Discovery of Next-Generation Antimicrobials through Bacterial Self-Screening of Surface-Displayed Peptide Libraries

Abstract

Peptides have great potential to combat antibiotic resistance. While many platforms can screen peptides for their ability to bind to target cells, there are virtually no platforms that directly assess the functionality of peptides. This limitation is exacerbated when identifying antimicrobial peptides because the phenotype, death, selects against itself and has caused a scientific bottleneck that confines research to a few naturally occurring classes of antimicrobial peptides. We have used this seeming dissonance to develop Surface Localized Antimicrobial Display (SLAY), a platform that allows screening of unlimited numbers of peptides of any length, composition, and structure in a single tube for antimicrobial activity. Using SLAY, we screened ∼800,000 random peptide sequences for antimicrobial function and identified thousands of active sequences, dramatically increasing the number of known antimicrobial sequences. SLAY hits present with different potential mechanisms of peptide action and access to areas of antimicrobial physicochemical space beyond what nature has evolved. VIDEO ABSTRACT.

Keywords: antibiotic resistance; bacteria; drug discovery; high-throughput screening; infectious diseases.

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 hours 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) and 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 hours. (G) Neighboring cells are unaffected by surface expression of antimicrobial peptides. White and blue cells with empty plasmid and cecropin P1 respectively. Input cultures (left) were collected, serial diluted, and spotted before induction of 1 mM IPTG. Cells were induced at a total starting OD 600nm of 0.01. After 3 hours of surface expression, cells were collected, serial diluted, and spotted (right). All growth curves were performed in triplicate. Data are represented as mean ± SEM.

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 (colored 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 methods section.

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 Fig. 2 and methods over a period of 4 hours with plasmids isolation at 0, 2, 3 and 4 hour time points in duplicate. Reads were normalized to the input counts and plotted as a function of time.

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 < or = −1 are plotted in green. Peptides with lfcMLE > −1 were considered inactive 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 vs hydrophobicity plot comparing SLAY active peptides and known active peptides. Known antimicrobial peptides complied from six available online databases are colored in black, active peptides from our screen are colored 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 (standard error of the mean) and the asterisks correspond to Bonferroni adjusted p-values (*, **, and *** denote p-value <0.05, <0.01, and <0.001 respectively) derived from Tukey’s range test performed in conjunction with an ANOVA.

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.