Volume 172, Issue 3, Pages 618-628.e13 (January 2018) Discovery of Next-Generation Antimicrobials through Bacterial Self-Screening of Surface-Displayed Peptide Libraries  Ashley T. Tucker, Sean P. Leonard, Cory D. DuBois, Gregory A. Knauf, Ashley L. Cunningham, Claus O. Wilke, M. Stephen Trent, Bryan W. Davies  Cell  Volume 172, Issue 3, Pages 618-628.e13 (January 2018) DOI: 10.1016/j.cell.2017.12.009 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell 2018 172, 618-628.e13DOI: (10.1016/j.cell.2017.12.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 SLAY Platform Demonstrated in Gram-Negative Bacteria (A) Diagram of surface display system. Antimicrobial peptide surface display system composed of (1) Lpp signal sequence, (2) OmpA (46–159) transmembrane protein, (3) flexible tether, (4) C-terminal peptide. The Lpp signal sequence is shown for clarity but is removed prior to insertion into the outer membrane. (B) Optical density plot over a period of 6 hr of a control peptide, tandem influenza hemagglutinin peptide 2xHA (top), and an antimicrobial peptide, cecropin P1 (bottom), expressed in the surface display system induced with 0 mM, 0.1 mM, and 1 mM IPTG. (C) Surface display expression of cecropin P1 as in (B) reported as colony forming units (cfu/mL) over time. (D) Expression of cecropin P1 at 0.1 mM IPTG in the parent strain W3110 (blue), CAMP-resistant W3110 strain WD101 (purple), and eptA deletion in WD101 (red). (E) The surface display is amenable to disulfide-forming peptides. Expression of protegrin 1 (top), defensin HNP-1 (middle), and a defensin cysteine mutant (bottom) plotted as optical density versus time in the E. coli strain W3110. (F) The surface display system functions across many gram-negative species, such as Acinetobacter baumannii and Pseudomonas aeruginosa. Each strain is displaying protegrin 1 at 0 mM, 0.1 mM, and 1 mM IPTG. Plotted are recorded as optical density over 6 hr. (G) Neighboring cells are unaffected by surface expression of antimicrobial peptides. White and blue cells with empty plasmid and cecropin P1, respectively, are displayed. Input cultures (left) were collected, serial diluted, and spotted before induction of 1 mM IPTG. Cells were induced at a total starting OD 600 nm of 0.01. After 3 hr of surface expression, cells were collected, serial diluted, and spotted (right). All growth curves were performed in triplicate. Data are represented as mean ± SEM. See also Figures S1, S2, S3, and S4. Cell 2018 172, 618-628.e13DOI: (10.1016/j.cell.2017.12.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 SLAY Workflow Batch screening of peptides using our surface display system can be achieved by first constructing a random library using random PCR primers that flank the peptide region (i), followed by collection of transformants, plasmid isolation, and subsequent transformation into a bacterial strain of interest. Next, the library is grown in culture and induced (ii). Peptides with antimicrobial activity (red) will drop out of the population (iii). Next-generation sequencing of the initial input at time zero and output (iv) at a pre-defined number of hours provides a read out of sequencing counts (v). From this information, top hits can be identified and tested. Further libraries can be constructed based on the identified top hits and the process can be repeated. A more detailed explanation of our workflow can be found in the STAR Methods section. Cell 2018 172, 618-628.e13DOI: (10.1016/j.cell.2017.12.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 SLAY Platform Demonstrated with a Small, Defined Library A defined set of 5 peptides were cloned and pooled into a small library. The library was tested as described in Figure 2 and STAR Methods over a period of 4 hr with plasmids isolation at 0-, 2-, 3-, and 4-hr time points in duplicate. Reads were normalized to the input counts and plotted as a function of time. Cell 2018 172, 618-628.e13DOI: (10.1016/j.cell.2017.12.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Computational Analysis of the Random Peptide Library Screen Results (A) Mean normalized input and output counts of total peptide library. Peptides considered active with lfcMLE ≤−1 are plotted in green. Peptides with lfcMLE >−1 were considered inactive and are plotted in orange. Peptides removed from further analysis contained initial reads in either replicate of less than or equal to 50 and are plotted in yellow. (B) Screened peptides are plotted according to their hydrophobicity and charge properties. Active peptides are colored in green, and inactive peptides are colored in orange. Ellipses represent a 95% confidence interval assuming a t distribution. (C) A charge versus hydrophobicity plot comparing SLAY active peptides and known active peptides. Known antimicrobial peptides complied from six available online databases are colored in black, and active peptides from our screen are colored in green. Ellipses represent a 95% confidence interval assuming a t distribution. (D) Plot of amino acid frequencies of known, active, and inactive peptides from our screen. The error bars represent the SEM, and the asterisks correspond to Bonferroni-adjusted p values (∗, ∗∗, and ∗∗∗ denote p values <0.05, <0.01, and <0.001, respectively) derived from Tukey’s range test performed in conjunction with an ANOVA. See also Figures S5 and S6. Cell 2018 172, 618-628.e13DOI: (10.1016/j.cell.2017.12.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Mechanism of Action of Select Peptides (A) The membrane damage of E. coli treated by peptides as measured by an increase in fluorescence intensity of PI. E. coli was treated with 25μM peptide. Controls were processed without peptides. (B) Time-kill analysis of selected active peptides from our screen and cecropin P1. (C) Hemolytic activity of selected peptides at 50 μM. Cell 2018 172, 618-628.e13DOI: (10.1016/j.cell.2017.12.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure S1 Intracellular Expression of 2× Tether with Cecropin P1 in E. coli W3110, Related to Figure 1B Cecropin P1 is expressed intracellularly with the 2x tether (without lpp-ompA for localization to the cell surface) at 0 mM, 0.01 mM, 0.1 mM and 1 mM IPTG. Growth curves were performed in triplicate over 6 hours. Data are represented as mean ± SEM. Cell 2018 172, 618-628.e13DOI: (10.1016/j.cell.2017.12.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure S2 Cecropin P1 Activity Increases with Tether Length, Related to Figure 1B Cecropin P1 is displayed with no tether, 1x tether and a 2x at 0 mM and 0.1 mM IPTG. Growth curves were performed in triplicate over 6 hours. Data are represented as mean ± SEM. Cell 2018 172, 618-628.e13DOI: (10.1016/j.cell.2017.12.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure S3 Altered Cecropin P1 Activity with the Addition of Mg2+ (top) and Trypsin (bottom), Related to Figure 1 Cecropin P1 is displayed with increasing concentrations of magnesium and trypsin. Controls were cecropin P1 expressed with no added magnesium or trypsin enzyme. Growth curves were performed in triplicate over 6 hours. Data are represented as mean ± SEM. Cell 2018 172, 618-628.e13DOI: (10.1016/j.cell.2017.12.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure S4 Dermaseptin Surface Display Activity Expressed in E. coli W3110, Related to Figure 1E Dermaseptin S4L7K is displayed on the surface. IPTG concentrations tested were 0mM, 0.01mM, 0.1mM and 1mM. Growth curves were performed in triplicate over 6 hours. Data are represented as mean ± SEM. Cell 2018 172, 618-628.e13DOI: (10.1016/j.cell.2017.12.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure S5 Logo Sequences of a Computational Library, the Total Library, and the 7,968 Top Hits, Related to Figure 4A Sequence logo comparison between a randomly generated 20 amino acid library, our library, and the top hits generated from our library. Sequence logos were generated from either the entire set of possible killing peptides (7,968 sequences), 10,000 randomly sampled sequences of the total library, or an amino acid translation of 10,000 randomly generated nucleotide sequences of a repeated “NNB” motif. All logos are plotted in units of probability. Cell 2018 172, 618-628.e13DOI: (10.1016/j.cell.2017.12.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure S6 Dendrogram Representing Clustering of 6,565 Top Killing Peptides from Our Screen, Related to Figure 4A The resulting clustering dendrogram was divided into 81 subgroups representing different groups of similar peptides identified in this analysis. Cell 2018 172, 618-628.e13DOI: (10.1016/j.cell.2017.12.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure S7 Bacterial Resistance Assay, Related to Figure 5B Serial passage of E. coli W3110 with 15 days of sub-inhibitory concentrations of peptides P1 and P7. Passages were done in triplicate. Resistance is not observed with cationic P1 peptide. One replicate of the anionic P7 peptide developed 64-fold resistance over the 15-day period. Cell 2018 172, 618-628.e13DOI: (10.1016/j.cell.2017.12.009) Copyright © 2017 Elsevier Inc. Terms and Conditions