VersaTile Platform to create engineered proteins active against the main pathogens implicated in burn wounds

Phage lytic proteins are a novel class of antibiotics with recently demonstrated clinical efficacy in a phase II trial. The main advantages of this type of proteins is that they rapidly kill the bacterial via peptidoglycan degradation followed by osmotic lysis. Moreover, most of them present a modular structure, which allows further engineering of the different activity domains to create proteins with enhanced activity against specific bacterial pathogens. However, this progress is empirical and hampered by the tedious and time consuming cloning procedures and the further activity assays. To address this technical barrier, our research group has developed the VersaTile Platform, which allows users to generate unlimited numbers of (combinatorial) shuffled proteins of any length, composition and structure in a single tube. A major expansion of this modularity principle is the fusion of endolysins to outer membrane permeabilizing peptides, facilitating efficient transfer across the outer membrane of Gram-negative bacteria, followed by rapid cell lysis. These peptides also increase the protein activity in the case of gram positive bacteria. In this context, we have used the VersaTile Platform to create engineered lysins against bacterial pathogens implicated in burn wound infections. Burn wound patients are particularly vulnerable to bacterial infections since a burn wound constitutes a perfect gateway for bacterial entrance and is also accompanied by several alterations that revoke the normal self-defense mechanisms. Two of the main pathogens implicated in burn wound infections are Staphylococcus aureus and Acinetobacter baumannii. First, we used the VersaTile Cloning to create a tile repository (more than 200 tiles) that includes different single domains of several phage lytic proteins and a collection of diverse antimicrobial peptides. From this repository, a library of engineered lysins (more than 10000) was created by VersaTile Shuffling combining the different domains and cloned into E. coli for protein expression. Finally, the activity of ~1400 clones was tested. A total of 29 proteins showed enhanced activity against A. baumannii and 37 against S. aureus. From all these proteins, 1D10, active against A. baumannii was selected for further testing its activity in an ex vivo model of pig burn wound infection. This protein is highly stable in human serum, highly thermostable and can prevent biofilm formation in an ex vivo model of pig skin, keeping the bacterial number under 105 CFU per pig skin explant when applying repeated doses of treatment with 50 μg of protein. In fact, after a single protein dose, we observed a 4 log reduction of the bacteria in 5 min. Overall our results indicate that the VersaTile Platform is a rapid, hit to lead methodology that allow the creation and testing of thousands of engineered proteins active against bacterial pathogens, constituting a source of almost infinite novel antibacterials.