Antimicrobial Microwebs of DNA-Histone Inspired from Neutrophil Extracellular Traps

Abstract

Neutrophil extracellular traps (NETs) are decondensed chromatin networks released by neutrophils that can trap and kill pathogens but can also paradoxically promote biofilms. The mechanism of NET functions remains ambiguous, at least in part, due to their complex and variable compositions. To unravel the antimicrobial performance of NETs, a minimalistic NET-like synthetic structure, termed "microwebs," is produced by the sonochemical complexation of DNA and histone. The prepared microwebs have structural similarity to NETs at the nanometer to micrometer dimensions but with well-defined molecular compositions. Microwebs prepared with different DNA to histone ratios show that microwebs trap pathogenic Escherichia coli in a manner similar to NETs when the zeta potential of the microwebs is positive. The DNA nanofiber networks and the bactericidal histone constituting the microwebs inhibit the growth of E. coli. Moreover, microwebs work synergistically with colistin sulfate, a common and a last-resort antibiotic, by targeting the cell envelope of pathogenic bacteria. The synthesis of microwebs enables mechanistic studies not possible with NETs, and it opens new possibilities for constructing biomimetic bacterial microenvironments to better understand and predict physiological pathogen responses.

Keywords: DNA nanofiber networks; antibiotic resistance; bacteria E. coli; biomimetic materials; neutrophil extracellular traps.

Figure 1.

Synthesis and characterization of NET-like microwebs. a) Scheme and b) Scanning electron microscopy (SEM) illustrating NETs. c) Scheme and d) SEM illustrating microwebs. Scale bars = 1 μm. e) Zeta potentials and f) size distribution (DLS measurement) of microwebs. Size distribution of sonicated NETs is shown by the red curve. g) DNase-induced degradation of microwebs in HBSS. Microwebs were stained by SYTOX (E_x/E_m= 488/523 nm).

Figure 2.

Entrapment of E. coli on microwebs. SEM images showing E. coli trapped on microwebs with a) ζ=−8 mV, b) ζ=0 mV and c) ζ=+6.5 mV. Scale bars = 10 μm. d) Percentage of trapped E. coli on microwebs as a function of incubation time. e) The number of planktonic E. coli escaped from microwebs were counted by plating and colony forming unit (CFU) enumeration. Percentage of planktonic E. coli is calculated from the ratio of E. coli in supernatants relative to that in NET-free control group. ** P < 0.01.

Figure 3.

Viability assessment of E. coli cultured with microwebs. Fluorescence microscopy images of E. coli in a) tryptic soy broth (TSB); b) 100 μg ml⁻¹ microwebs (ω_his = 50%), c) 100 μg ml⁻¹ NETs; d) 50 μg ml⁻¹ DNA solution and e) 50 μg ml⁻¹ histone solution. (Live cells, green; dead cells, red) Scale bars = 10 μm. The corresponding SEM images of E. coli cells cultured in indicated solutions are shown below, from f) to j). Scale bars = 1μm. k) Microwebs reduce colony forming units of E. coli in HBSS. 10⁵ E. coli cells were incubated in 100 μl microwebs (50 μg ml⁻¹ DNA, ω_his = 50%), NETs (contains 50 μg ml⁻¹ DNA, triggered by phorbol 12-myristate 13-acetate), 50 μg ml⁻¹ DNA, and 50 μg ml⁻¹ histone respectively for 1 hour before transferred to agar plates. **P<0.01; *** P < 0.001) Growth curves of E. coli in a mixture of 100 μl microweb suspension and 100 μl TSB plus 1% glucose.

Figure 4.

Microwebs can work cooperatively with antibiotics against E. coli. (a,b) CFU counting of E. coli (Inoculum = 10⁵ cells) after culture in a 100μl HBSS containing 100 μg ml⁻¹ microwebs(ω_his = 50%) plus antibiotics (a) colistin; (b) amoxicillin for 1 hour at 37°C. Comparisons on the growth curves of E. coli (Inoculum = 10⁶ cells) cultured in a mixture solution of 100μl TSB, 100μl HBSS, microwebs and antibiotics: (c) with colistin, without microwebs; (d) with colistin, with 50 μg ml⁻¹ microwebs; (e) with amoxicillin, without microwebs; and (f) with amoxicillin, with 50 μg ml⁻¹ microwebs. ** P < 0.01; ***P<0.001.