Publish date: 11.03.2018
Bacterial resistance extended to all existing classes of antibiotics and became a global health problem. The discovery of new classes of antibiotics becomes more difficult and expensive every year, therefore, antimicrobial peptides are of great interest to scientists in academia and in pharmaceutical industry due to their almost unlimited variety. However, peptide screening for antimicrobial activity is an extremely difficult task.
The authors, of an article published in the journal Cell, (Tucker AT, Leonard SP, DuBois CD, Knauf GA, Cunningham LA, Wilke CO, Trent MS Davies BW., Discovery of Next-Generation Antimicrobials through Bacterial Self-Screening of Surface-Displayed Peptide Libraries Cell 172, 618-628, January 25, 2018) developed a new approach to search for new antimicrobial peptides - a surface-localized antimicrobial display (Surface Localized Antimicrobial Display, SLAY).
The peptides (in this paper they are random sequences of 20 amino acids) are expressed in E. coli as part of a fusion protein consisting of a murein lipoprotein signal sequence (that directs proteins for export from the cytoplasm and subsequently cleaved), five transmembrane domains of the corresponding membrane protein (for outer membrane localization), and a peptide connected to the 180 Å linker (allowing the peptide to interact freely with the external membrane and to penetrate into the periplasm and into the cytoplasmic membrane of gram-negative bacteria).
This simulates the situation in vivo when the antimicrobial drug first interacts with the surface of the bacterium, penetrates through the outer membrane into the periplasm and then passes through the cytoplasmic membrane to bind to its target.
To test the developed screening system, the authors cloned the known antimicrobial peptide in this vector and showed that the induction of expression causes the death of bacteria. If a peptide sequence without antimicrobial activity was cloned in the given vector, the induction did not affect the growth of bacteria in any way.
Further, the authors showed that the antimicrobial peptide expressed according to this method affects only the growth of the host cell, but not the adjacent cells. This means that the screening of a library of different peptides can be done in a single tube.
After induction, those cells in which the antimicrobial peptides were expressed, ceased to grow and divide, while cells where peptides without antimicrobial activity were expressed continued to grow. Sequencing the entire DNA of the mixture (next generation sequencing) before and after the induction establishes which sequences are disappearing, or the relative amount sequences are decreasing, and thus allows the detection of antimicrobial peptides.
In order to prove the concept of this new screening method a control library was constructed that included three peptides with known antimicrobial activity and two peptides without activity. All of them were correctly identified. Then, a library of 800,000 random sequences of 20 amino acids was constructed and about 7,000 antimicrobial peptides were found. From this group of peptides, the authors selected 22 substances with different properties, synthesized them and tested for activity against both E. coli and against other pathogens resistant to antibiotics (Pseudomonas, Klebsiella).
Some of the peptides showed significant activity, and, what is particularly interesting, their amino acid sequence and mechanism of action differed from cationic antimicrobial peptides found in nature.
Undoubtedly, like any screening, this method is only the first step on the way to developing a new drug. Identified peptides must be synthesized and tested for antimicrobial activity and toxicity. The next step, as a rule, is to optimize the lead compounds in order to expand the spectrum of activity and reduce side effects. After the most promising compounds have been identified, a long and costly animal-testing program followed by clinical trials begins.
The system developed by the authors of the article can make a significant contribution to the fight against bacteria resistant to antibiotics, as it gives access to previously unexplored chemical space and allows us to hope for the discovery of new antimicrobial peptides, as well as new targets and mechanisms of action for antimicrobial drugs.
Maria Debabova, journalist, founder of the popular science YouTube channel "Ratiomania"