Research ArticleHEALTH AND MEDICINE

Ω76: A designed antimicrobial peptide to combat carbapenem- and tigecycline-resistant Acinetobacter baumannii

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Science Advances  24 Jul 2019:
Vol. 5, no. 7, eaax1946
DOI: 10.1126/sciadv.aax1946
  • Fig. 1 An illustration of a single step of AMP design using Heligrapher, showing how the maximum common subgraph scoring scheme functions.

  • Fig. 2 In vitro and in vivo toxicity assessment of Ω76 and controls.

    (A) HeLa cell line toxicity for peptides Ω17 and Ω76. (B) HaCaT cell line toxicity for peptides Ω17 and Ω76. (C) Human blood hemolysis assay for peptides Ω17 and Ω76. (A to C) All experiments were performed in three to five replicates. Lines and shaded regions indicate means and SD, respectively. (D) In vivo LD50 (median lethal dose) determination for Ω76, pexiganan, and colistin using a BALB/c mouse model. (E) Multidose cumulative toxicity determination for Ω76, pexiganan, and colistin using a BALB/c mouse model. (F) Row 1: Ω76-colistin coadministration experiment. All data relevant to this experiment have been highlighted in yellow across all panels. Row 2: Ω76 multidose survival experiment [repeated from (E) for completeness]. Rows 3 to 5: Cumulative toxicity determination for Ω76 administered at different time intervals. Row 6: Ω76 single-dose survival experiment [repeated from (D) for completeness]. BALB/c mouse kidney and liver histological sections after treatment with Ω76 and controls. (G) Kidney from untreated mouse, displaying no damage. (H) Kidney from Ω76-treated mouse, displaying no damage. (I) Kidney from colistin-treated mouse. Cast formation (red arrowheads) and tubular necrosis (green arrowhead, dislodged cellular material) are both visible. (J) Liver from untreated mouse, displaying no damage. (K) Liver from Ω76-treated mouse, displaying no damage. (L) Liver from colistin-treated mouse, displaying no damage. Scale bar, 20 μm. All raw data for this figure are provided in dataset S1.

  • Fig. 3 In vivo efficacy of Ω76 using a BALB/c mouse peritoneal model of infection.

    (A) Protocol for the survival experiment to determine the efficacy of Ω76. (B) Protocol for peritoneal and spleen CFU estimation to determine the efficacy of Ω76. (C) Results of the survival experiment to determine the efficacy of Ω76, along with untreated, meropenem, and tigecycline controls (P values were calculated using Fisher’s exact test). (D) Results of the peritoneal CFU estimation experiment to determine the efficacy of Ω76, along with untreated, meropenem, and tigecycline controls (P values were calculated using the Welch two-sample t test). (E) Results of the spleen CFU estimation experiment to determine the efficacy of Ω76, along with untreated, meropenem, and tigecycline controls (P values were calculated using the Welch two-sample t test). All raw data for this figure are provided in dataset S1.

  • Fig. 4 Confocal microscopy experiments performed on E. coli (K12 MG1655) and A. baumannii (P1270).

    (Top) FITC-labeled Ω76-treated E. coli. Ω76 colocalized with Nile red, indicating a membrane-binding propensity. (Bottom) FITC-labeled Ω76-treated A. baumannii (P1270). Ω76 again colocalized with Nile red, indicating a membrane-binding propensity. (Table) Pearson’s correlation coefficients given for all combinations of stains (DAPI/FITC peptide/Nile red). Better stain colocalization is denoted by higher correlation values. In both cases, the Nile red/FITC peptide pair was the most strongly correlated. Scale bar, 2 μm. Note that these images have been digitally magnified by 3× for clarity. All original images are provided in dataset S1.

  • Fig. 5 The time-kill kinetics and elimination kinetics of Ω76.

    (A and B) Kinetic experiments showing the rapid action of Ω76 on A. baumannii (P1270). All experiments in these panels were performed in triplicate. (A) Time-kill curves performed on A. baumannii (P1270) treated with clinically relevant doses of Ω76, colistin, and an untreated control. These experiments were performed in ex vivo whole human blood. (B) PO43-32 release radioassay to detect the leakage of small molecules upon incubation of A. baumannii (P1270) with Ω76, colistin, and an untreated control. (C) Pharmacokinetic experiments performed on mice to determine the bloodstream absorption and elimination kinetics of peritoneally injected Nselmet-Ω76. Curve fitting was performed using spline interpolation. The shaded area corresponds to Nselmet-Ω76 serum concentrations above the MBC. Inset: Fold reduction curves for Ω76, performed on A. baumannii (P1270) in ex vivo whole human blood. The fold reduction curve for Ω76 at 32 mg/liter is derived from the same data displayed in (A). The fold reduction curve for Ω76 at MBC (4 mg/liter) closely follows the trend at 32 mg/liter up to 6 min. However, Ω76 at MBC is unable to continue reducing A. baumannii (P1270) CFU counts, diverging from the 32 mg/liter trend at 8 min. For all experiments, lines and shaded regions indicate means and SD, respectively. All raw data for this figure are provided in dataset S1.

  • Fig. 6 The molecular response of A. baumannii (P1270) to Ω76 challenge.

    Up- and down-regulated (GO-up and Go-down) genes are colored green and red, respectively. For clarity, only DEGs belonging to GO terms with biological functions relevant to this study are shown. A full list of DEGs can be found in tables S4 and S5. Note that some genes can have multiple functions and belong to multiple GO terms. Note that some genes do not have corresponding gene names assigned. In these cases, the truncated Agilent ID has been used. For example, “2251” mentioned in the above figure corresponds to Agilent ID ABAYE2251.

  • Fig. 7 Calculation of the solution structure of Ω76 in the presence of DPC micelles.

    Sequential assignments were carried out using the HN- Hα region of the 1H,1H-NOESY spectra of Ω76 (A) in 100% CD3OH and (B) in 25 mM DPC (D38) in 90%/10% H2O/D2O. The labels are of the form x-y or z, where x is the residue number of the Hα resonance, y is the residue number of the HN resonance, and z is an intra-residue correlation (x = y). (C) HN-HN region of the 1H,1H-NOESY spectra of Ω76 in 100% CD3OH and (D) in 25 mM DPC (D38) in 90%/10% H2O/D2O. The labels indicate the residue numbers of the HN resonances involved in the correlation. Eighteen HiN-Hi+1N correlations are observed and labeled in bold, while 14 of 16 weaker HiN-Hi+2N correlations are also shown. The remaining two HiN-Hi+2N correlations are seen at lower contour levels and are therefore not labeled. (E) Summary of the NOE and chemical shift data used for assigning the secondary structure as a function of residue number. (F) Ensemble of 30 calculated structures (left) and view down the helix axis (right). The side-chain colors indicate hydrophobic (white), acidic (red), and basic (blue) residues.

  • Table 1 The Ω-family peptides.

    (Top) Names, sequences, and I_scores of all Ω-family peptides synthesized for this study. Note that pexiganan was used as a toxicity control. (Bottom) Sequence alignment between Ω76 and pexiganan. Despite some similarities, the two peptides display vastly different toxicological profiles, as described in the “In vitro and in vivo toxicity of designed AMPs” section.

    PeptideSequenceI_score
    Ω03KLGKKLRKKLKKIGKGLKAI3
    Ω13KAIKRIGKRIKKLLLKLKKK5
    Ω17RKKAIKLVKKLVKKLKKALK19
    Ω76FLKAIKKFGKEFKKIGAKLK6
    Ω93IKALGKKLRKGKKKIGKKVK1
    PexigananGIGKFLKKAKKFGKAFVKILKKNot tested
    Ω76-shuf1AFLLKKKKGIIFFEKAKKGKNot tested
    Ω76-shuf2AKKKKFIFIKEKAFLLKGKGNot tested
    Ω76-shuf3KKKKGFILILKEAFAFKKGKNot tested
    Ω76-shuf4AKFKKEKILLFAKGKFIKGKNot tested
    Ω76––––FLKAIKKFGKEFKKIGAKLK
    PexigananGIGKFLKKAKKFGKAFVKILKK––
  • Table 2 MBC values for Ω17 and Ω76 against clinical isolates.

    (Top) MBC values for Ω17 against clinical isolates. This table depicts a frequency distribution. Taking E. coli as an example, Ω17 inhibits six isolates with an MBC of 1 mg/liter, seven isolates with an MBC of 2 mg/liter, three isolates with an MBC of 4 mg/liter, and three more isolates with an MBC of 8 mg/liter. Therefore, the median MBC value (MBC50) for E. coli is 2 mg/liter. (Bottom) MBC values for Ω76 against clinical isolates. Resistance phenotypes are also mentioned. CRE, carbapenem-resistant Enterobacteriaceae; ESBL, extended-spectrum beta lactamase producers; MRSA, methicillin-resistant S. aureus; MRCN, methicillin-resistant coagulase-negative Staphylococci.

    Ω17: OrganismResistance0.250.51248163264128>128MBC50
    E. coliCRE2223
    E. coliESBL441
    E. coli1
    Total67332
    A. baumanniiCRE111
    A. baumannii1
    Total11112
    K. pneumoniaeCRE11111
    K. pneumoniaeESBL12
    K. pneumoniae1
    Total111111364
    P. aeruginosaCRE121
    P. aeruginosaESBL1
    P. aeruginosa2
    Total31218
    E. faecalis23>128
    CoNSMRCN13
    CoNS1111
    Total12414
    S. aureusMRSA111
    S. aureus132
    Total11133128
    Gram −ve7125631144
    Gram +ve13421113616
    Total815984124104
    Ω76: OrganismResistance0.250.51248163264128>128MBC50
    E. coliCRE2313
    E. coliESBL5211
    E. coli1
    Total5432234
    A. baumanniiCRE141
    A. baumannii1
    Total1514
    K. pneumoniaeCRE1211
    K. pneumoniaeESBL111
    K. pneumoniae1
    Total12132128
    P. aeruginosaCRE13
    P. aeruginosaESBL1
    P. aeruginosa11
    Total121316
    E. faecalis113>128
    CoNSMRCN1221
    CoNS121
    Total114232
    S. aureusMRSA12
    S. aureus6
    Total18>128
    Gram −ve55107433328
    Gram +ve125311>128
    Total5510868631316

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaax1946/DC1

    Fig. S1. The origins of common subgraphical motifs shared by five Ω-family AMPs.

    Fig. S2. Checkerboard assay to determine whether Ω76 and colistin in combination display a synergistic, additive, or antagonistic effect in vitro.

    Fig. S3. Blood tests performed for multidose Ω76-treated mice in comparison to untreated control mice.

    Fig. S4. Pilot experiments to determine the in vivo efficacy of Ω76 against A. baumannii (P1270) using a BALB/c mouse peritoneal model of infection.

    Fig. S5. Independent replication experiment to confirm Ω76 activity against A. baumannii (P1270) in BALB/c mice, performed by A.D.

    Fig. S6. SEM experiments performed for A. baumannii (B4505).

    Fig. S7. SEM experiments performed for A. baumannii (B4505) protoplasts.

    Fig. S8. SEM experiments performed for E. coli (K12 MG1655).

    Fig. S9. SEM experiments performed for S. flexneri (MTCC1457).

    Fig. S10. The classification of genes into GO terms after enrichment analysis.

    Fig. S11. Ramachandran plot analysis of the best 30 structures of Ω76.

    Fig. S12. The electrostatic surface potential and hydrophobic moment of Ω76.

    Fig. S13. An illustration for all the steps required to determine the hemolytic activity of an AMP using human blood.

    Fig. S14. Titration to determine the dose of A. baumannii (P1270) to be used for later efficacy experiments.

    Fig. S15. The protocols used for generating time-kill curves are illustrated in detail.

    Fig. S16. The protocol used for the PO43-32 leakage radioassay is illustrated in detail.

    Table S1. A detailed tabulation depicting the origin of common subgraphical motifs shared by five Ω-family AMPs.

    Table S2. MIC values expressed in micrograms per liter for 30 cultures tested against five designed peptides.

    Table S3. MBC values expressed in micrograms per liter for Gram-positive cultures tested against two designed peptides displaying the highest I_scores (Ω17 and Ω76).

    Table S4. MBC values expressed in micrograms per liter for Gram-negative cultures tested against two designed peptides displaying the highest I_scores (Ω17 and Ω76).

    Table S5. MBC values expressed in micrograms per liter for seven clinical isolates of A. baumannii.

    Table S6. 16S rRNA gene sequencing data for A. baumannii (P1270), confirming the genus and species.

    Table S7. List of significantly up-regulated genes for A. baumannii (P1270) exposed to 0.1×, 0.25×, and 0.5× MBC concentrations of Ω76.

    Table S8. List of significantly down-regulated genes for A. baumannii (P1270) exposed to 0.1×, 0.25×, and 0.5× MBC concentrations of Ω76.

    Table S9. NMR restraints and structure evaluation statistics for Ω76 (30 structures).

    Text S1. Coordinates of the 30 lowest-scoring NMR models of Ω76.

    Dataset S1. Raw data for all figures.

    Dataset S2. All original images acquired during confocal microscopy experiments are provided.

    Dataset S3. Raw NOESY and TOCSY spectra.

    Dataset S4. A. baumannii gene to GO term mappings.

    Protocol S1. HELIGRAPHER.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. The origins of common subgraphical motifs shared by five Ω-family AMPs.
    • Fig. S2. Checkerboard assay to determine whether Ω76 and colistin in combination display a synergistic, additive, or antagonistic effect in vitro.
    • Fig. S3. Blood tests performed for multidose Ω76-treated mice in comparison to untreated control mice.
    • Fig. S4. Pilot experiments to determine the in vivo efficacy of Ω76 against A. baumannii (P1270) using a BALB/c mouse peritoneal model of infection.
    • Fig. S5. Independent replication experiment to confirm Ω76 activity against A. baumannii (P1270) in BALB/c mice, performed by A.D.
    • Fig. S6. SEM experiments performed for A. baumannii (B4505).
    • Fig. S7. SEM experiments performed for A. baumannii (B4505) protoplasts.
    • Fig. S8. SEM experiments performed for E. coli (K12 MG1655).
    • Fig. S9. SEM experiments performed for S. flexneri (MTCC1457).
    • Fig. S10. The classification of genes into GO terms after enrichment analysis.
    • Fig. S11. Ramachandran plot analysis of the best 30 structures of Ω76.
    • Fig. S12. The electrostatic surface potential and hydrophobic moment of Ω76.
    • Fig. S13. An illustration for all the steps required to determine the hemolytic activity of an AMP using human blood.
    • Fig. S14. Titration to determine the dose of A. baumannii (P1270) to be used for later efficacy experiments.
    • Fig. S15. The protocols used for generating time-kill curves are illustrated in detail.
    • Fig. S16. The protocol used for the 32PO34 leakage radioassay is illustrated in detail.
    • Table S1. A detailed tabulation depicting the origin of common subgraphical motifs shared by five Ω-family AMPs.
    • Table S2. MIC values expressed in micrograms per liter for 30 cultures tested against five designed peptides.
    • Table S3. MBC values expressed in micrograms per liter for Gram-positive cultures tested against two designed peptides displaying the highest I_scores (Ω17 and Ω76).
    • Table S4. MBC values expressed in micrograms per liter for Gram-negative cultures tested against two designed peptides displaying the highest I_scores (Ω17 and Ω76).
    • Table S5. MBC values expressed in micrograms per liter for seven clinical isolates of A. baumannii.
    • Table S6. 16S rRNA gene sequencing data for A. baumannii (P1270), confirming the genus and species.
    • Table S7. List of significantly up-regulated genes for A. baumannii (P1270) exposed to 0.1×, 0.25×, and 0.5× MBC concentrations of Ω76.
    • Table S8. List of significantly down-regulated genes for A. baumannii (P1270) exposed to 0.1×, 0.25×, and 0.5× MBC concentrations of Ω76.
    • Table S9. NMR restraints and structure evaluation statistics for Ω76 (30 structures).

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Text S1 (.pdb format). Coordinates of the 30 lowest-scoring NMR models of Ω76.
    • Dataset S1 (Microsoft Excel format). Raw data for all figures.
    • Dataset S2 (.zip format). All original images acquired during confocal microscopy experiments are provided.
    • Dataset S3 (.zip format). Raw NOESY and TOCSY spectra.
    • Dataset S4 (.csv format). A. baumannii gene to GO term mappings.
    • Protocol S1 (.zip format). HELIGRAPHER.

    Files in this Data Supplement:

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