Health care authorities are calling for new antibacterial therapies to cope with the global emergence of antibiotic-resistant bacteria. Bacteriophage-encoded lysins are a unique class of antibacterials with promising (pre)clinical progress. Custom engineering of lysins allows for the creation of variants against potentially any bacterial pathogen. We here present a high-throughput hit-to-lead development platform for engineered lysins. The platform is driven by VersaTile, a new DNA assembly method for the rapid construction of combinatorial libraries of engineered lysins. We constructed approximately 10,000 lysin variants. Using an iterative screening procedure, we identified a lead variant with high antibacterial activity against Acinetobacter baumannii in human serum and an ex vivo pig burn wound model. This generic platform could offer new opportunities to populate the preclinical pipeline with engineered lysins for diverse (therapeutic) applications.

Bacteriophage-encoded endolysins degrading the bacterial peptidoglycan are promising antibacterials for combating antibiotic-resistant bacteria. However, endolysins have limited use against Gramnegative bacteria, since the outer membrane prevents access to the peptidoglycan. Here, we present innolysins, an innovative concept for engineering endolysins to exert antibacterial activity against Gram-negative bacteria. innolysins combine the enzymatic activity of endolysins with the binding capacity of phage receptor binding proteins (RBPs). As proof-of-concept, we constructed 12 Innolysins by fusing phage T5 endolysin and RBP Pb5 in different configurations. One of these, Innolysin Ec6 displayed antibacterial activity against Escherichia coli only in the presence of Pb5 receptor FhuA, leading to 1.22 ± 0.12 log reduction in cell counts. Accordingly, other bacterial species carrying FhuA homologs such as Shigella sonnei and Pseudomonas aeruginosa were sensitive to Innolysin Ec6. To enhance the antibacterial activity, we further constructed 228 novel Innolysins by fusing 23 endolysins with Pb5. High-throughput screening allowed to select Innolysin Ec21 as the best antibacterial candidate, leading to 2.20 ± 0.09 log reduction in E. coli counts. Interestingly, Innolysin Ec21 also displayed bactericidal activity against E. coli resistant to third-generation cephalosporins, reaching a 3.31 ± 0.53 log reduction in cell counts. Overall, the Innolysin approach expands previous endolysinengineering strategies, allowing customization of endolysins by exploiting phage RBPs to specifically target Gram-negative bacteria. Development of novel antibacterials against Gram-negative bacteria is challenging because they possess an outer membrane that prevents many compounds from reaching their intracellular targets 1. Bacteriophages (phages), viruses that infect bacteria, have naturally evolved mechanisms to overcome the outer membrane to infect their bacterial hosts 2,3. In the first step of infection, phages bind to host cells and inject their genetic material across the outer and inner membrane of the bacterial cells into the cytoplasm 4,5. Also, during the final stage of the lytic infection cycle, phages produce proteins within the cell, which destroy the bacterial cell wall, leading to cell lysis 6,7. Thus, the molecular tools developed during phage evolution may be exploited to develop novel phagebased antibacterials that are able to pass the outer membrane and to kill Gram-negative bacteria. Phages recognize their host bacteria by binding to specific surface receptors that may be outer membrane proteins, lipopolysaccharides or components of bacterial capsules, pili and flagella 8-10. The adhesion specificity is mediated by receptor binding proteins (RBPs) that form fibers or spikes at the distal phage tail. A wellcharacterized RBP is the monomeric Pb5, located at the tail tip of the phage T5, which binds irreversibly to the bacterial receptor FhuA during infection of the E. coli host 11,12. FhuA is an outer membrane protein that ...

To meet the needs of synthetic biologists, DNA assembly methods have transformed from simple 'cut-and-paste' procedures to highly advanced, standardised assembly techniques. Implementing these standardised DNA assembly methods in biotechnological research conducted in non-model hosts, including Pseudomonas putida and Pseudomonas aeruginosa, could greatly benefit reproducibility and predictability of experimental results. SEVAtile is a Type IIs-based assembly approach, which enables the rapid and standardised assembly of genetic partsor tilesto create genetic circuits in the established SEVA-vector backbone. Contrary to existing DNA assembly methods, SEVAtile is an easy and straightforward method, which is compatible with any vector, both SEVA-and non-SEVA. To prove the efficiency of the SEVAtile method, a threevector system was successfully generated to independently co-express three different proteins in P. putida and P. aeruginosa. More specifically, one of the vectors, pBGDes, enables genomic integration of assembled circuits in the Tn7 landing site, while self-replicatory vectors pSTDesX and pSTDesR enable inducible expression from the XylS/Pm and RhaRS/PrhaB expression systems, respectively. Together, we hope these vector systems will support research in both the microbial SynBio and Pseudomonas field.

The high specificity of bacteriophages is driven by their receptor-binding proteins (RBPs). Many Klebsiella bacteriophages target the capsular exopolysaccharide as the receptor and encode RBPs with depolymerase activity. The modular structure of these RBPs with an N-terminal structural module to attach the RBP to the phage tail, and a C-terminal specificity module for exopolysaccharide degradation, supports horizontal transfer as a major evolutionary driver for Klebsiella phage RBPs. We mimicked this natural evolutionary process by the construction of modular RBP chimeras, exchanging N-terminal structural modules and C-terminal specificity modules. All chimeras strictly follow the capsular serotype specificity of the C-terminal module. Transplanting chimeras with a K11 N-terminal structural RBP module in a Klebsiella phage K11 scaffold results in a capsular serotype switch and corresponding host range modification of the synthetic phages, demonstrating that horizontal transfer of C-terminal specificity modules offers Klebsiella phages an evolutionary highway for rapid adaptation to new capsular serotypes. IMPORTANCE The antimicrobial resistance crisis has rekindled interest in bacteriophage therapy. Phages have been studied over a century as therapeutics to treat bacterial infections, but one of the biggest challenges for the use of phages in therapeutic interventions remains their high specificity. In particular, many Klebsiella phages have a narrow spectrum constrained by the high diversity of exopolysaccharide capsules that shield access to the cells. In this work, we have elaborated how Klebsiella phages deal with this high diversity by exchanging building blocks of their receptor-binding proteins.

The prevalence of extensively and pandrug-resistant Acinetobacter baumannii leaves little or no therapeutic options for treatment for this bacterial pathogen. Bacteriophages and their lysins represent attractive alternative antibacterial strategies in this regard. We used the extensively drug-resistant A. baumannii strain MK34 to isolate the bacteriophage PMK34 (vB_AbaP_PMK34). This phage shows fast adsorption and lacks virulence genes, nonetheless its narrow host spectrum based on capsule recognition limits broad application. PMK34 is a Fri1virus member of the Autographiviridae and has a 41.8 kb genome (50 ORFs), encoding an endolysin (LysMK34) with potent muralytic activity (1499.9 ± 131 U/μM), a typical mesophilic thermal stability up to 55 °C and a broad pH activity range (4-10). LysMK34 has an intrinsic antibacterial activity up to 4.8 and 2.4 log units for A. baumannii and Pseudomonas aeruginosa strains, respectively, but only when a high turgor pressure is present. Addition of 0.5 mM EDTA or application of an osmotic shock after treatment can compensate for the lack of a high turgor pressure. The combination of LysMK34 and colistin results in up to 32-fold reduction of the MIC of colistin, and colistin-resistant strains are re-sensitized in both Mueller-Hinton broth and 50 % human serum. As such, LysMK34 may be used to safeguard the applicability of colistin as a last-resort antibiotic. Importance A. baumannii is one of the most challenging pathogens for which development of new and effective antimicrobials is urgently needed. Colistin is a last resort antibiotic and even colistin-resistant A. baumannii strains exist. Here, we present a lysin that sensitizes A. baumannii for colistin and can revert colistin resistance to colistin susceptibility. The lysin also shows a strong, turgor pressure dependent intrinsic antibacterial activity, providing new insights in the mode-of-action of lysins with intrinsic activity against Gram-negative bacteria.