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

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.

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 lg/ml) in the presence of 100 lM 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 lg/ml) in the presence of 100 lM 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 AE 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. 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 lM peptide P2 and CP2. The isogenic SLO mutant strain was used as a negative control. Data are mean AE 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.
▸ Figure EV4. The MRC-1 peptide P2 inhibits CDC-induced cytolysis and intracellular survival of bacteria in human DCs.
A, B (A) IL-12 and (B) TNF-a release by human THP-1 macrophages stimulated with purified PLY, LLO, or SLO (0.5 lg/ml) in the presence or absence of 100 lM peptides P2, P3, or control peptide CP2 for 18 h. Cholesterol (100 lM) was used as positive control to inhibit hemolysis. Data are mean AE s.e.m. from three independent experiments. **P < 0.01, ***P < 0.001, and ****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. C The loss of the GFP signal intensity owing to cell death at 3 h post-stimulation of GFP-expressing 3D lung epithelial models (n = 3) was quantified relative to 1 h time point. Data are mean AE s.e.m. from two independent experiments with n = 3 model/condition. *P < 0.05 by paired t-test. Exact P values are shown in Appendix Table S4. D Cytotoxicity of the lung epithelial model stimulated with 1 lg/ml PLY in the presence or absence of 100 lM peptide P2 or the control peptide CP2 at 18 h. Data are mean AE s.e.m. from two independent experiments with n = 3 model/condition. ****P < 0.0001 by one-way ANOVA with Dunnett's post hoc test for multiple comparisons. Exact P values are shown in Appendix Table S4. E, F (E) TNF-a and (F) IL-8 release by the lung epithelial model stimulated with 1 lg/ml PLY in the presence or absence of 100 lM peptide P2 or the control peptide CP2 at 18 h. **P < 0.01 and ****P < 0.0001 by one-way ANOVA with Dunnett's post hoc test for multiple comparisons. Exact P values are shown in Appendix Table S4. Data are mean AE s.e.m. from two independent experiments with n = 3 model/condition. G Immunofluorescence microscopy images showing intracellular pneumococci of type 4 or type 2 in infected human DCs treated or not with 100 lM peptide P2 or the control peptide CP2. Anti-PLY antibody was used as control to block PLY. DCs infected with D39 or T4 alone possessed intracellular bacteria (green) that colocalized with MRC-1 (red). Arrows indicate MRC-1 co-localized intracellular bacteria (yellow). DCs treated with peptide P2, but not CP2, were devoid of intracellular bacteria. Images are representative of two independent experiments. Scale bars, 10 lm. A-C DCs were infected with (A) the PLY mutant strain T4RDply only, or in the presence of 100 lM peptides, (B) P2 or (C) CP2 at MOI of 10 for 2 h. Immunofluorescence microscopy images show that peptide P2 had no effect on the co-localization of T4RDply (green) which co-localized with LC3B (cyan), but not with MRC-1 (red). Images are representative of three independent experiments. Scale bars, 10 lm.