOSCN reductase activity, which we term Har (hypothiocyanous acid reductase), in Streptococcus pneumoniae, a bacterium previously found to be tolerant of HOSCN. S. pneumoniae generates large amounts of hydrogen peroxide that can be converted to HOSCN in the respiratory tract. Using deletion mutants, we demonstrate that the HOSCN reductase is dispensable for growth of S. pneumoniae in the presence of lactoperoxidase and thiocyanate. However, bacterial growth in the HOSCN-generating system was completely crippled when deletion of HOSCN reductase activity was combined with disruption of GSH import or recycling. Our findings identify a new bacterial HOSCN reductase and demonstrate a role for this protein in combination with GSH utilization to protect S. pneumoniae from HOSCN.

Hypothiocyanous acid (HOSCN) is an antimicrobial oxidant produced from hydrogen peroxide and thiocyanate anions by heme peroxidases in secretory fluids such as in the human respiratory tract. Some respiratory tract pathogens display tolerance to this oxidant, which suggests that there might be therapeutic value in targeting HOSCN defense mechanisms. However, surprisingly little is known about how bacteria protect themselves from HOSCN. We hypothesized that tolerant pathogens have a flavoprotein disulfide reductase that uses NAD(P)H to directly reduce HOSCN, similar to thioredoxin reductase in mammalian cells. Here, we report the discovery of a previously uncharacterized flavoprotein disulfide reductase with HOSCN reductase activity, which we term Har (hypothiocyanous acid reductase), in Streptococcus pneumoniae, a bacterium previously found to be tolerant of HOSCN. S. pneumoniae generates large amounts of hydrogen peroxide that can be converted to HOSCN in the respiratory tract. Using deletion mutants, we demonstrate that the HOSCN reductase is dispensable for growth of S. pneumoniae in the presence of lactoperoxidase and thiocyanate. However, bacterial growth in the HOSCN-generating system was completely crippled when deletion of HOSCN reductase activity was combined with disruption of GSH import or recycling. Our findings identify a new bacterial HOSCN reductase and demonstrate a role for this protein in combination with GSH utilization to protect S. pneumoniae from HOSCN.
We recently reported that S. pneumoniae is relatively tolerant of HOSCN, when compared with another respiratory microbe Pseudomonas aeruginosa (20). This raises the question as to what mechanisms are responsible for HOSCN tolerance. We first investigated the contribution of the low molecular weight thiol GSH in S. pneumoniae (21). The genes required for the biosynthesis of GSH are absent in S. pneumoniae, so the bacteria rely on its import from their environment (22)(23)(24). The ability to import and recycle GSH was shown to be important for the ability of S. pneumoniae to cope with HOSCN; however, even when they lacked GSH, the bacteria could still survive HOSCN exposure for up to 1 h. Also, they were able to grow, albeit more slowly, in the presence of LPO, where in the presence of SCN − , bacterially produced H 2 O 2 is converted to HOSCN (21). These findings suggested the presence of additional HOSCN protective mechanisms in S. pneumoniae.
HOSCN is known to be well tolerated by mammalian cells, owing to the presence of thioredoxin reductase (TrxR), a flavoprotein disulfide reductase (FDR) that can directly reduce HOSCN using NADPH (25). Interestingly, an enzyme that consumes NAD(P)H in the presence of HOSCN has also been reported for streptococcal species present in human oral cavities (26). The HOSCN oxidoreductase activity was correlated with the ability of oral streptococci to recover from exposure to HOSCN in vitro (26). Because SCN − levels reach low millimolar levels (17,18) and LPO is an abundant peroxidase in saliva (10)(11)(12), the ability of oral streptococci to tolerate HOSCN is vital for their survival in this niche. The aim of the present study was to identify enzymes in S. pneumoniae with HOSCN reductase activity and to determine if they have any role in protecting this serious human pathogen from HOSCN.

Evidence for reduction of HOSCN by an FDR in S. pneumoniae
To investigate whether S. pneumoniae has an enzyme that uses NADPH to reduce HOSCN, we added NADPH to bacterial lysates and observed its consumption by the loss of absorbance at 340 nm upon the addition of HOSCN (Fig. 1A). NADH was also oxidized when HOSCN was added to lysates (Fig. 1B), and NAD(P)H consumption was not observed when either lysate or HOSCN was omitted from the reaction mixture (Fig. 1, A and B). NADH was consumed 20-fold faster than NADPH and while its consumption was significantly higher in the presence of HOSCN, it was also rapidly oxidized in the absence of HOSCN (Fig. 1B). NADH consumption in the absence of oxidant is possibly because of the oxygen-reducing NADH oxidase present in S. pneumoniae (27).
To confirm that HOSCN reduction was occurring in this system, we monitored the rate of HOSCN decline in S. pneumoniae lysates (Fig. 1, C and D). There was no significant difference between the rates of HOSCN and NADPH consumption measured concurrently on the same lysates (0.11 ± 0.03 and 0.09 ± 0.01 μM/min/mg, mean ± SD; n = 4; paired two-tailed t test). As a control, no appreciable loss of HOSCN was observed over the same period in the absence of the bacterial lysate or NADPH (Fig. 1, C and D). Lysates consumed HOSCN slightly more rapidly in the presence of NADH instead of NADPH (0.15 ± 0.02 versus 0.09 ± 0.01, mean ± SD, p < 0.01, unpaired two-tailed t test).
Varying the concentrations of HOSCN and NADPH at fixed concentrations of either NADPH (200 μM) or HOSCN (100 μM), respectively, produced Michaelis-Menten saturation curves consistent with an enzymatic reduction of HOSCN by NADPH (Fig. 1, E and F). The apparent K M values for HOSCN and NADPH added to lysates were 5 (2.5-9.0, 95% confidence interval) and 107 (76-151) μM and the V max 0.12 (0.1-0.14) and 0.13 (0.12-0.15) μM/min/mg, respectively. When no NADPH was added, HOSCN was still consumed at approximately 15% of the maximal measured rate, which is likely facilitated by endogenous NAD(P)H present in the lysates.
HOSCN oxidizes GSH to GSSG, which is reduced by glutathione reductase (GR) at the expense of NADPH (21,22). To test whether the observed NADPH consumption in response to HOSCN in S. pneumoniae lysates was mediated by GSH, we used ΔgshT and Δgor mutants of S. pneumoniae, which lack GSH or GR, respectively (22). NADPH utilization still occurred in lysates from the mutant strains, indeed to a greater extent than in WT, indicating that it was independent of GSH oxidation and recycling by the GR system ( Fig. 2A). To rule out that the observed HOSCN reductase activity involved another low molecular weight thiol such as coenzyme A, we passed lysates through a centrifugal filter with a molecular weight cutoff (MWCO) of 3 kDa and demonstrated that NADPH consumption in response to HOSCN still occurred in the retentate (Fig. 2B). Next, we tested the involvement of TrxR in reducing HOSCN in bacterial lysates as mammalian TrxR is known to have HOSCN reductase activity (25). The TrxR inhibitor auranofin did not affect HOSCN reductase activity in S. pneumoniae lysates when added at 20 μM (Fig. 2B), despite having previously been shown to inhibit recombinant bacterial TrxR at a 25-fold lower concentration (28). Similarly, the activity was not inhibited by 500 μM of 1chloro-2,4-dinitrobenzene (Fig. 2B), a thiol-alkylating reagent that reacts preferentially with TrxR rather than with GSH and fully inhibits human TrxR at 100 μM (29).
We hypothesized that the observed HOSCN reductase activity in S. pneumoniae was due to direct reduction by another member of the FDR/pyridine nucleotide-disulfide reductase family. FDRs, such as GR or TrxR, shuttle electrons from NADPH to a tightly bound flavin adenine dinucleotide and then to a redox-active disulfide (Fig. 3A). One of the two resulting reduced thiols then reacts with an oxidized substrate, for example, GSSG or oxidized thioredoxin, to form a mixed disulfide. Subsequent reaction with the adjacent active site thiol results in regeneration of the active site disulfide and release of the reduced substrate (Fig. 3A). HOSCN reacts with thiols to produce sulfenyl thiocyanate derivatives (30,31). We propose that in the case of a HOSCN reductase, a sulfenyl thiocyanate intermediate is formed, which is then attacked by the other active site thiol resulting in the release of SCN − (Fig. 3A). S. pneumoniae lysates lost their ability to consume NADPH in response to HOSCN when they were boiled or passed over Cibacron Blue beads, an affinity absorbent for proteins with a dinucleotide fold (32) (Fig. 3B). These results suggested that HOSCN reduction was due to a heat-sensitive enzyme with a dinucleotide-binding site. To further probe the involvement of an FDR-type reduction mechanism, any potential thiol-containing FDR substrates were alkylated with iodoacetamide (IAM), rendering them unavailable for oxidation by HOSCN. For this, S. pneumoniae lysates were incubated with IAM prior to monitoring reductase activity (Fig. 3B). Only a partial inhibition by IAM (25%) was observed when it was incubated with S. pneumoniae lysates for 10 min prior to the addition of NADPH and HOSCN, and this effect was not significant. In contrast, the alkylating reagent significantly inhibited the reductase activity when it was coincubated with the lysate in the presence of NADPH to generate the IAM-reactive di-thiol state in the active site (Fig. 3, A and B). These results are consistent with the direct reduction of HOSCN by an FDR (Fig. 3A).
Given that S. pneumoniae cope with H 2 O 2 generated during their metabolism, we tested whether S. pneumoniae lysates can also use NADPH to reduce H 2 O 2. NADPH consumption in S. pneumoniae lysates was not observed in response to H 2 O 2 (Fig. 3B). MPO can use H 2 O 2 not only to produce HOSCN but also to oxidize chloride to the potent bactericidal hypochlorous acid (HOCl) (14,33). HOCl rapidly oxidizes NADPH directly and could therefore not be tested as a possible substrate for the bacterial HOSCN-reducing enzyme. Ammonia chloramine, a less reactive secondary oxidant derived from HOCl (34,35), did not appear to act as a substrate as the rate of NADPH oxidation was not enhanced in the presence of S. pneumoniae lysates (Supporting information, Fig. S1).  ). A, representative absorbance traces for the consumption of NADPH are shown. B, NADPH/NADH consumption was expressed as the amount consumed (ΔA/6220 M −1 cm −1 /1 cm) per min relative to the protein content as determined by Bradford. Each symbol represents a separate experiment using lysates obtained from independent cultures and the bar the mean ± SD; * denotes a significant difference (p < 0.01) between consumption rates in the absence ("−," open circles) and presence ("+," closed circles) of HOSCN for each respective dinucleotide and # (p < 0.001) between the consumption rates in the presence of HOSCN and NADH versus NADPH as determined by one-way ANOVA with Tukey's multiple comparisons test. C, HOSCN under conditions described in (A) was quantified by measuring TNB oxidation by the reaction mixture either immediately (0 min) or 20 min after mixing. Symbols represent the mean ± SD of four independent experiments. D, consumption of HOSCN in the absence ("−," open circles) or presence ("+," closed circles) of NADPH or NADH was measured as in (C) and expressed as the amount consumed per minute relative to the protein content. Each symbol represents a separate experiment using lysates obtained from independent cultures and the bar the mean ± SD; * denotes a significant difference (p < 0.05) between consumption rates in the absence and presence of each respective dinucleotide and # (p < 0.01) between the consumption rate in the presence of NADHP versus NADH as determined by one-way ANOVA with Tukey's multiple comparisons test. E, consumption of NADPH (200 μM) or (F) HOSCN (100 μM) by S. pneumoniae lysates in the presence of increasing concentrations of HOSCN or NADPH, respectively. Symbols in E and F represent the mean ± SD of at least five independent experiments. A Michaelis-Menten curve fit was applied to derive apparent K M and V max values ± 95% confidence interval (CI). F, the point for 0 μM added NADPH was excluded from the curve fit.

Identification of a candidate HOSCN reductase in S. pneumoniae by LC-MS and confirmation by gene deletion
To enrich the HOSCN reductase for identification by LC-MS, we performed pull-down experiments on S. pneumoniae lysates using the Cibacron agarose beads. Attempts at eluting the activity off the beads using high salt, NADPH, and/or flavin adenine dinucleotide were unsuccessful. Instead, we directly loaded the beads onto an SDS-PAGE gel and following Coomassie staining, excised the protein bands for in-gel tryptic digestion and LC-MS/MS analysis (Fig. S2). Numerous proteins were identified when searching the peptide spectra against the S. pneumoniae proteome using database search engines (Supporting information, Protein hits.xlsx). Protein hits were manually filtered for enzymes with oxidoreductase activity, provided they had a high confidence score and the predicted molecular weight was consistent with their position on the SDS-PAGE gel (Table S1). Our IAM results suggested that a redox active disulfide was involved in the reduction of HOSCN. Of the shortlisted oxidoreductases, only an uncharacterized class I pyridine nucleotide reductase, GR and TrxR, belong to the family of FDRs, the latter two of which we had already ruled out using inhibitors and mutant bacteria. The class I pyridine nucleotide reductase that appeared in band 8 (37-50 kDa) is encoded by gene SPD_1415 in S. pneumoniae strain D39. In the S. pneumoniae TIGR4 and R6 strains, this gene is annotated as SP1588 and spr1442, respectively. To confirm that the enzyme encoded by S. pneumoniae strain D39 gene SPD_1415 is responsible for the HOSCN reductase measured in lysates, we targeted the gene for deletion. This was done by replacing the gene with a spectinomycin resistance gene using overlap extension PCR (Fig. S3A) (36). Genetic replacement was confirmed by restriction digest (Fig. S3B) and sequencing (Fig. S4). Lysates of the constructed S. pneumoniae mutant strain no longer consumed NADPH in response to HOSCN confirming that a HOSCN reductase is encoded by this gene (Fig. 4A). We therefore named the gene har and the protein it encodes Har (hypothiocyanous acid reductase).
The HOSCN reductase in S. pneumoniae combines with GSH to confer tolerance to HOSCN To investigate the contribution of our newly identified HOSCN reductase to the ability of S. pneumoniae to resist HOSCN, we grew the WT and Δhar strains in the presence of LPO and SCN − , where LPO uses bacterial H 2 O 2 and SCN − to produce HOSCN. The growth of both strains was retarded to a small degree in the presence of the HOSCN-generating system (Fig. 4, B and C); however, the har deletion did not make the bacteria more sensitive to HOSCN.
We have previously shown that the ability of S. pneumoniae to import and recycle GSH is important for HOSCN tolerance (21). To test whether the GSH antioxidant system and the HOSCN reductase combine to protect S. pneumoniae, we generated double deletion mutants of har with the gene gshT that enables GSH import or with gor that is responsible for recycling GSSG. The absence of HOSCN reductase activity in the double deletion mutants was confirmed using the NADPH consumption assay (Fig. 4A). Bacterial growth in the presence of the HOSCN-generating system was completely inhibited in both double deletion mutants (Fig. 4, D and E). In contrast, while growth was significantly decreased in single gor and gshT deletion mutants when exposed to this system, they eventually reached the same absorbance as untreated cells (21). Catalase protected the double deletion mutants from the LPO system by removing H 2 O 2 and preventing HOSCN generation but not completely (Fig. 4, D and E). In the absence of LPO, catalase had no significant effect on the growth of any of the strains (data not shown), confirming that H 2 O 2 alone does not affect bacterial growth. Collectively, these data highlight the importance of both HOSCN reductase activity and GSH utilization for S. pneumoniae to grow in the presence of a HOSCNgenerating system.

Discussion
We have identified a reductase enzyme in S. pneumoniae that combines with GSH utilization to protect these bacteria from HOSCN. When both components were compromised, S. pneumoniae growth was completely inhibited in the presence of LPO and SCN − , where H 2 O 2 generated during the normal metabolism of these bacteria was converted to HOSCN. The LPO-catalyzed production of HOSCN in epithelial lining fluids has long been considered an integral part of the innate immune response (8,31,37). In addition to bacteria such as S. pneumoniae, sources for H 2 O 2 include the epithelial cell dual oxidase (38) and the neutrophil NADPH oxidase (39). When these white blood cells infiltrate a site of infection, they also provide another HOSCN-producing enzyme, MPO (14). As such, combined pharmacological Figure 2. HOSCN reductase activity in Streptococcus pneumoniae is not carried out by glutathione reductase or thioredoxin reductase. A, reductase activity in lysates from S. pneumoniae WT, GSH transporter substrate-binding protein, and glutathione reductase mutants (ΔgshT and Δgor) was measured as described for Figure 1, A and B. B, lysates from WT S. pneumoniae were passed through a MWCO 3 kDa centrifugal filter before measuring the reductase activity of the filter retentate. Auranofin (20 μM) and DNCB (500 μM) were preincubated with WT lysates for 5 min in the presence of NADPH before measuring the reductase activity. Reductase activity in each case was expressed relative to that obtained for untreated lysate. Each symbol represents a separate experiment using lysates obtained from independent cultures, and the bar is the mean ± SD. A significant difference compared with WT in (A) and to the 100% control in (B) was determined by one-way ANOVA with Dunnett's multiple comparison test and is indicated by *p < 0.05.
inhibition of HOSCN reductase activity and GSH import provides a powerful opportunity to disrupt S. pneumoniae colonization and infection.
HOSCN is well tolerated by host epithelial cells, at least in part owing to the HOSCN-metabolizing action of mammalian TrxR (25). In contrast, the Gram-negative bacteria Escherichia coli and P. aeruginosa are sensitive to killing by HOSCN (25,31,37), their lysates do not consume NADPH in response to HOSCN, and the TrxR from E. coli does not reduce HOSCN (25). Collectively, these studies suggested that HOSCN is selectively toxic for bacteria. However, only a few studies have investigated HOSCN tolerance and defense mechanisms of bacteria that successfully colonize niches in the host where HOSCN is continuously produced, such as oral cavities and the nasopharynx. The ability of bacterial FDRs to act as HOSCN reductases has been largely overlooked. Initial insight came from a study on H 2 O 2 -producing oral streptococci, where some species were shown to have an uncharacterized HOSCN oxidoreductase activity that correlated with their ability to survive HOSCN (26). We noted that S. pneumoniae survives HOSCN exposure much better than the opportunistic and HOSCN reductase-negative respiratory pathogen P. aeruginosa (20), which unlike S. pneumoniae, does not colonize and rarely causes disease in healthy people. We have now identified and characterized a HOSCN reductase in S. pneumoniae that contributes to the ability of this deadly pathogen to tolerate HOSCN.
The previous observation that lysates from some oral streptococcal species consume NADPH in the presence of HOSCN under similar conditions to those reported here suggests that HOSCN reductases exist in other bacteria (26). An amino acid sequence alignment revealed that species that exhibited HOSCN reductase activity, that is, Streptococcus sanguinis, Streptococcus mitis, and Streptococcus salivarius, had Har homologs that share 82 to 99% sequence identity with the S. pneumoniae enzyme. Furthermore, the E. coli enzyme RclA, thought to function as a copper reductase protecting E. coli against a combined HOCl and intracellular copper stress (40,41), has recently been shown to have HOSCN reductase activity (42). RclA has high sequence homology with Har (47% sequence identity, 64% positives) and reduces HOSCN with an apparent K M value of 2 μM HOSCN, which is A, proposed mechanism for the reduction of HOSCN analogous to other known FDRs, in which NADPH reduces the tightly bound flavin adenine dinucleotide, which transfers electrons to a redox-active disulfide present in the conserved CXXXC motif. In the case of low molecular weight thiol disulfide reductases or TrxR, the thus reduced Nterminal thiol can undergo a dithiol-disulfide exchange reaction with a bound oxidized substrate RS-SR (e.g., GSSG or oxidized thioredoxin). We propose that in the case of a HOSCN reductase, the thiol reacts with HOSCN to form a sulfenyl thiocyanate intermediate, which is then attacked by the C-terminal thiol to generate the disulfide resulting in the release of SCN − . B, consumption of 200 μM NADPH by S. pneumoniae lysates in the absence of oxidant (lysate alone) and after the addition of 100 μM H 2 O 2 or HOSCN. Lysates were boiled for 5 min at 95 C or incubated with Cibacron (C) Blue agarose beads before measuring reductase activity of the boiled lysates and bead supernatant in response to 100 μM HOSCN. IAM (20 mM) was preincubated with the lysates for 10 min in the presence of NADPH before measuring the reductase activity. IAM # (20 mM) and lysates were preincubated for 10 min in the absence of NADPH before monitoring the reductase activity. NADPH consumption, that is, reductase activity, in each case was expressed relative to that obtained for the control system (untreated lysate, 100 μM HOSCN). Each symbol represents a separate experiment using lysates obtained from independent cultures and the bar the mean ± SD. A significant difference compared with the 100% control was determined by one-way ANOVA with Dunnett's multiple comparison test and is indicated by *p < 0.05. similar to that found in the present study of 5 μM for lysates of S. pneumoniae. In addition, SCN − is reported to be generated during the reaction cycle of RclA (42), which supports the mechanism we propose in Figure 3A. RclA is known to be conserved among bacteria that colonize epithelial surfaces (40), so it is conceivable that HOSCN reductase enzymes provide bacteria with the means to colonize this niche. Future work is needed to determine whether these enzymes protect other human pathogens from HOSCN.
While HOSCN is the sole product of LPO activity in epithelial lining fluids, the neutrophil-derived enzyme MPO, while preferentially oxidizing SCN − , can also oxidize chloride A, reductase activity, that is, NADPH consumption, was measured in lysates of S. pneumoniae D39 WT, Δhar, ΔgshT-Δhar, and Δgor-Δhar in the presence ("+," closed symbols) and absence ("−," open symbols) of HOSCN as described for Figure 1. Each symbol represents a separate experiment using lysates obtained from independent cultures and the bar the mean ± SD. A significant difference between activities in the absence versus presence of HOSCN for each strain was determined by unpaired t test and is indicated by * for p < 0.05. A significant difference of activities in the presence of HOSCN between mutant strains and WT was determined by one-way ANOVA with Dunnett's multiple comparison test is indicated by # for p < 0.05. B-E, WT, Δhar, ΔgshT-Δhar, and Δgor-Δhar (starting absorbance at 620 nm = 0.1) were grown for 6 h in the presence of 800 μM SCN − (control), 800 μM SCN − + 50 nM LPO (LPO system), or 800 μM SCN − + 50 nM LPO + 100 μg/ml catalase (LPO system + CAT) at 37 C with 5% CO 2 in BHI media. Measurements were taken every 30 min on a plate reader. Data are presented as mean + SD from four independent experiments. to produce HOCl (14,33). At average SCN − and chloride airway fluid concentrations of 400 μM and 100 mM, respectively (8,19), MPO will use the available H 2 O 2 to produce HOSCN and HOCl at a ratio of 3:2 (14). Because of the reaction of HOCl with NADPH, we have been unable to obtain experimental evidence for the HOCl reductase activity of Har. Furthermore, HOCl reacts rapidly with a myriad of other biological targets (43), including with SCN − to form HOSCN (44). It is unlikely that sufficient amounts of HOCl enter bacteria to react directly with Har. However, HOCl reacts with amines and amino acids to form chloramines, which like HOSCN are more selective for thiol groups and are more likely to be substrates for intracellular Har and related bacterial FDRs (45). Glycine and taurine chloramine were poor substrates for RclA compared with HOSCN, and no other amino acid chloramines were reduced by the enzyme (42). We investigated ammonia chloramine, which is derived from the reaction of chloramines/HOCl with ammonia or the breakdown of dichloramines (34,35), but saw no evidence of reductase activity in S. pneumoniae lysates. Collectively, these studies suggest that this family of FDR enzymes has a high substrate specificity for HOSCN.
While har deletion did not sensitize the bacteria to HOSCN, we have previously observed a significant decrease in growth when GSH import and recycling was interfered with (21). This suggests that GSH can compensate for the lack of the HOSCN-metabolizing capability by reacting with HOSCN to form GSSG, followed by GR-mediated recycling of GSSG back to GSH as described before (21). However, when both the HOSCN reductase and either GSH import or recycling was blocked, we observed complete suppression of S. pneumoniae growth in the LPO system. Even high concentrations of catalase to remove H 2 O 2 failed to completely protect the double deletion mutants suggesting that only small amounts of HOSCN are required to inhibit growth. This finding provides insight into the exquisite HOSCN sensitivity achieved by the dual targeting strategy. Catalases and other peroxidases are likely going to compete with LPO for the H 2 O 2 in vivo; therefore, our results highlight that there may be therapeutic value in targeting both components of the S. pneumoniae HOSCN defense in order to maximally sensitize these bacteria to this oxidant.
Although deletion of the SPD_1415/har alone was not associated with an increased sensitivity to a HOSCNgenerating system, it is conceivable that the HOSCN reductase becomes more important in protecting S. pneumoniae from HOSCN stress in situations where host GSH is limiting. Under physiological conditions, GSH is present in very high concentrations in the alveolar epithelial lining fluid (46), but it can become oxidized during airway inflammation and infection (47,48). While S. pneumoniae can still assimilate GSH through importing its oxidized form GSSG, the relative uptake efficiency is unknown. Isothermal titration calorimetry studies using a Streptococcus mutans homolog of the substratebinding protein GshT responsible for GSH utilization suggest that GSSG is a 25-fold less preferred ligand compared with GSH (49). A shift in GSH/GSSG ratio in the epithelial lining fluid during S. pneumoniae infection may thus determine the efficiency of host-GSH acquisition and consequently the relative importance of the HOSCN reductase in S. pneumoniae HOSCN defense.
Further support for the HOSCN reductase being a redundant protective mechanism comes from the fact that we found its activity to be significantly higher in the ΔgshT and Δgor strains compared to WT. This finding suggests that the reductase enzyme may be upregulated when GSH utilization is compromised. Alternatively, increased reductase activity in the GSH mutants could point to higher NADPH/NADH levels as previously observed by deleting GshT in Streptococcus pyogenes (50). Increased HOSCN reductase activity in the S. pneumoniae Δgor mutant might also explain why we previously found this strain to survive equally well as the WT following exposure to reagent HOSCN in Hank's buffered saline, despite its inability to recycle oxidized GSH (21). Collectively, these studies highlight the interconnectedness of the bacterial HOSCN defense systems, a better understanding of which might hold the key to successfully crippling S. pneumoniae's HOSCN tolerance.
In conclusion, the present study is the first to identify a HOSCN reductase in the human pathogen S. pneumoniae and to investigate its role in protecting the bacteria against this oxidant. Future work should focus on understanding whether the HOSCN reductase contributes to the bacteria's virulence using animal models and endeavor to find ways to inhibit the ability S. pneumoniae to protect itself from HOSCN.

Media
Brain heart infusion (BHI) medium (Oxoid; Thermo Fisher Scientific) and Bacto Todd Hewitt medium supplemented with 1.5% yeast extract (THY; BD) were made from dehydrated culture media according to the manufacturers' instructions. Columbian sheep blood agar medium was made from defibrinated sheep's blood (Fort Richard Laboratories) and Difco Columbia Blood Agar Base (BD) following the manufacturer's instructions, and spectinomycin was added to give a final concentration of 150 μg/ml. All media were sterilized by autoclaving, and plates were poured using aseptic technique, cooled, sealed, and stored at 4 C for up to 10 weeks.

Bacterial strains and culture
All bacterial strains were stored under standard conditions and maintained on Columbian or tryptic soy broth sheep blood agar plates (Fort Richard Laboratories). S. pneumoniae strain D39, serotype 2 (NCTC 7466) mutant strains ΔgshT (unable to import GSH) and Δgor (unable to recycle oxidized GSH) were generated as described previously (22). All strains were statically grown overnight at 37 C with 5% CO 2 in BHI media, then diluted in fresh BHI, and grown to an absorbance of 0.4 to 0.7 at 620 nm before experiments.

HOSCN reductase activity in bacterial lysates
Bacteria were pelleted by centrifugation at 10,000g for 50 min at 4 C, washed once with PBS, then resuspended in 2 to 5 ml of 100 mM phosphate (pH 7.4), and 1 mM EDTA. S. pneumoniae was lysed by pulse sonication on ice for 10 min. Bacterial debris was removed by centrifugation at 10,000g for 10 min at 4 C. The consumption of NADPH or NADH (200 μM) by bacterial lysates (200 μl) after the addition of HOSCN (100 μM) or buffer was measured in 100 mM phosphate (pH 7.4) containing 1 mM EDTA (reductase buffer) by monitoring the loss of absorbance at 340 nm over 5 min in a 1 ml cuvette using an UV-visible spectrophotometer (Agilent 8453). The protein concentration in bacterial lysates was determined by Bradford assay (54). Reductase activity was defined as the rate of NAD(P)H consumption and was calculated using e 340 = 6220 M −1 cm −1 for NAD(P)H (55) and the following formula Reductase activityðμmol = min = mgÞ ¼ ΔAbs 340 Δt Reductase activity in S. pneumoniae lysates was also measured in the presence of increasing concentrations of NADPH at a fixed HOSCN concentration (100 μM) and in the presence of increasing concentrations of HOSCN at a fixed concentration of NADPH (200 μM) as well as after the addition of 100 μM H 2 O 2 instead of HOSCN. S. pneumoniae lysates were boiled for 5 min at 95 C or buffer-exchanged using MWCO centrifugal filters (3 kDa Vivaspin or 10 kDa Amicon, at least three additions of the original lysate volume of reductase buffer) before being added to the assay. Cibacron Blue 3GA agarose beads (500 μl) were pelleted at 10,000g and washed with reductase buffer before adding lysate and incubating for 30 to 60 min at 4 C with end-over-end rotation. Beads were removed by centrifugation, and the supernatant was added to the NADPH consumption assay. For inhibitor studies, auranofin (20 μM), 1-chloro-2,4-dinitrobenzene (500 μM), and IAM (20 mM) were incubated with the lysate and NADPH (200 μM) in reductase buffer. After 5 min, HOSCN (100 μM) was added, and the NADPH consumption was measured. IAM (20 mM) was also incubated with lysates in the absence of NADPH, then NADPH (200 μM) and HOSCN (100 μM) were added, and the NADPH consumption measured straight away.

HOSCN consumption in bacterial lysates
HOSCN was monitored using its ability to oxidize yellow TNB to the colorless dimer 5,5 0 -dithiobis-(2-nitrobenzoic acid) (56). S. pneumoniae lysates were mixed with reductase buffer, NADPH (200 μM), and HOSCN (100 μM) in a total of 1 ml. Straight away and after 20 min, 30 μl of the reaction mixture was added to 970 μl of 15 μM TNB. The absorbance change at 412 nm was used to measure the HOSCN concentration present in the mixture using e 412 = 14,100 M −1 cm −1 for TNB and accounting for the fact that 1 mol HOSCN causes the loss of 2 mol of TNB. The difference in HOSCN concentrations measured at 0 and 20 min was used to calculate the rate of HOSCN consumption relative to the amount of protein present in the lysate. HOSCN was also monitored in response to varying amounts of NADPH (0-500 μM NADPH).

LC-MS on Cibacron Blue agarose-bound S. pneumoniae proteins
Ammonium sulphate (65%) was added to S. pneumoniae lysate (5 ml) and incubated at 4 C for 30 min with end-overend rotation followed by centrifugation at 10,000g for 10 min at 4 C. The supernatant was removed, the pellet was resuspended in 1 ml of 100 mM phosphate (pH 7.4), 1 mM EDTA, and dialyzed using centrifugal filter units (MWCO = 10 kDa). The sample was added to 500 μl washed Cibacron Blue 3GA agarose beads and incubated for 30 min at 4 C with end-overend rotation. At each step of the purification procedure, a sample was removed, mixed with 5× reducing SDS-PAGE sample buffer, heated at 95 C for 5 min, and separated on a TGX 4 to 15% stain-free gel (Bio-Rad), which was stained with Coomassie Blue R250. Bands were excised and cut into smaller pieces and then washed by adding alternately 80% acetonitrile in water and 100 mM ammonium bicarbonate. When gel pieces were destained, they were dehydrated in 80% acetonitrile, then reduced with 10 mM DTT in 50 mM ammonium bicarbonate for 1 h at 45 C, dehydrated again, and alkylated with 20 mM IAM for 15 min in the dark. After washing, 0.5 μg trypsin in 50 mM bicarbonate buffer was added and incubated at 37 C overnight. The supernatant was removed, combined with the eluate of a subsequent elution with 80% acetonitrile, dried in a vacuum evaporator, and resuspended in 0.1% formic acid in water. Samples were separated by a Jupiter 4u Proteo 90 Å column (150 mm × 2 mm; Phenomenex) coupled in line to a Velo Pro ion trap mass spectrometer (Thermo Fisher Scientific). Collision-induced dissociation MS/MS spectra in positive-ion mode were acquired for the ten most abundant precursors with a signal intensity of greater than 500, as described previously (57). Fragment spectra were searched against protein sequences of the unreviewed TrEMBL proteome of S. pneumoniae deposited on UniProt using Proteome Discoverer 2.5 (Thermo Fisher Scientific) using Sequest HT and MS Amanda 2.0 search engines using methionine sulfoxide and carbamidomethyl-cysteines as dynamic medications. Protein hits for each band can be found in the supporting information.

Generation of S. pneumoniae deletion mutants
Deletion mutants (Δhar, ΔgshT-Δhar, and Δgor-Δhar) were generated by allelic replacement with a spectinomycin resistance marker using splicing overlap extension (SOE) PCR based on a previously described method (36). Primers used for the generation of S. pneumoniae Δhar mutant are listed in Table 1. Briefly, primers PP196/PP197 were used to amplify the spectinomycin resistance marker (aad), and PP198/PP194 and PP195/PP199 were used to generate overlapping PCR fragments of the 5 0 and 3 0 flanking regions of har, respectively. The 5 0 and 3 0 fragments were then assembled either side of the resistance gene by SOE PCR of the overlapping fragments and amplified to one construct using PP198/PP199 (Fig. S3A). A NotI restriction site was incorporated into the spectinomycin resistance cassette. The resulting SOE DNA product was amplified and transformed into WT, Δgor, and ΔgshT S. pneumoniae D39 strains.
S. pneumoniae D39 WT, ΔgshT, and Δgor were transformed using the protocol (for transforming S. pneumoniae with transposon DNA) as described (58), with alterations. Briefly, S. pneumoniae starter cultures with bacteria prepared for competence induction were generated and frozen at −80 C. When needed, these cultures were supplemented with glycine in THY, pH 6.8, and grown to an absorbance of 0.03 to 0.1 at 600 nm at 37 C with 5% CO 2 . The volume of culture needed to get a total of 1 ml of absorbance of 0.03 at 600 nm was centrifuged at 12,000g for 5 min. The supernatant was replaced with THY, pH 7.8, supplemented with 0.2% bovine serum albumin, and 1 mM CaCl 2 . Bacterial competence was stimulated using 0.23 μM competence stimulating peptide-1 and incubated for 15 min. Har mutant construct DNA (1 μg) was then added and incubated at 37 C with 5% CO 2 for 45 min. The sample was centrifuged for 8 min at 3000g, suspended in 1/ 10th volume of the supernatant, and plated onto blood agar plates containing 150 μg/ml spectinomycin. Plates were incubated overnight at 37 C with 5% CO 2 .
Spectinomycin-resistant strains Δhar, ΔgshT-Δhar, and Δgor-Δhar were confirmed by PCR and sequencing. Genomic DNA was isolated from candidate strains using a DNeasy Blood & Tissue Kit (Qiagen), following the protocol for Grampositive bacteria and subjected to PCR using primers PP198 and PP199. PCR of WT clones produced a 2920 bp product, whereas strains with har replacement produced a 2415 bp product. Clones were also digested with NotI to confirm the presence of the spectinomycin gene (which carries a 3 0 -end NotI site unique to the construct) (Fig. S3B). Sequencing along the 5 0 and 3 0 arms through the upstream (SPD_1414) and downstream (SPD_1416) genes flanking har revealed no har sequence and confirmed complete replacement of har with spectinomycin resistance (aad) (Fig. S4).
Bacterial growth in the presence of the LPO/SCN − system S. pneumoniae WT, Δhar, ΔgshT-Δhar, and Δgor-Δhar strains (starting absorbance at 620 nm = 0.1) were grown for 6 h at 37 C with 5% CO 2 in the presence or the absence of an enzymatic HOSCN-generating system using LPO (50 nM), SCN − (800 μM), and bacterially produced H 2 O 2 as described before (21). Additional treatments containing catalase (100 μg/ ml), that is, bacteria + SCN − + CAT and bacteria + SCN − + LPO + CAT, were included to rule out any bacterial growth inhibition because of H 2 O 2 .

Statistical analyses
Graphs were plotted, and the statistical analyses stated in the figure legends were performed using GraphPad Prism (version 8.2.1; GraphPad Software, Inc). A p value of <0.05 was considered significant.

Data availability
Raw mass spectrometry and sequencing data will be available upon request.
Supporting information-This article contains supporting information.
Acknowledgments-We acknowledge Professors Anthony J. Kettle and Haike Antelmann for scholarly input and helpful discussions. We also thank Professor Andrew Camilli from the Tufts University School of Medicine, MA, USA for the kind gift of the plasmid pMagellan6.  Table 1 List of primers used in this study Overlapping primers showing sequence that is homologous to spectinomycin (italics) and restriction sites AscI and NotI introduced for analyses (underlined).