You get what you test for: The killing effect of phage lysins is highly dependent on buffer tonicity and ionic strength.

The phage lysin field has done nothing but grow in the last decades. As a result, many different research groups around the world are contributing to the field, often with certain methodological differences that pose a challenge to the interpretation and comparison of results. In this work, we present the case study of three Acinetobacter baumannii-targeting phage lysins (wild-type endolysin LysMK34 plus engineered lysins eLysMK34 and 1D10) plus one lysin with broad activity against Gram-positive bacteria (PlySs2) to provide exemplary evidence on the risks of generalization when using one of the most common lysin evaluation assays: the killing assay with resting cells. To that end, we performed killing assays with the aforementioned lysins using hypo-, iso- and hypertonic buffers plus human serum either as the reaction or the dilution medium in a systematic manner. Our findings stress the perils of creating hypotonic conditions or a hypotonic shock during a killing assay, suggesting that hypotonic buffers should be avoided as a test environment or as diluents before plating to avoid overestimation of the killing effect in the assayed conditions. As a conclusion, we suggest that the nature of both the incubation and the dilution buffers should be always clearly identified when reporting killing activity data, and that for experimental consistency the same incubation buffer should be used as a diluent for posterior serial dilution and plating unless explicitly required by the experimental design. In addition, the most appropriate buffer mimicking the final application must be chosen to obtain relevant results.

The Specific Capsule Depolymerase of Phage PMK34 Sensitizes Acinetobacter baumannii to Serum Killing.

The rising antimicrobial resistance is particularly alarming for Acinetobacter baumannii, calling for the discovery and evaluation of alternatives to treat A. baumannii infections. Some bacteriophages produce a structural protein that depolymerizes capsular exopolysaccharide. Such purified depolymerases are considered as novel antivirulence compounds. We identified and characterized a depolymerase (DpoMK34) from Acinetobacter phage vB_AbaP_PMK34 active against the clinical isolate A. baumannii MK34. In silico analysis reveals a modular protein displaying a conserved N-terminal domain for anchoring to the phage tail, and variable central and C-terminal domains for enzymatic activity and specificity. AlphaFold-Multimer predicts a trimeric protein adopting an elongated structure due to a long α-helix, an enzymatic ß-helix domain and a hypervariable 4 amino acid hotspot in the most ultimate loop of the C-terminal domain. In contrast to the tail fiber of phage T3, this hypervariable hotspot appears unrelated with the primary receptor. The functional characterization of DpoMK34 revealed a mesophilic enzyme active up to 50 °C across a wide pH range (4 to 11) and specific for the capsule of A. baumannii MK34. Enzymatic degradation of the A. baumannii MK34 capsule causes a significant drop in phage adsorption from 95% to 9% after 5 min. Although lacking intrinsic antibacterial activity, DpoMK34 renders A. baumannii MK34 fully susceptible to serum killing in a serum concentration dependent manner. Unlike phage PMK34, DpoMK34 does not easily select for resistant mutants either against PMK34 or itself. In sum, DpoMK34 is a potential antivirulence compound that can be included in a depolymerase cocktail to control difficult to treat A. baumannii infections.

PhaLP: A Database for the Study of Phage Lytic Proteins and Their Evolution.

Phage lytic proteins are a clinically advanced class of novel enzyme-based antibiotics, so-called enzybiotics. A growing community of researchers develops phage lytic proteins with the perspective of their use as enzybiotics. A successful translation of enzybiotics to the market requires well-considered selections of phage lytic proteins in early research stages. Here, we introduce PhaLP, a database of phage lytic proteins, which serves as an open portal to facilitate the development of phage lytic proteins. PhaLP is a comprehensive, easily accessible and automatically updated database (currently 16,095 entries). Capitalizing on the rich content of PhaLP, we have mapped the high diversity of natural phage lytic proteins and conducted analyses at three levels to gain insight in their host-specific evolution. First, we provide an overview of the modular diversity. Secondly, datamining and interpretable machine learning approaches were adopted to reveal host-specific design rules for domain architectures in endolysins. Lastly, the evolution of phage lytic proteins on the protein sequence level was explored, revealing host-specific clusters. In sum, PhaLP can act as a starting point for the broad community of enzybiotic researchers, while the steadily improving evolutionary insights will serve as a natural inspiration for protein engineers.

Predicting bacteriophage hosts based on sequences of annotated receptor-binding proteins.

Nowadays, bacteriophages are increasingly considered as an alternative treatment for a variety of bacterial infections in cases where classical antibiotics have become ineffective. However, characterizing the host specificity of phages remains a labor- and time-intensive process. In order to alleviate this burden, we have developed a new machine-learning-based pipeline to predict bacteriophage hosts based on annotated receptor-binding protein (RBP) sequence data. We focus on predicting bacterial hosts from the ESKAPE group, Escherichia coli, Salmonella enterica and Clostridium difficile. We compare the performance of our predictive model with that of the widely used Basic Local Alignment Search Tool (BLAST). Our best-performing predictive model reaches Precision-Recall Area Under the Curve (PR-AUC) scores between 73.6 and 93.8% for different levels of sequence similarity in the collected data. Our model reaches a performance comparable to that of BLASTp when sequence similarity in the data is high and starts outperforming BLASTp when sequence similarity drops below 75%. Therefore, our machine learning methods can be especially useful in settings in which sequence similarity to other known sequences is low. Predicting the hosts of novel metagenomic RBP sequences could extend our toolbox to tune the host spectrum of phages or phage tail-like bacteriocins by swapping RBPs.

Synthetic biology of modular endolysins.

Endolysins and their derivatives have emerged in recent years as a novel class of antibacterials, which have now entered the clinical phases. Their rapid mode-of-action and proteinaceous nature differentiates them from any other class of antibiotics. A key feature of endolysins is their modularity and the opportunities that emerge thereof to customize properties such as specificity, activity, stability and solubility. Extensive protein engineering efforts have expanded the activity spectrum to (pan)drug-resistant Gram-negative bacteria or have improved the activity against Gram-positive pathogens. In addition, specific cell wall binding domains derived from endolysins are exploited for the development of diagnostics.