Surface Localized Antimicrobial Display, or SLAY, is a high-throughput screening platform that identifies novel antimicrobial peptides to combat multi-drug-resistant gram-negative bacteria.
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Antibiotic resistance is one of the biggest threats to human life, with antibiotic resistant bacteria estimated to cause 30 million deaths by 2050.
Antibiotic resistance occurs when bacteria change and become non-responsive to antibiotic medicines. There is an urgent need to develop new class of antibiotics that can target the diverse range of antibiotic resistant bacteria that have now emerged.
Antibiotic development
The last new class of antibiotics was developed over 40 years ago, however, several research groups have developed antimicrobial peptides that supplement old antibiotics.
Antimicrobial peptides or host defense peptides are broad spectrum antibiotics which can target bacteria, enveloped viruses, and fungi. However, techniques to screen peptide libraries and discover newer peptides are currently lacking, with most studies focusing on naturally occurring cationic antimicrobial peptides or CAMPs.
Due to their cationic charge and size they attach and insert into the bacterial membrane bilayers to form pores. Although CAMPs have been shown to be effective in vitro, in vivo their effects are less potent. Thus, new techniques for antimicrobial peptide exploration beyond naturally available templates are required.
Fundamentals of SLAY
Creating a peptide library and transforming bacterial cells
For achieving surface localized antimicrobial display, a random peptide library is created using random PCR primers that flank the peptide region. Plasmids containing these peptide sequences are then transformed or integrated into the genome of bacteria (of interest).
The peptides are fusion proteins consisting of the following: a) murein lipoprotein (lpp) signal sequence that directs proteins for export from the cytoplasm; b) five transmembrane domains (residues 46–159) of the OmpA membrane protein for outer membrane localization; c) flexible tether that allows spatial freedom; d) C-terminal peptide.
The transmembrane domain is added to the peptide as during infections, antibiotics first interact with a bacterium at its cell surface. Thus to recapitulate this, the peptides are targeted to the bacterial cell surface.
Expression of antimicrobial peptides in the bacteria
Subsequently, the gram negative bacteria expressing the fusion peptide proteins are grown in culture and induced by IPTG. The peptides with antibacterial properties will lead to bactericidal or bacteriostatic effects and the expressing bacteria will be eliminated from the population.
Next generation sequencing of input and output populations
Using next generation sequencing techniques, sequences from plasmid libraries are collected pre- and post-induction. In silico techniques are then used to compare each peptide and its abundance pre- and post-induction. This leads to identification of potential antimicrobial peptides.
Validation of antimicrobial peptides
For validation, three antimicrobial peptides discovered in SLAY and two control peptides are transformed or integrated in to the E.coli genome. Subsequently they are induced and harvested at 0,2,3,4 hours.
Next generation library construction and sequencing is done and the reads are normalized to the input counts. While the control peptides should show a near neutral log change fold in the input and output sequences, test peptides should ideally exhibit a log2 fold change of -1 or lower indicating their removal from the population over time.
SLAY uncovers the chemically diverse pool of antibiotics
The antibiotics discovered till now are dominated by peptides that are cationic (positively charged) and amphipathic (containing both hydrophilic and hydrophobic parts). However, the peptides identified using SLAY have neutral charge and neutral hydrophobicity on average.
Although in traditional peptides positively charged lysine is found in much higher frequency, there is no enrichment of any specific amino acid in active versus inactive sequences discovered through SLAY. They also have equally frequent presence of hydrophobic amino acids, such as alanine, isoleucine, leucine, and valine. Thus, SLAY helps uncover vast and unexplored landscape of potential antimicrobial peptides.
SLAY hits have different mechanisms of antibacterial action
Although traditional antimicrobial peptides act on the bacteria through membrane disruption with bactericidal effects, peptides identified by SLAY peptides have been proposed to possess non-pore forming and diverse mechanisms of action.
The mechanism of action of peptides identified through SLAY is still unclear. Also, the SLAY peptides and other cationic peptides have a significant difference in their toxic properties. Cationic peptides cause seizure-like activity at 25 mg/kg and immediate mortality at 35 mg/kg showing its toxic effects. However, anionic peptides discovered in SLAY do not show any toxic effects upto 50 mg/kg.
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