Phage lytic enzymes

Phage lytic enzymes are enzymes produced by bacterial viruses, either as part of their virion to facilitate bacterial infection through local peptidoglycan degradation, or as soluble proteins to induce massive cell lysis at the end of the lytic replication cycle [...

Modeling the architecture of depolymerase-containing receptor binding proteins in Klebsiella phages

Klebsiella pneumoniae carries a thick polysaccharide capsule. This highly variable chemical structure plays an important role in its virulence. Many Klebsiella bacteriophages recognize this capsule with a receptor binding protein (RBP) that contains a depolymerase domain. This domain degrades the capsule to initiate phage infection. RBPs are highly specific and thus largely determine the host spectrum of the phage. A majority of known Klebsiella phages have only one or two RBPs, but phages with up to 11 RBPs with depolymerase activity and a broad host spectrum have been identified. A detailed bioinformatic analysis shows that similar RBP domains repeatedly occur in K. pneumoniae phages with structural RBP domains for attachment of an RBP to the phage tail (anchor domain) or for branching of RBPs (T4gp10-like domain). Structural domains determining the RBP architecture are located at the N-terminus, while the depolymerase is located in the center of protein. Occasionally, the RBP is complemented with an autocleavable chaperone domain at the distal end serving for folding and multimerization. The enzymatic domain is subjected to an intense horizontal transfer to rapidly shift the phage host spectrum without affecting the RBP architecture. These analyses allowed to model a set of conserved RBP architectures, indicating evolutionary linkages

Bacteriophage-encoded virion-associated enzymes to overcome the carbohydrate barriers during the infection process

Bacteriophages are bacterial viruses that infect the host after successful receptor recognition and adsorption to the cell surface. The irreversible adherence followed by genome material ejection into host cell cytoplasm must be preceded by the passage of diverse carbohydrate barriers such as capsule polysaccharides (CPSs), O-polysaccharide chains of lipopolysaccharide (LPS) molecules, extracellular polysaccharides (EPSs) forming biofilm matrix, and peptidoglycan (PG) layers. For that purpose, bacteriophages are equipped with various virion-associated carbohydrate active enzymes, termed polysaccharide depolymerases and lysins, that recognize, bind, and degrade the polysaccharide compounds. We discuss the existing diversity in structural locations, variable architectures, enzymatic specificities, and evolutionary aspects of polysaccharide depolymerases and virion-associated lysins (VALs) and illustrate how these aspects can correlate with the host spectrum. In addition, we present methods that can be used for activity determination and the application potential of these enzymes as antibacterials, antivirulence agents, and diagnostic tools

Synthetic biology of modular enzymes: From enzymes to enzybiotics

Over the past few years antimicrobial resistance has evolved from a rare event to an everyday occurring problem in health care. The future looks even more grim due to the increase of antimicrobial resistance against antibiotics and the unprecedented discovery void of new antibiotic classes. Enzyme-based antimicrobials or enzybiotics represent a novel class of antibacterials. Specifically, endolysins encoded by bacterial viruses (bacteriophages) that degrade the peptidoglycan layer, have gained tremendous interest with many proof-of-concept studies up to clinical phase studies. Initially, native endolysins were only considered for Gram-positive bacteria as the peptidoglycan layer of Gram-negative bacteria is protected by the outer membrane. However, Gram-negative pathogens constitute the largest threat for health care given the higher extent of multi- and pan-drug resistance and lower number of recently developed antibiotics or antibiotics in the pipeline. Using enzyme engineering, we have expanded the spectrum to Gram-negative bacteria. This is achieved by fusing outer membrane permeabilizing peptides via a linker to endolysins. The peptide locally destabilizes the outer membrane and transfers the endolysin moiety across the outer membrane, followed by active peptidoglycan degradation. Exposure of Gram-negative bacteria to these engineered enzybiotics (Artilysin®s) results in a prompt, highly bactericidal effect, which has been confirmed in in vitro keratinocyte cultures and nematodes. Case studies in wound care treatment of dogs have shown a successful outcome. Enzymes are an unusual source for the development of antibacterials, but we have shown that exactly this enzymatic nature provides these engineered enzybiotics with novel features for antibacterials. First, they are rapid and act immediately upon contact. Real-time time-lapse microscopy shows that cells are killed within seconds. Second, they show no cross-resistance with existing antibiotics due to the novel mode-of-action and do not provoke resistance development. Third, they actively degrade all bacterial cells regardless if they are metabolically active or not, whereas traditional antibiotics require an active metabolism. Therefore, engineered enzybiotics are able to kill metabolically dormant persisters that cause recurrent, chronic infections (Briers et al., 2014; Gerstmans et al., 2016). A unique feature of this class of enzybiotics is the engineering potential. They are modular proteins, comprising different domains: an outer membrane permeabilizing peptide, a linker sequence and an endolysin, which in turn comprises a cell wall binding domain and an enzymatically active domain. Depending on the modular composition and the specific order of modules, its enzymatic and antibacterial properties, expression level and stability can be modulated. Using combinatorial shuffling in a synthetic biology approach, we show how targeted antibacterials with diverse properties can be constructed. In sum, engineered enzybiotics provide a platform approach for customized development of antibacterials with unique features based on their enzymatic nature. Briers, Y., Walmagh, M., Van Puyenbroeck, V., Cornelissen, A. Cenens, W., Aertsen, A., Oliveira, H., Azeredo, J., Verween, G., Pirnay, J.-P, Volckaert, G., and Lavigne, R. (2014a) Engineered endolysin-based ‘Artilysins’ to combat multidrug-resistant Gram-negative pathogens. MBio 5:e01379-14. Gerstmans, H., Rodriguez-Rubio, L., Lavigne, R., Briers, Y. (2016) From endolysins to Artilysins: novel enzyme-based approaches to kill drug-resistant bacteria. Biochem Soc T 44:123-12

Quality control and statistical evaluation of combinatorial DNA libraries using nanopore sequencing

Protein engineering and synthetic biology applications increasingly rely on the assembly of modular libraries composed of thousands of different combinations of DNA building blocks. At present, the validation of such libraries is performed by Sanger sequencing analysis on a small subset of clones on an ad hoc basis. Here, we implement a systematic procedure for the comprehensive evaluation of combinatorial libraries, immediately after their creation in vitro, using long reads sequencing technology. After an initial step of nanopore sequencing, we use straightforward bioinformatics tools to tabulate the composition and synteny of the building blocks in each read. We subsequently use exploratory statistics to assess the library and validate its diversity before carrying downstream cloning and screening assays

The preclinical and clinical progress of bacteriophages and their lytic enzymes : the parts are easier than the whole

The therapeutic potential of phages has been considered since their first identification more than a century ago. The evident concept of using a natural predator to treat bacterial infections has, however, since then been challenged considerably. Initially, the vast success of antibiotics almost eliminated the study of phages for therapy. Upon the renaissance of phage therapy research, the most provocative and unique properties of phages such as high specificity, self-replication and co-evolution prohibited a rapid preclinical and clinical development. On the one hand, the typical trajectory followed by small molecule antibiotics could not be simply translated into the preclinical analysis of phages, exemplified by the need for complex broad spectrum or personalized phage cocktails of high purity and the more complex pharmacokinetics. On the other hand, there was no fitting regulatory framework to deal with flexible and sustainable phage therapy approaches, including the setup and approval of adequate clinical trials. While significant advances are incrementally made to eliminate these hurdles, phage-inspired antibacterials have progressed in the slipstream of phage therapy, benefiting from the lack of hurdles that are typically associated with phage therapy. Most advanced are phage lytic enzymes that kill bacteria through peptidoglycan degradation and osmotic lysis. Both phages and their lytic enzymes are now widely considered as safe and have now progressed to clinical phase II to show clinical efficacy as pharmaceutical. Yet, more initiatives are needed to fill the clinical pipeline to beat the typical attrition rates of clinical evaluation and to come to a true evaluation of phages and phage lytic enzymes in the clinic

Rapid and high-throughput evaluation of diverse configurations of engineered lysins using the VersaTile technique

Bacteriophage-encoded lysins are an emerging class of antibacterial enzymes based on peptidoglycan degradation. The modular composition of lysins is a hallmark feature enabling optimization of antibacterial and pharmacological properties by engineering of lysin candidates based on lysin and non-lysin modules. In this regard, the recent introduction of the VersaTile technique allows the rapid construction of large modular lysin libraries based on a premade repository of building blocks. In this study, we perform a high-throughput construction and screening of five combinatorial lysin libraries with different configurations, targeting Klebsiella pneumoniae. An elaborate analysis of the activity distribution of 940 variants and sequencing data of 74 top hits inhibiting the growth of Klebsiella pneumoniae could be associated with specific design rules. Specific outer membrane permeabilizing peptides (OMPs) and enzymatically active domains (EADs) are significantly overrepresented among the top hits, while cell wall binding domains (CBDs) are equally represented. Especially libraries with the configuration (OMP–linker–CBD–EAD) and the inverse configuration (CBD–EAD–linker–OMP) yield the most active variants, with discernible clusters of variants that emerge above the remaining variants. The approach implemented here provides a blueprint for discovery campaigns of engineered lysins starting from libraries with different configurations and compositions