Abstract
Human enteric α-defensin 5 (HD5) is an immune system peptide that acts as an important antimicrobial factor but is also known to promote pathogen infections by enhancing adhesion of the pathogens. The mechanistic basis of these conflicting functions is unknown. Here we show that HD5 induces abundant filopodial extensions in epithelial cells that capture Shigella, a major human enteroinvasive pathogen that is able to exploit these filopodia for invasion, revealing a mechanism for HD5-augmented bacterial invasion. Using multi-omics screening and in vitro, organoid, dynamic gut-on-chip and in vivo models, we identify the HD5 receptor as P2Y11, a purinergic receptor distributed apically on the luminal surface of the human colonic epithelium. Inhibitor screening identified cAMP-PKA signalling as the main pathway mediating the cytoskeleton-regulating activity of HD5. In illuminating this mechanism of Shigella invasion, our findings raise the possibility of alternative intervention strategies against HD5-augmented infections.
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Data availability
The data supporting the findings of this study are available within the Article and its supplementary files. Publicly available datasets can be accessed at: HGNC (https://www.genenames.org/), Reactome v.86 (https://reactome.org/), The Human Protein Atlas 23.0 (https://www.proteinatlas.org/), Protein Data Bank (https://www.rcsb.org/), UCSC Genome Browser (https://genome.ucsc.edu/), Sequence Read Archive (SRA) (https://www.ncbi.nlm.nih.gov/sra), Genome Sequence Archive (GSA) (https://ngdc.cncb.ac.cn/gsa/). RNA-seq data generated in this study have been deposited at the Genome Sequence Archive (https://ngdc.cncb.ac.cn/search/specific?db=hra&q=HRA006201)82,83. Mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium via the PRIDE partner repository, with dataset identifiers PXD057832 and PXD057885. Data for the analysis are available on GitHub84 and Zenodo85. All bacterial strains, original microscopy images and more relevant data are available from the corresponding author (K.Y., kaiye@xjtu.edu.cn) on reasonable request. Material transfer agreements may be required to distribute resources and materials generated in this study. Source data are provided with this paper.
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Acknowledgements
We thank A. Jegou and L. Cao from Institute Jacques Monod of CNRS and Université Paris Cité for scientific advice and technical assistance on pyrene assay of in vitro actin polymerization; Y. Hao and Z. Ren at the Instrument Analysis Center of Xi’an Jiaotong University for technical assistance with fluorescence and electron microscopic analysis; Y. Wu from Institut Pasteur for providing the Sleeping Beauty transposon system; Guangzhou CSR Biotech Co. Ltd for cell imaging using their commercial super-resolution microscope (HIS-SIM), data acquisition, SR image reconstruction, analysis and discussion; Q. Lu and M. Xia from Shanghai Jiaotong University for suggestions on the molecular docking simulation analysis. This work was supported by National Key R&D Program of China grants 2022YFC3400300 to K.Y.; National Natural Science Foundation of China grants 32125009 to K.Y., 32070134 to D.X., 32270188 to D.X., 82030062 to W.L. and 32222022 to Y.J.; and the Fundamental Research Funds of Xi’an Jiaotong University (xtr052023012 to D.X.).
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D.X., K.Y., P.J.S., W.L. and N.S. conceptualized the project. D.X., M.G., X.X., G.L., S.J.B., C.W. and Y.J. developed the methodology. D.X., M.G., X.X., G.L., Yaxin Liu, C.W., T.X., C.L., Q.W., Wenying Zhao, W. Zeng, Yuezhuangnan Liu, S.L., S.Z. and Wei Zhao conducted investigations. D.X., M.G., X.X., G.L. and S.Z. performed visualization. K.Y., W.L., D.X. and Y.J. acquired funding. D.X. and K.Y. administered the project. K.Y., P.J.S., W.L. and N.S. supervised the project. D.X., M.G. and S.J.B. wrote the original draft. D.X., K.Y., P.J.S., W.L. and S.J.B. reviewed and edited the manuscript.
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Nature Microbiology thanks Gill Diamond and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 Influence of HD5 and Shigella on cytoskeleton in HeLa cells and schematic illustration of experimental procedure of Shigella infection and FRET assay.
a, Fluorescence microscopy analysis of cytoskeleton of HeLa cells in the presence of a serial concentration of HD5. F-actin is red, and nuclei are blue (DAPI). The bars represent 20 μm. b, Influence of Shigella at MOI of 100 on the cytoskeleton of HeLa cells. F-actin is red, GFP-expressing Sf301 is green and nuclei are blue (DAPI). The bars represent 10 μm. c, Schematic illustration of experimental procedure of different pretreatment combinations and co-incubation. To further distinguish the effects of HD5 on bacteria and host cell respectively, HD5 was allowed to interact with Shigella or HeLa cells separately for 15 min, followed by 3 washes to thoroughly remove unbound defensin molecules. Different pretreatment combinations result in different levels of bacterial adhesion and invasion as shown in Fig. 2c. d, Illustration of Förster resonance energy transfer (FRET) assay and its application to analyze the T3SS activity of Shigella in its invasion in the absence of presence of HD5. e, Influence of 2 μM HD5 on the adhesion and invasion of Shigella ΔmxiD mutant in comparison with wildtype Sf301 strain. Significance compared with solvent control (0 μM) group was calculated using a one-way ANOVA (Tukey’s multiple comparison Test), and indicated as the p value. ***p < 0.001 and ****p < 0.0001. Data are presented as mean±s.d. Results are representative of at least three independent experiments.
Extended Data Fig. 2 Influence of HD5 on cytoskeleton of different cell lines and HeLa mutants.
a, Fluorescence microscopy analysis and quantification (n = 10) of cytoskeleton alterations in 10 adherent epithelial cell lines, primary colonocytes and 8 suspension cell lines in the absence (control) or presence of 2 μM HD5. In the upper panel, for epithelial cell lines and primary cells, the bar represents 10 μm; for suspension cells, the bar represents 20 μm. HIFE are indicated with arrows. F-actin is grey and nuclei are blue (DAPI). In the lower panel, bars indicate mean; errors, s.d. b, Fluorescence microscopy analysis of cytoskeleton of HeLa cells microinjected with HD5. HD5 was microinjected into HeLa cells to produce intracellular concentrations of 0.5 to 5 μM, which failed to produce any significant alteration to the cytoskeleton. Injected cells are indicated in red (with Texas Red as microinjection tracker), F-actin is green and nuclei are blue (DAPI). The bar represents 50 μm. c, Fluorescence absorbance analysis of in vitro actin polymerization dynamics of 4 μM actin in the presence of 4 μM HD5 or 1 nM formin. The addition of up to 4 μM HD5 into an in vitro actin polymerization system had minimal effect on the nucleation and elongation of actin filaments. Each data point represents the fluorescence value at that time point. d, Immunoblotting examination of β1 integrin and CD44 knockout in HeLa cells. e, Influence of β1 integrin and CD44 knockout on Shigella adhesion/invasion (n = 6). Data are presented as mean±s.d. f, Influence of β1 integrin and CD44 knockout on the cytoskeleton of HeLa cells in the presence or absence of 4 μM HD5. The bar represents 20 μm. Depletion of β1 integrin subunit and CD44 from HeLa cells by the CRISPR/Cas9 system neither affected the association of HD5 with Shigella adhesion and invasion, nor the formation of filopodia. Significance was determined by a two-tailed unpaired Student’ s t-test, and indicated as the p value. ns: no signification, **p < 0.01, ***p < 0.001 and ****p < 0.0001. All results are representative of at least three independent experiments.
Extended Data Fig. 3 Human phospho-kinase antibody array analysis and phosphoproteomic analysis of HeLa cells upon HD5 treatment.
a, Human phospho-kinase antibody array analysis of the phosphorylation of major kinases in the absence and presence of 4 μM HD5 as instructed by the manufacturer. Significance compared with solvent control (0 μM) group was calculated using two-tailed unpaired Student’ s t-test, and indicated as the p value. ns: no signification, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Data are presented as mean±s.d. b, Top 50 proteins in phosphoproteomic analysis phosphorylated upon HD5 stimulation and dephosphorylated upon suramin treatment. Many of those proteins are involved in cytoskeleton related pathway such as Ras signaling pathway (RALGAPA1), Focal adhesion (CAV2), tight junction (CTTN), PI3K-Akt signaling pathway (PKN2), intermediate-sized filaments formation (KRT8), MAPK signaling (MAPK1), determining cell morphology (MISP), Rap1 interacting factor 1(RIF1), microtubule motor activity (KIF4B),cell junction organization (PLEC) and GTPase activator activity (USP6NL). c, GO terms in cellular component (CC) ontology of the phosphoproteome altered by HD5. A total of 363 phosphorylated sites, including 316 pSer (pS), 30 pThr (pT), and 17pTyr (pY) were selected as high-confidence targets from phosphoproteomic analysis for subsequent gene ontology (GO) enrichment analysis. Results are representative of three independent experiments.
Extended Data Fig. 4 Fluorescence microscopy analysis of influence of widely-recognized cytoskeleton-regulating pathways inhibitors on cytoskeleton of HeLa cells in the presence of 2 μM HD5.
The concentrations of inhibitors used as: 20 μM H89, 20 μM PKI 14-22, 200 μM IBMX, 90 μM RO 20-1724, 50 μM KH7, 10 μM AZD4547, 2 μM AZD6244, 50 μM SP600125, 10 μM SB2035804, 10 μM Gefitinib, 10 μM Lapatinib, 1 μM Bisindolylmaleimide I, 50 μM Rapamycin, 5 μM PI-103, 2.5 μM U-73122, 5 μM Bapta-AM, 10 μM Bapta-AM. F-actin is grey, and nuclei are blue (DAPI). The bar represents 10 μm. Results are representative of three independent experiments.
Extended Data Fig. 5 P2Y11 was identified as the main receptor of HD5 for HIFE generation.
a, Expression analysis of Gs-coupled GPCRs in the tested cell lines using HPA database. b, Transcriptome analysis of the expression of GPCRs in the tested cell lines. c, Mass spectrometry analysis of the biotin-HD5-pulldown proteins using P2Y11-target searching strategy and two peptides of P2Y11 are present in the HD5-associated protein complex. d, 100-ns dynamic trajectory of simulated interaction between HD5 and P2Y11. e, MD simulation showing that NF340 exhibited -2.99 kcal/mol binding free energy to weakly occupy the receptor P2Y11. f-g, Fluorescence microscopy analysis and quantification of influence of NF157 on cytoskeleton of HeLa cells when added after HD5 addition or before HD5 addition. F-actin is red and nuclei are blue (DAPI). The bar represents 20 μm. Data are presented as mean±s.d. Significance between the indicated groups was calculated using a one-way ANOVA (Tukey’ s multiple comparison Test), and p values are as follows: **p < 0.01, ***p < 0.001 and ****p < 0.0001.h, Immunoblotting analysis of P2Y11 expression after CRISPR/Cas9 knockout. All results are representative of at least three independent experiments.
Extended Data Fig. 6 Influence of P2Y11 agonists on HeLa cytoskeleton and the influence of HD5 on caspase activities of HeLa cells.
a, b, Effects of ATPγs and NAD+ on filopodia generation capacity and bacterial invasion efficiency (n = 8). In a, F-actin is red and nuclei are blue (DAPI), the bar represents 10 μm; in b, Data are presented as mean±s.d. c, In non-infected (left panel) and infected (right panel) conditions, the caspase activities (Caspase 3/7) of HeLa cells were assessed in the presence or absence of 4 μM HD5 (n = 3). Significance between indicated groups was calculated using a one-way ANOVA (Dunnett’ s multiple comparison Test for b and Tukey’ s multiple comparison for c), and indicated as the p values. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. All results are representative of at least three independent experiments.
Extended Data Fig. 7 MD analysis of HD5-P2Y11 interaction and distribution of P2Y11 in HeLa cells and human colonic samples.
a, Root-mean-square deviation variance analysis of the steady engagement of HD5 with P2Y11 after 50 ns in the 100-ns dynamic trajectory. b, Fluorescence microscopy analysis of P2Y11 distribution in the absence or presence of 2 μM HD5 in HeLa cells. P2Y11 is green, F-actin is red and nuclei are blue (DAPI). Cell outlines are indicated by the dash lines and arrows. The bar represents 10 μm. c, Fluorescence microscopy analysis of P2Y11 distribution in two human colonic tissue (samples 2 and 3). P2Y11 is green, F-actin is red, and nuclei are blue (DAPI). The bar represents 100 μm. The right panels show gray values of P2Y11 signals in the apical and basolateral regions in human colonic crypt in the samples. All results are representative of at least three independent experiments.
Extended Data Fig. 8 Influence of HD5 on cell adhesion, cell viability, TEER, DSS-damage and microvilli generation of epithelial cells.
a, b, Fluorescence microscopy analysis of influence of initial adherence on HeLa, CACO-2 and HT29-MTX cells in the presence of 2 μM HD5 for 30 min (a) and quantification of cell attachment on petri dishes assessed by crystal violet staining 1 h post-seeding (b). F-actin is grey and nuclei are blue (DAPI). The bars represent 1 μm. c, Effects of different concentrations of HD5 on cell viability after 48-hour treatment (n = 5). d, The influence of different concentrations of HD5 on the trans-epithelial electrical resistance (TEER) during the polarization process of CACO-2 cells. e, Fluorescence microscopy analysis of the protective effect of HD5 on intestinal epithelial barrier upon 10% DSS damage. ZO-1 is green, F-actin is red and nuclei are blue (DAPI). The bars represent 100 μm. f, SEM analysis (n = 5) of the microvilli on polarized CACO-2 cells cultured in transwell for 5 days in the absence (control) or presence of 2 μM HD5. The bars represent 1 μm. Data are presented as mean±s.d. (b-d, f). Significance between indicated groups was calculated using a one-way ANOVA (Dunnett’s multiple comparison Test), and indicated as the p value. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All results are representative of at least three independent experiments.
Extended Data Fig. 9 Influence of P2Y11 antagonists on Shigella infection in epithelial cells and macrophage.
a, Effect of P2Y11 antagonists (suramin, NF449 and NF157) on Shigella adhesion in the absence or presence of 2 μM HD5 (n = 4). b, Influence of suramin, NF449, NF157 on the invasion efficiency of Sf301 in non-polarized CACO-2 cells in the presence of 2 μM HD5 for 30 min (n = 4). Adhesion is defined as the total number (colony-forming unit, CFU) of CACO-2 cell-associated bacteria after 10-minute centrifuge. Invasion is expressed as the CFU of intracellular bacteria in CACO-2 cells in the next 30 min. Invasion efficiency is presented as ratio of invasion/adhesion. c, Fluorescence microscopy analysis examination of NF157 on cytoskeleton of PMA-differentiated THP-1 cells in the presence of 2 μM HD5. F-actin is red, P2Y11 is green and nuclei are blue (DAPI). The bar represents 10 μm. d, Influence of NF157 on the adhesion and invasion efficiency of Shigella in PMA-differentiated THP-1cells in the presence or absence of 2 μM HD5 (n = 6). Data are presented as mean±s.d. (a, b, d). Significance was determined by a one-way ANOVA (Dunnett’ s multiple comparison Test for a, b and Tukey’ s multiple comparison Test for d), and indicated as the p value. ns: no signification, **p < 0.01, ***p < 0.001 and ****p < 0.0001. All results are representative of at least three independent experiments.
Extended Data Fig. 10 Influence of NF157 on Shigella infection in CACO-2 cells and P2Y11 expression in corneal epithelial cells of guinea pigs.
a, Fluorescence microscopy analysis of influence of NF157 on the TSAR activation of Sf301 in non-polarized CACO-2 cells. F-actin is grey, Shigella bacteria are red, GFP-expressing bacteria are green and nuclei are stained with DAPI (blue). The bar represents 100 μm. b-d, Influence of NF157 on intracellular CFU (n = 6) (b), trans-epithelial electrical resistance (n = 3) (TEER, c) and epithelial integrity (d) of polarized CACO-2 cells infected with Shigella in the absence or presence of HD5. F-actin is red. Bacteria are green and nuclei are stained with DAPI (blue), and the bars represent 100 μm. Data are presented as mean±s.d. (b, c). Significance was determined by a two-tailed unpaired Student’ s t-test (b, c), and indicated as the p value. ns: no signification, **p < 0.01, ***p < 0.001 and ****p < 0.0001. e, Influence of NF157 on the Shigella T3SS activation (TSAR activity) on gut-on-chip with constant flow at the rate to 20 μL/h. The bars represent 50 μm. f. Immunofluorescence analysis of expression of P2Y11 in corneal epithelial cells of guinea pigs in the absence (upper panels) or presence (lower panels) of HD5 (Rhodamine-tagged). F-actin is red, P2Y11 is green, HD5 is grey and nuclei are stained with DAPI (blue), and the bars represent 100 μm. All results are representative of at least three independent experiments.
Supplementary information
Supplementary Table 1
Cell lines information; gene expression (GPCR) of different cell lines; oligo sequence and commercial reagents.
Supplementary Video 1
Cytoskeleton alteration of HeLa cells within 25 min in the presence of 4 μM HD5. F-actin is grey. Bar indicates 5 μm. Result is representative of at least 3 independent experiments.
Supplementary Video 2
Shigella invasion of HeLa cells within 25 min in the absence of HD5. F-actin is grey, GFP-expressing Sf301 is green. Bar indicates 5 μm. Result is representative of at least 3 independent experiments.
Supplementary Video 3
Shigella invasion of HeLa cells within 25 min in the presence of 4 μM HD5. F-actin is grey, GFP-expressing Sf301 is green. Bar indicates 5 μm. Result is representative of at least 3 independent experiments.
Supplementary Video 4
Vertical 3D view of fluorescence microscopy analysis of P2Y11 distribution in gut-on-chip built with CACO-2 cells. P2Y11 is green, F-actin is red and nuclei are blue (DAPI). Bar represents 25 μm. Result is representative of at least 3 independent experiments.
Supplementary Video 5
Vertical 3D view of fluorescence microscopy analysis of Shigella infection in gut-on-chip built with CACO-2 cells. Shigella is green, F-actin is red and nuclei are blue (DAPI). Bar represents 25 μm. Result is representative of at least 3 independent experiments.
Supplementary Video 6
Vertical 3D view of fluorescence microscopy analysis of Shigella infection in gut-on-chip built with CACO-2 cells in the presence of 20 μM NF157. F-actin is red, actively-invading bacteria are green (TSAR-positive) and nuclei are stained with DAPI (blue). Bar represents 25 μm. Result is representative of at least 3 independent experiments.
Supplementary Video 7
Vertical 3D view of fluorescence microscopy analysis of Shigella infection in gut-on-chip built with CACO-2 cells in the presence of 4 μM HD5. F-actin is red, actively-invading bacteria are green (TSAR-positive) and nuclei are stained with DAPI (blue). Bar represents 25 μm. Result is representative of at least 3 independent experiments.
Supplementary Video 8
Vertical 3D view of fluorescence microscopy analysis of Shigella infection in gut-on-chip built with CACO-2 cells in the presence of 20 μM NF157 and 4 μM HD5. F-actin is red, actively-invading bacteria are green (TSAR-positive) and nuclei are stained with DAPI (blue). Bar represents 25 μm. Result is representative of at least 3 independent experiments.
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Xu, D., Guo, M., Xu, X. et al. Shigella infection is facilitated by interaction of human enteric α-defensin 5 with colonic epithelial receptor P2Y11. Nat Microbiol 10, 509–526 (2025). https://doi.org/10.1038/s41564-024-01901-9
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DOI: https://doi.org/10.1038/s41564-024-01901-9
