Hornerin contains a Linked Series of Ribosome-Targeting Peptide Antibiotics

Abstract

Cationic intrinsically disordered antimicrobial peptides (CIDAMPs) belong to a novel class of epithelial peptide antibiotics with microbicidal activity against various pathogens, including Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Here we show that treatment of distinct bacteria with different hornerin (HRNR)-derived CIDAMPs cause formation of unique cytoplasmic protein aggregates, suggesting a common intracellular mode of action. We further found that, unlike most amphipathic antimicrobial peptides, HRNR traverses bacterial membranes energy-dependently and accumulates within the cytoplasm. Strikingly, certain structurally different, HRNR-based CIDAMPs were found to bind to an identical panel of distinct bacterial ribosomal proteins, thereby manifesting features of several known classes of antibiotics. This may cause the formation of aberrant proteins and toxic protein aggregates in HRNR-treated pathogens which eventually may induce its death. Our study reveals evidence that structurally distinct CIDAMPs of an abundant body surface protein simultaneously target multiple sites of the bacterial protein synthesis machinery.

Figure 1

TEM analyses of HRNR-treated E. coli and P. aeruginosa. (a,b) Transmission electron microscopy (TEM) analyses of 6.25 × 10⁷/ml E. coli ATCC 11775 in 10 mM NaP, pH 5.5, 1 h treatment with 312.5 µg/ml rHRNR_2591–2684. (c) E. coli control. Note the absence of membrane perturbation and the presence of intracytoplasmic electron-dense aggregates in rHRNR_2591–2684-treated bacteria (a,b). The hyperhydrated looking periplasmic space of many cells in the control (a), is similar as seen for E. coli treated with low ion strength and acidic buffers. TEM of 6.25 × 10⁷/ml P. aeruginosa ATCC 10145, in 10 mM NaP, pH 5.5, 1 h treatment with 312.5 µg/ml rHRNR_2591–2684 (d,e). (f) P. aeruginosa control. Note condensation of electron-dense cytoplasmic material and blebs of the outer membrane with an occasional ballooning (d,e). 1 h treatment of 6.25 × 10⁷/ml P. aeruginosa ATCC 10145 with 469 µg/ml rSUMO3-HRNR_2591–2684 in 10 mM NaP, pH 5.5 revealed widespread peeling of the outer membrane (g,h). (i) P. aeruginosa control. Images are representative of two independent experiments, sampling on average 10 images per condition and species in each experiment.

Figure 2

Ultrastructural analyses of rHRNR_2591–2684-treated S. aureus. TEM analyses of 6.25 × 10⁷/ml S. aureus ATCC 6538, in 10 mM NaP, pH 5.5, treated with 312.5 µg/ml rHRNR_2591–2684 for 2 h. Note the condensation of electron-dense cytoplasmic material and formation of membrane blebs (a–c). Occasionally ballooning (c) and aggregated cells (d), connected via electron-dense contacts, were found upon rHRNR_2591–2684-treatment. (e,f) Control. Images are representative of two independent experiments, sampling on average 10 images.

Figure 3

Ultrastructural analyses of HRNR-treated C. albicans. TEM analyses of 6.25 × 10⁷/ml C. albicans ATCC 244433, treated for 2 h with 312.5 µg/ml rHRNR_2591–2684 in 10 mM NaP, pH 5.5 (a–d). (e,f) Control. Note the release of electron dense vesicles (a–c) and marked changes of the intracytoplasmic morphology. Images are representative of two independent experiments, sampling on average 10 images in each experiment.

Figure 4

rHRNR_2591–2684 is a non-permeabilizing, energy-dependently translocating CIDAMP. (a) rHRNR_2591–2684 does not permeabilize the bacterial membrane. Lysis by 50 μg/mL lysozyme (expressed as OD₅₉₅ against time ± s. e. m., n = 3) of chloramphenicol-treated P. aeruginosa PAO1 cells (gray line) in the presence of polymyxin B (PMB, 10 μg/mL; dotted line) or rHRNR_2591–2684 (5 μg/mL; black line). (b) rHRNR_2591–2684 is translocated into bacterial cytosol. HRNR-Western blot of fractionated, rhHRNR_2591–2684-treated PAO1, Ctrl: control (untreated PAO1). S: sample supernatant, OM: outer membrane-, P: periplasmic-, IM: inner membrane-, C: cytoplasmic fraction, HR: Hornerin fragment rHRNR_2591–2684. (c) rHRNR_2591–2684 translocation is energy-dependent. PAO1 was treated with rHRNR_2591–2684 in the presence of NaN₃, fractionated and analyzed by HRNR-Western blot.

Figure 5

HRNR binds to intracytoplasmic aggregates in P. aeruginosa. P. aeruginosa ATCC 10145 was treated for 5 min with rHRNR_2591–2684 (45 µg/ml 10 mM NaP, pH 5.5), and then cellular localization of this CIDAMP was analyzed by immunocytochemistry with a HRNR_2591–2684-specific polyclonal antibody, followed by incubation with a gold-conjugated secondary antibody (a). Bacteria, treated with rHRNR_2591–2684 (b) or buffer (c), followed by incubation with the gold-conjugated secondary antibody, served as controls. Note accumulation of intracytoplasmic immuno-gold (a), which is corresponding to electron dense cytoplasmic aggregates seen upon TEM-analyses of CIDAMP-treated P. aeruginosa (Fig. 1c,d). Images are representative of two independent experiments, sampling on average 10 images in each experiment.

Figure 6

Distinct HRNR-derived CIDAMPs show similar ribosomal protein binding patterns. (a) SulfoLink®–column-bound proteins of an E. coli-extract were separated on a Jupiter® C18 RP-HPLC column with a Prp-gradient. HPLC fractions containing UV-absorbing peaks (C1–D10) were divided into five aliquots and adjusted in parallel to five PAGE-gels and separated. (b) Silver-stained proteins. (c) HRNR-Far-Western blot for probing with biotinylated HR1-18 (HRNR_2556–2677) using Strep-Tactin®, (d) HRNR-Far-Western blot for probing with biotinylated rSumo3-HRNR_2591–2684 using Strep-Tactin®, (e) HRNR-Far-Western blot for probing with rHRNR_2591–2684 using anti-HRNR_2591–2684 antibodies, (f) HRNR-Far-Western blot for probing with rHRNR_1075–1172 using anti-HRNR_1075–1172 antibodies. Note similarities of the staining patterns, irrespective the CIDAMP AA-sequence or biotin-labeling and irrespective whether a Strep-Tactin®- or antibody-detectable CIDAMP was used to probe and detect the target protein on the membrane. Note the presence of 70 kDa bands upon HRNR-Far-Western blot analyses in most of the investigated HPLC fractions with highest intensity for rHRNR_1075–1172 binding (f). The most intensive band, corresponding to a 37 kDa protein in fraction number C13, was identified as E. coli ribosomal protein L2.

Figure 7

TEM of Gentamicin-treated P. aeruginosa at pH 5.5. (a,b) TEM of P. aeruginosa ATCC 11145, treated with 30 µg/mL Gentamicin in 10 mM NaP, pH 5.5, for 2 h at 37 °C. (c,d) Buffer control (also Supplementary Fig. S1g,h). Note condensation of electron-dense cytoplasmic material in Gentamicin-treated bacteria (a,b). Images are representative of two independent experiments, sampling on average 10 images.