Mannose receptor‐derived peptides neutralize pore‐forming toxins and reduce inflammation and development of pneumococcal disease

Article28 September 2020Open Access Source DataTransparent process Mannose receptor-derived peptides neutralize pore-forming toxins and reduce inflammation and development of pneumococcal disease Karthik Subramanian Karthik Subramanian orcid.org/0000-0002-4381-5037 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Federico Iovino Federico Iovino Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Vasiliki Tsikourkitoudi Vasiliki Tsikourkitoudi orcid.org/0000-0003-0820-3585 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Padryk Merkl Padryk Merkl Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Sultan Ahmed Sultan Ahmed Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden Search for more papers by this author Samuel B Berry Samuel B Berry Department of Medicine, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, Sweden Search for more papers by this author Marie-Stephanie Aschtgen Marie-Stephanie Aschtgen Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Mattias Svensson Mattias Svensson Department of Medicine, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, Sweden Search for more papers by this author Peter Bergman Peter Bergman Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden The Immunodeficiency Unit, Department of Infectious Diseases, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Georgios A Sotiriou Georgios A Sotiriou orcid.org/0000-0001-5040-620X Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Birgitta Henriques-Normark Corresponding Author Birgitta Henriques-Normark [email protected] orcid.org/0000-0002-5429-4759 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden Lee Kong Chian School of Medicine (LKC) and Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore, Singapore Search for more papers by this author Karthik Subramanian Karthik Subramanian orcid.org/0000-0002-4381-5037 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Federico Iovino Federico Iovino Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Vasiliki Tsikourkitoudi Vasiliki Tsikourkitoudi orcid.org/0000-0003-0820-3585 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Padryk Merkl Padryk Merkl Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Sultan Ahmed Sultan Ahmed Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden Search for more papers by this author Samuel B Berry Samuel B Berry Department of Medicine, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, Sweden Search for more papers by this author Marie-Stephanie Aschtgen Marie-Stephanie Aschtgen Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Mattias Svensson Mattias Svensson Department of Medicine, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, Sweden Search for more papers by this author Peter Bergman Peter Bergman Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden The Immunodeficiency Unit, Department of Infectious Diseases, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Georgios A Sotiriou Georgios A Sotiriou orcid.org/0000-0001-5040-620X Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Birgitta Henriques-Normark Corresponding Author Birgitta Henriques-Normark [email protected] orcid.org/0000-0002-5429-4759 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden Lee Kong Chian School of Medicine (LKC) and Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore, Singapore Search for more papers by this author Author Information Karthik Subramanian1, Federico Iovino1, Vasiliki Tsikourkitoudi1, Padryk Merkl1, Sultan Ahmed2, Samuel B Berry3, Marie-Stephanie Aschtgen1, Mattias Svensson3, Peter Bergman2,4, Georgios A Sotiriou1 and Birgitta Henriques-Normark *,1,5,6 1Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden 2Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden 3Department of Medicine, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, Sweden 4The Immunodeficiency Unit, Department of Infectious Diseases, Karolinska University Hospital, Stockholm, Sweden 5Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden 6Lee Kong Chian School of Medicine (LKC) and Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore, Singapore *Corresponding author. Tel: +46 852480000; E-mail: [email protected] EMBO Mol Med (2020)12:e12695https://doi.org/10.15252/emmm.202012695 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cholesterol-dependent cytolysins (CDCs) are essential virulence factors for many human pathogens like Streptococcus pneumoniae (pneumolysin, PLY), Streptococcus pyogenes (streptolysin O, SLO), and Listeria monocytogenes (Listeriolysin, LLO) and induce cytolysis and inflammation. Recently, we identified that pneumococcal PLY interacts with the mannose receptor (MRC-1) on specific immune cells thereby evoking an anti-inflammatory response at sublytic doses. Here, we identified the interaction sites between MRC-1 and CDCs using computational docking. We designed peptides from the CTLD4 domain of MRC-1 that binds to PLY, SLO, and LLO, respectively. In vitro, the peptides blocked CDC-induced cytolysis and inflammatory cytokine production by human macrophages. Also, they reduced PLY-induced damage of the epithelial barrier integrity as well as blocked bacterial invasion into the epithelium in a 3D lung tissue model. Pre-treatment of human DCs with peptides blocked bacterial uptake via MRC-1 and reduced intracellular bacterial survival by targeting bacteria to autophagosomes. In order to use the peptides for treatment in vivo, we developed calcium phosphate nanoparticles (CaP NPs) as peptide nanocarriers for intranasal delivery of peptides and enhanced bioactivity. Co-administration of peptide-loaded CaP NPs during infection improved survival and bacterial clearance in both zebrafish and mice models of pneumococcal infection. We suggest that MRC-1 peptides can be employed as adjunctive therapeutics with antibiotics to treat bacterial infections by countering the action of CDCs. Synopsis Cholesterol-dependent cytolysins (CDCs) are major virulence factors for many pathogenic bacteria. Pneumolysin (PLY) is a CDC produced by the human respiratory pathogen Streptococcus pneumoniae. This study identifies peptides from the mannose receptor MRC-1 that inhibit toxin function and reduce pneumococcal disease severity. The C-type lectin domain 4 (CTLD4) of MRC-1 interacts with the cholesterol binding loop of PLY. Peptides from CTLD4 of MRC-1 competitively inhibit toxin-induced lysis of host cells and reduce inflammation. Pre-treatment of cells with peptides inhibits bacterial entry via MRC-1, results in targeting of bacteria to autophagosomes, and enhances bacterial killing. Administration of peptide-loaded calcium phosphate nanoparticles improves survival and reduces bacterial load in mice and zebrafish models of pneumococcal disease. The paper explained Problem Cholesterol-dependent cytolysins (CDCs) are bacterial toxins that bind to cholesterol on target cell membrane and form pores resulting in cell lysis and inflammation. CDCs are major virulence factors for several bacteria such as the human respiratory pathogen, S. pneumoniae. The structures of CDCs are conserved and hence represent attractive targets for novel treatments that neutralize toxin function. We recently identified that the human mannose receptor, MRC-1, binds to pneumococcal toxin, pneumolysin. In this study, we developed peptides from the region of interaction between MRC-1 and CDCs to inhibit toxin activity and treat pneumococcal disease. Results Using computational docking, we designed peptides from the CTLD4 domain of MRC-1 that bind to the cholesterol-binding loop of the major CDCs: PLY, LLO, and SLO. We screened peptides for toxin inhibition and identified peptides that dose-dependently bind and inhibit toxin-induced cytolysis and inflammation. Also, the peptides inhibit bacterial internalization into DCs via MRC-1 and target intracellular bacteria to autophagy killing. Further, we developed biocompatible calcium phosphate nanoparticles conjugated to peptides for enhanced bioactivity and intranasal delivery. We found that the peptide-conjugated nanoparticles enhanced survival and reduced the bacterial burden and inflammation in mouse and zebrafish models of pneumococcal infection. Impact Our study suggests that the nanoparticles conjugated to MRC-1 peptides could be used as an adjunctive therapy together with antibiotics to treat bacterial infections. Introduction Bacterial infections are leading causes of mortality and morbidity worldwide, and the emergence of resistance to many antibiotics is a major threat to society (Fischbach & Walsh, 2009). A common characteristic of many pathogenic bacteria, including some that have evolved drug resistance, is that they employ pore-forming toxins (PFTs) as virulence factors (Dal Peraro & van der Goot, 2016). PFTs constitute more than one-third of all cytotoxic toxins, making them the largest category of bacterial virulence factors (Gonzalez et al, 2008). Cholesterol-dependent cytolysins (CDCs) are a subclass of ß-PFTs that bind to cholesterol on eukaryotic cells and form barrel-shaped pores to mediate cytolysis (Los et al, 2013). CDCs promote bacterial virulence in many ways such as (i) induction of epithelial barrier dysfunction (Witzenrath et al, 2006), (ii) lysis of phagocytic immune cells (Domon et al, 2016), and (iii) facilitating bacterial invasion of host cells and intracellular survival (Subramanian et al, 2019b) (Birmingham et al, 2008). Prominent examples of bacterial CDCs include pneumolysin (PLY) from Streptococcus pneumoniae, streptolysin O (SLO) from Streptococcus pyogenes, listeriolysin O (LLO) from Listeria monocytogenes, intermedilysin (ILY) from Streptococcus intermedius, anthrolysin O (ALO) from Bacillus anthracis, and perfringolysin O (PFO) from Clostridium perfringens (Gilbert et al, 1999; Park et al, 2004; Boyd et al, 2016; Savinov & Heuck, 2017; Nguyen et al, 2019; Ogasawara et al, 2019; Subramanian et al, 2019a,b; Vogele et al, 2019). The structures of CDCs are conserved, consisting of four domains, and domain 4 is known to bind to the eukaryotic cell membrane (Tweten, 2005). Specifically, the highly conserved tryptophan-rich undecapeptide loop in domain 4 has been shown to bind cholesterol on eukaryotic membranes (van Pee et al, 2017). The binding triggers oligomerization of membrane-bound monomeric toxins into pre-pore structure. Conformational change triggers two α-helices in domain 3 to unfold into ß-hairpins which then insert into the membrane to form 250–300 Å pores. Due to their ubiquitous expression in bacterial pathogens, CDCs are attractive targets for development of novel broadly applicable antimicrobial therapeutics. The application of antibiotics to treat bacteremic patients is known to cause release of CDCs from lysed bacteria (Spreer et al, 2003), and hence adjunctive therapies to ameliorate the tissue damage caused by the released toxins are needed. In our recent study, we identified that the human mannose receptor (MRC-1/CD206) expressed on dendritic cells and lung alveolar macrophages binds to PLY at sublytic doses. MRC-1 is an endocytic receptor that binds and internalizes glycoproteins with terminal mannose, fucose, or N-acetylglucosamine residues, as well as pathogens bearing high mannose structures. However, at sublytic doses, MRC-1 binds to PLY, independent of the capsular polysaccharides, resulting in an anti-inflammatory response and enhanced intracellular survival of pneumococci (Subramanian et al, 2019b). Here, we used computational docking to predict the sites of interaction between MRC-1 and CDCs. By constructing peptides from the region of interaction, we studied whether we may inhibit the action of CDCs. Using human macrophages, DCs, and 3D lung epithelial tissue models, we investigated the effect of treatment with the peptides on toxin-induced cytolysis, epithelial damage, inflammation, and bacterial invasion. Finally, we explored the use of biocompatible calcium phosphate nanoparticles (CaP NPs) as peptide nanocarriers for intranasal delivery of the peptides to the lungs and studied their effect on survival and bacterial clearance using two in vivo infection models, mice and zebrafish. Results MRC-1 co-localizes with the bacterial CDCs, PLY, LLO, and SLO, in human dendritic cells To test whether MRC-1 could serve as a common receptor for structurally conserved bacterial CDCs, we incubated human monocyte-derived DCs with a non-cytolytic dose (0.2 μg/ml) of the purified toxins, PLY, LLO, and SLO, for 45 min, and performed immunofluorescence staining. We found that MRC-1 co-localized with all the three CDCs in DCs (Fig 1A–C). To verify uptake by DCs, we co-stained for the early endosomal antigen, EEA-1, and found that MRC-1 co-localized with the three CDCs along with EEA-1 (Fig 1A–C). The extent of co-localization of MRC-1 is quantified in Fig EV1A. To test whether the cholesterol-binding loop in domain 4 of the CDCs, that binds to the host cell membrane, is also involved in the interaction with MRC-1, we used toxoid derivates of PLY (W433F) and LLO (W489F) bearing point mutations in a key tryptophan residue of the cholesterol-binding loop. In contrast to the wild-type toxins, the toxoids showed drastically reduced binding to the DCs and did not co-localize with MRC-1, indicating that the cholesterol-binding loop of PLY and LLO is essential for the interaction with MRC-1 on DCs (Fig EV1B and C). Further, as shown previously for PLY (Subramanian et al, 2019b), antibody blockade of MRC-1 also impaired the binding of LLO and SLO in DCs (Fig EV1D and E) and impaired co-localization with EEA-1 (Fig EV1F). Figure 1. MRC-1 co-localizes with the bacterial CDCs, PLY, LLO, and SLO, in human dendritic cells A–C. Human DCs were incubated with a non-cytolytic dose (0.2 μg/ml) of purified (A) PLY, (B) LLO, or (C) SLO for 45 min. Immunofluorescence staining shows that PLY, LLO, and SLO (green) co-localize with MRC-1 (red) in DCs and EEA-1 (purple, early endosomes). All scale bars, 5 μm. Images are representative of three independent experiments. Source data are available online for this figure. Source Data Figure 1 [emmm202012695-sup-0014-SDataFig1.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Co-localization of PLY, LLO, and SLO with MRC-1 and EEA-1 in DCs and effect of MRC-1 blockade A. Pearson's correlation coefficient showing extent of co-localization of MRC-1 with PLY, LLO, and SLO. Data represent mean ± s.d. from three experiments. B, C. Human DCs were incubated with a non-cytolytic dose (0.2 μg/ml) of purified toxoid derivatives, (B) PLY(W433F) and (C) LLO(W489F) for 45 min. Immunofluorescence staining shows that both PLY(W433F) and LLO(W489F) (green) show weak binding to DCs and do not co-localize with MRC-1 (red) in DCs. Scale bars, 5 μm. Images are representative of three independent experiments. D, E. Human DCs were incubated with 0.2 μg/ml of purified (D) LLO and (E) SLO for 60 min upon pre-treatment with 1 μg/ml of anti-MRC-1. Immunofluorescence microscopy shows that upon MRC-1 blockade, the binding of LLO and SLO (green) to DCs is diminished and do not co-localize with the early endosomal antigen, EEA-1 (purple). Scale bars, 10 μm. F. Pearson's correlation coefficient showing extent of co-localization of EEA-1 with PLY, LLO, and SLO with or without MRC-1 antibody blockade. Upon antibody blockade of MRC-1, the extent of co-localization with EEA-1 was reduced. Data represent mean ± s.d. from three experiments. *P < 0.05; **P < 0.01 by two-way ANOVA with Bonferroni post-test. Exact P values are shown in Appendix Table S4. Download figure Download PowerPoint The CTLD4 domain of MRC-1 interacts with the cholesterol-binding loop of bacterial CDCs Next, we wanted to determine the specific sites of interaction between MRC-1 and the CDCs. We recently showed that the CTLD4 domain of MRC-1 interacts with the membrane-binding domain 4 of PLY (Subramanian et al, 2019b). Hence, we performed computational docking of the crystal structures of PLY, LLO, and SLO with the CTLD4 domain of MRC-1 on the ClusPro 2.0 docking server (Kozakov et al, 2017), based on least energy configuration. The structures of PLY, LLO, and SLO are conserved and consist of four domains, D1–D4, wherein domain 4 binds to cholesterol on the eukaryotic cell membrane (Fig 2A–C). The tryptophan-rich undecapeptide loop (highlighted in yellow) in domain 4 binds to the cell membrane and is highly conserved among the CDCs. The cholesterol-binding loop has been shown to alter the avidity of cholesterol binding by altering oligomerization (Dowd et al, 2012). Modeling results suggest that the CTLD4 of MRC-1 (red) interacts with the cholesterol-binding loop (yellow) in domain 4 of PLY, LLO, and SLO, respectively (Fig 2A–C). Particularly, modeling predicted that the tryptophan residues, W433 of PLY and W489 of LLO, located in the cholesterol-binding loop of domain 4, are involved in hydrogen bonding interactions with CTLD4 of MRC-1. This is in line with our earlier data, where we observed that the mutant derivatives, PLY (W433F) and LLO (W489F), did not interact with MRC-1 (Fig EV1B and C). Figure 2. The CTLD4 domain of MRC-1 interacts with the cholesterol-binding loop of bacterial CDCs A–C. Computational docking of (A) PLY, (B) LLO, and (C) SLO (in green) with the CTLD4 domain of MRC-1 (in red) was performed using the ClusPro 2.0 docking server. Modeling based on least energy configurations indicate that the unstructured loop of MRC-1 docks to the conserved cholesterol-binding loop (in yellow) of PLY, LLO, and SLO. The amino acid residues predicted to involve in hydrogen bonding interactions are zoomed in below. D, E. 3D view of the CTLD4 domain of MRC-1 showing the surface location of the peptides P2 and P3 (in pink). Acidic and basic amino acid residues are shown in red and blue, respectively. Indicated in green is the calcium binding site. Source data are available online for this figure. Source Data Figure 2 [emmm202012695-sup-0015-SDataFig2.zip] Download figure Download PowerPoint MRC-1 peptides bind to bacterial CDCs and inhibit their induction of hemolysis and cytolysis of macrophages Since our results indicated that MRC-1 interacts with the cholesterol-binding loop of the CDCs, we hypothesized that peptides from the region of interaction could inhibit toxin interactions with eukaryotic cells. Therefore, we constructed overlapping 13-mer peptides from the CTLD4 of MRC-1, and two negative control peptides from the fibronectin type II domain and intracellular tail (Fig EV2A and Appendix Table S1). The peptides were commercially synthesized to > 95% purity and screened for their ability to inhibit hemolysis of red blood cells induced by purified bacterial toxins. Addition of peptides P1-P6 inhibited PLY- and LLO-induced hemolysis as opposed to the control peptides, CP1 and CP2, as evident by the residual red blood cell pellet at the end of the hemolytic assay (Fig EV2B). Peptides P2 and P3 were the most potent, inhibiting hemolysis by up to 50% (Fig EV2C). In agreement, both peptides P2 and P3 contain a continuous stretch of ≥ 3 amino acids that formed hydrogen bonding interactions with the amino acids in the cholesterol-binding loop of the CDCs. The amino acids involved in hydrogen bonding interactions are indicated in Appendix Table S2. We found that both peptides, P2 and P3, were surface localized on the MRC-1 CTLD4 domain, which is ideal for interactions with bacterial toxins (Fig 2D and E). Cholesterol, a known inhibitor of PLY-induced hemolysis, was used as a positive control. Bovine serum albumin (BSA) was used as a negative control to verify that the inhibition of PLY-mediated hemolysis by the MRC-1 peptides was specific. Click here to expand this figure. Figure EV2. Location and activity of MRC-1 peptides against PLY and LLO A. Domain architecture and location of peptides (P1–P6, CP1, and CP2) on the MRC-1 protein. P1–P6 are from the CTLD4 domain which binds to the membrane-binding loop of CDCs while the control peptides, CP1 and CP2, are from regions that do not bind CDCs. B. Red blood cell hemolysis assay showing the residual cell pellet after hemolysis by purified PLY and LLO (1 μg/ml) in the presence of 100 μM of MRC-1 peptides. Complete hemolysis indicated by absence of red pellet was achieved by PLY and LLO alone as well as the control peptides, CP1 and CP2, while peptides P1–P6 conferred protection to various extents. Cholesterol was used as positive control to block hemolysis. C. Quantification of hemolysis induced by PLY (1 μg/ml) in the presence of 100 μM of MRC-1 peptides, P1–P6, and control peptides, CP1 and CP2. BSA was used as negative control to show specificity while cholesterol was used as positive control to block hemolysis. Data are mean ± s.e.m. from two experiments with triplicates. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA with Tukey's post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4. Download figure Download PowerPoint Next, we set up an ELISA to ascertain the binding of MRC-1 peptides to the purified CDCs and focused on peptides P2 and P3 that showed the highest potency. Increasing doses of purified PLY or BSA (negative control) were added to peptides P2, P3, or control peptides CP1 or CP2, that were immobilized on the plates. Peptides P2 and P3 were found to bind dose-dependently to PLY, but not to BSA, suggesting that the binding was specific (Fig 3A). The control peptides, CP1 and CP2, showed only background levels of binding. Peptides P2 and P3 also bound dose-dependently to LLO and SLO (Fig EV3A and B). To determine the optimal working concentration of the peptides, we performed a hemolysis assay by titrating increasing concentrations of the peptides in the presence of 1 μg/ml PLY. To verify that the peptide sequence rather than its amino acid composition is crucial for inhibiting toxin activity, we used a scrambled version of peptide P2 as a control. We found that both peptides P2 and P3 dose-dependently inhibited hemolysis induced by the purified toxins PLY, LLO, and SLO, resulting in up to 50% inhibition at concentrations ranging from 10 to 100 μM (Figs 3B, and EV3C and D). However, the scrambled peptide P2 did not have any significant effect on hemolysis. Using regression analysis, we determined the ED50 of the peptides P2 and P3 against the purified toxins (Appendix Table S3). Figure 3. MRC-1 peptides bind to bacterial CDCs and inhibit their induction of hemolysis and cytolysis of macrophages A. ELISA showing the dose-dependent binding of plate-bound MRC-1 peptides P2, P3, and the control peptides CP1 and CP2 to PLY (0–0.5 μM). BSA was used as negative control to show the binding specificity. Data are mean ± s.e.m. of two independent experiments, each containing three replicates per condition. B. Hemolysis assay (n = 4) of 1 μg/ml purified PLY in the presence of increasing concentrations of MRC-1 peptides, P2, scrambled P2, P3, and control peptide CP2 (1–1000 μM). Data represent mean ± s.e.m. *P < 0.05 by one-way ANOVA with Dunnett's post hoc test for multiple comparisons. Exact P values are shown in Appendix Table S4. C. LDH cytotoxicity assay in human THP-1 macrophages stimulated with purified PLY, LLO, or SLO (0.5 μg/ml) in the presence or absence of 100 μM peptides P2, scrambled P2, P3, or control peptide CP2 for 18 h. Cholesterol (100 μM) was used as positive control to inhibit hemolysis. Data are mean ± s.e.m. from 4 independent experiments. ****P < 0.0001 by two-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4. D. Binding of FITC-labeled peptides P2 and CP2 to wild-type pneumococci, TIGR4 (T4), and its isogenic PLY mutant (T4Δply) was visualized by fluorescence microscopy. Scale bars, 10 μm. In magnified images, scale bars, 1 μm. Images are representative of three independent experiments. E. The hemolytic activity of wild-type pneumococci, TIGR4 (T4) and the PLY mutant, T4Δply in the presence of 100 μM peptide P2 and CP2. Data are the mean ± s.e.m. of three independent experiments. ***P < 0.001 by one-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4. Source data are available online for this figure. Source Data Figure 3 [emmm202012695-sup-0016-SDataFig3.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Dose-dependent binding of peptides to LLO and SLO and inhibition of hemolysis and pro-inflammatory cytokine responses A, B. ELISA showing the dose-dependent binding of plate-bound MRC-1 peptides P2, P3, and the control peptides CP1 and CP2 to (A) purified LLO and (B) SLO (0–0.5 μM). BSA was used as negative control to show the binding specificity. Data are mean ± s.e.m. from two independent experiments with triplicates. C, D. Hemolysis assay of 1 μg/ml purified (C) LLO and (D) SLO in the presence of increasing concentrations of MRC-1 peptides, P2, P3, scrambled P2, and control peptide CP2 (1–1,000 μM). Data are mean ± s.e.m. from 4 independent experiments. *P < 0.05 by one-way ANOVA with Dunnett's post hoc test for multiple comparisons. Exact P values are shown in Appendix Table S4. E, F. Hemolytic activity of (E) S. pyogenes type M1T1 and (F) L. monocytogenes in the presence of 100 μM peptide P2 and CP2. The isogenic SLO mutant strain was used as a negative control. Data are mean ± s.e.m. from three independent experiments. **P < 0.01 and ***P < 0.001 by one-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4. Download figure Download PowerPoint To visualize inhibition of the bacterial CDCs by the peptides in real time, we performed live imaging using human THP-1 monocyte-derived macrophages upon addition of PLY in the presence or absence of peptide P2. The cells were pre-loaded with the live-dead stain comprising of Calcein AM and propidium iodide that differentially stain live and dead cells green and red, respectively. The cells were imaged for 20 min post-addition of 0.5 μg/ml PLY in the presence or absence of 100 μM peptide P2, or control peptide CP2. This dosage of PLY was chosen since at concentrations between 0.5 and 1 μg/ml PLY has been shown to activate the inflammasome and induce acute lung injury in acute pneumonia model (Stringaris et al, 2002; Witzenrath et al, 2006). In contrast to untreated cells, addition of PLY resulted in membrane blebbing and positive staining of cells by propidium iodide (Movies EV1 and EV2). However, in the presence of the peptide P2, but not of the control peptide CP2, most of the cells were intact and stained green, indicating protection from cytolysis (Movies EV3 and EV4). Cholesterol was used as positive control (Movie EV5) and BSA as a negative control to show the specificity of the peptides (Movie EV6). No cell death was observed when the toxoid form of PLY, Pdb (W433F), that is defective in pore-formation, was added (Movie EV7). Peptide P2 also protected the cells from cytolysis mediated by LLO and SLO (Movies EV8–EV11). To quantify cell death, we measured the release of lactate dehydrogenase (LDH) from lysed cells into the culture supernatant. Peptides P2 and P3 significantly reduced cell death of macrophages induced by PLY, LLO, or SLO (Fig 3C), but no significant effect was observed with the co