Research ArticleAPPLIED SCIENCES AND ENGINEERING

A biomimetic peptide recognizes and traps bacteria in vivo as human defensin-6

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Science Advances  08 May 2020:
Vol. 6, no. 19, eaaz4767
DOI: 10.1126/sciadv.aaz4767
  • Fig. 1 Antimicrobial mechanism of natural HD6 and artificial HDMP.

    The HDMP mimics the HD6 that (2) in situ self-assembles on microbial surfaces by ligand-receptor interactions, resulting in (3) self-assembled trapping fibrous networks for the inhibition of microbial invasion. Different from the HD6 that is (1) in situ secreted from Paneth cells, the HDMP is (1′) transported into the targeted region through intravenous administration as nanoparticle (NP) formulation, overcoming the barrier of self-assembling peptides to application in vivo.

  • Fig. 2 Preparation of HDMP NPs and its transformation into NFs.

    (A) Molecular structure of HDMP and schematic illustration of HDMP assembly into NPs, transforming into nanorods and nanofibers (NFs) upon the incubation of lipoteichoic acid (LTA). Transmission electron microscope (TEM) image of HDMP NPs (B), HDMP nanorods (C), and NFs (D) triggered by LTA at 24 and 48 hours, respectively. (E) Dynamic light scattering (DLS) and (F) circular dichroism (CD) of HDMP assembly from NPs into NFs in the presence of LTA in aqueous solutions. (G) Ultraviolet-visible (UV-vis) and fluorescence (FL) spectra of HDMP NPs and HDMP NFs, respectively. The TEM images are representative of three independent experiments. Scale bar, 50 nm. a.u., arbitrary units.

  • Fig. 3 HDMP NPs recognize and transform into NFs on S. aureus in vitro.

    (A) Schematic illustration of HDMP NP– and C-HDMP NP–treated bacteria procedure. The bacteria were first treated with HDMP (30 μM) or C-HDMP (30 μM) NPs for 4 hours, followed by washing with phosphate-buffered saline (PBS) for confocal imaging. (B) Confocal microscopy showing specific recognition of HDMP NPs to S. aureus (top) and low targeting of C-HDMP to S. aureus (bottom). Scale bar, 5 μm. (C) Confocal imaging of S. aureus and E. coli mixture treated by the HDMP NPs (30 μM), showing the specific recognition of HDMP NPs to S. aureus, not E. coli. The red and yellow arrows indicate S. aureus and E. coli, respectively. Scale bar, 10 μm. (D) The normalized intensity profile for regions of interest across the red line in (C). (E to H) TEM images of S. aureus and E. coli treated with HDMP NPs (30 μM) and C-HDMP NPs (30 μM), exhibiting transformed HDMP fibrous networks out of the S. aureus wall (white dashed line) (E) and remained C-HDMP NPs (white dashed circles) on the S. aureus wall (F); the E. coli only without HDMP (G) and C-HDMP NPs (H). Scale bar, 200 nm. The confocal images and TEM images are representative of three independent experiments.

  • Fig. 4 HDMP NPs transform into fibrous networks for trapping S. aureus and inhibiting its invasion in vitro.

    Schematic illustrations and SEM images of S. aureus treated with (A) HDMP NPs (30 μM) and bare S. aureus (B), indicating that the HDMP NPs transformed into fibrous networks trapping the S. aureus. The SEM images are representative of three independent experiments. Scale bar, 1 μm. (C to E). The time-dependent S. aureus agglutination assay, showing that HDMP NPs could trap bacteria effectively. (C) S. aureus suspension without treatment in 6 hours. (D) S. aureus suspension treated with HDMP NPs in 6 hours and (E) S. aureus suspension treated with C-HDMP NPs in 6 hours. (F and G) The invasion inhibition of bacteria to cells, showing the highly efficient inhibition rate of HDMP NPs. Invasion of human umbilical vein endothelial cells (HUVECs) (F) and human embryonic kidney 293 (293T) cells (G) by S. aureus [106 colony-forming units (CFU)/ml] with/without HDMP NPs (10 μM). (H) Three-dimensional confocal image of invasion of 293T cells by S. aureus. The confocal image is representative of three experiments. Scale bar, 1 unit = 9.5 μm. The * represents significant difference between blank and HDMP NP–treated groups (*P < 0.05, **P < 0.01).

  • Fig. 5 HDMP NPs recognize and transform into NFs on S. aureus in vivo.

    (A) In vivo fluorescence images of leg muscle inoculated with 108 CFU bacteria (right) and PBS (left), followed by HDMP NP intravenous administration. (B) The quantification of (A). The * represents significant difference between the PBS group and the experiment group (**P < 0.01). (C) Ex vivo confocal images of infected muscle tissue treated with HDMP NPs, showing specific recognition of HDMP to bacteria in vivo. Green fluorescence is HDMP. Red fluorescence is propidium iodide. Scale bar, 40 μm. (D) The TEM images of muscle tissue slices inoculated with S. aureus and treated with HDMP and C-HDMP NPs, showing transformed HDMP NFs and maintained C-HDMP NPs on bacterial surfaces, respectively. The TEM images are representative of three independent experiments.

  • Fig. 6 In vivo antibacterial effect of HDMP NPs.

    (A) Images of S. aureus inoculated in the right leg muscle in mice in the presence and absence of HDMP NPs (n = 6). (B) The representative hematoxylin and eosin (H&E) staining images of the leg muscle tissue of mice, indicating that the HDMP NP–treated S. aureus did not induce the bacterial infection. (C) Survival curve of the bacteremia mice infected by S. aureus treated with/without HDMP NPs (n = 6), showing the highly efficient antibacterial ability of HDMP NPs. Amount of (D) tumor necrosis factor–α (TNF-α) and (E) interleukin-6 (IL-6) in mouse sera in the bacteremia model during the experiment. The * represents significant difference between the control group and the experiment group (*P < 0.05, **P < 0.01). (F) Survival curve of the bacteremia mice infected by methicillin-resistant S. aureus (MRSA) treated with HDMP NPs, compared with vancomycin (n = 6). (G) Representative images of the MRSA-infected kidney (96 hours, untreated) and MRSA-infected kidney after 1-hour infection treated with vancomycin (5 mg/kg i.v.) or HDMP NPs (5 mg/kg i.v.) (96 hours). The white areas are indicated by black dashed circles. (H) CFU levels at 96 hours in kidney of mice intravenously infected with MRSA with treatment of vancomycin (5 mg/kg), HDMP NPs (5 mg/kg), or PBS 1 hour after infection. n = 3 per treatment group. The * represents significant difference between the control group and the experiment group (***P < 0.001). Photo credit: Yu Fan, NCNST.

Supplementary Materials

  • Supplementary Materials

    A biomimetic peptide recognizes and traps bacteria in vivo as human defensin-6

    Yu Fan, Xiang-Dan Li, Ping-Ping He, Xiao-Xue Hu, Kuo Zhang, Jia-Qi Fan, Pei-Pei Yang, Hao-Yan Zheng, Wen Tian, Zi-Ming Chen, Lei Ji, Hao Wang, Lei Wang

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