Sequence-Function Relationships in Phage-Encoded Bacterial Cell Wall Lytic Enzymes and Their Implications for Phage-Derived Product Design

Abstract

Phage (endo)lysins are thought to be a viable alternative to usual antibiotic chemotherapy to fight resistant bacterial infections. However, a landscape view of lysins’ structure and properties regarding their function, with an applied focus, is somewhat lacking. Current literature suggests that specific features typical of lysins from phages infecting Gram-negative bacteria (G−) (higher net charge, amphipathic helices) are responsible for improved interaction with the G− envelope. Such antimicrobial peptide (AMP)-like elements are also of interest for antimicrobial molecules design. Thus, this study aims to provide an updated view on the primary structural landscape of phage lysins to clarify the evolutionary importance of several sequence-predicted properties, particularly for the interaction with the G− surface. A database of 2,182 lysin sequences was compiled, containing relevant information such as domain architectures, data on the phages’ host bacteria, and sequence-predicted physicochemical properties. Based on such classifiers, an investigation of the differential appearance of certain features was conducted. Such analyses revealed different lysin architectural variants that are preferably found in phages infecting certain bacterial hosts. Particularly, some physicochemical properties (higher net charge, hydrophobicity, hydrophobic moment, and aliphatic index) were associated with G− phage lysins, appearing specifically at their C-terminal end. Evidence on the remarkable genetic specialization of lysins regarding the features of the bacterial hosts has been provided, specifically supporting the nowadays common hypothesis that lysins from G− usually contain AMP-like regions. IMPORTANCE Phage-encoded lytic enzymes, also called lysins, are one of the most promising alternatives to common antibiotics. The potential of lysins as novel antimicrobials to tackle antibiotic-resistant bacteria not only arises from features such as a lower chance to provoke resistance, but also from their versatility as synthetic biology parts. Functional modules derived from lysins are currently being used for the design of novel antimicrobials with desired properties. This study provides a view of the lysins diversity landscape by examining a set of phage lysin genes. This way, we have uncovered the fundamental differences between the lysins from phages that infect bacteria with different superficial architectures, and, thus, also the reach of their specialization regarding cell wall structures. These results provide clarity and evidence to sustain some of the common hypotheses in current literature, as well as make available an updated and characterized database of lysins sequences for further developments.

A five-fold expansion of the global RNA virome reveals multiple new clades of RNA bacteriophages

“…Closteroviridae and Rhabdoviridae(Walker et al, 2020;Wolf et al, 2020), and were further confirmed and extended in this study. Collectively, these observations emphasise the inherent 23 plasticity of RNA ancestral in all three virus groups.…”

“…3B). Numerous dsDNA bacteriophages and dsRNA viruses of the Cystoviridae family encode one or more lytic enzymes, known as endolysins, that possess diverse enzymatic activities and degrade bacterial peptidoglycan (Cahill and Young, 2019; Criel et al, 2021; Vázquez et al, 2021). In contrast, leviviruses induce host cell lysis by inhibiting the peptidoglycan biosynthesis via small membrane proteins that comprise the so-called single gene lysis (Sgl) system (Cahill and Young, 2019).…”

Potential Role of the Host-Derived Cell-Wall Binding Domain of Endolysin CD16/50L as a Molecular Anchor in Preservation of Uninfected Clostridioides difficile for New Rounds of Phage Infection

“…S4C). As our findings are consistent with other studies performed in several pairs of bacterial cells and endolysins, i.e., in C. difficile , B. anthracis , and L. monocytogenes (Table S1) ( 26 , 63 , 64 ), we indirectly infer that the CBD might have other physiologically beneficial impacts on the endolysins or phage biology besides substrate specificity; otherwise, the modular architecture of the endolysins would not be evolutionarily conserved across diverse bacteriophages ( 65 , 66 ).…”

Bacteriophage Lytic Enzyme P9ly as an Alternative Antibacterial Agent Against Antibiotic-Resistant Shigella dysenteriae and Staphylococcus aureus

“…Overall, it seems that the 64-residues at the N-terminus of P9ly are both necessary for cell lysis and crucial for peptidoglycan binding, which is consistent with the fact that 93.9% of the endolysins derived from phages infecting Gram-negative bacteria are globular ( Briers and Lavigne, 2015 ). Actually, this type of architecture carrying a single phage lysozyme domain also accounted for 50.8% of the discovered lysins from phages infecting Gram-negative bacteria in a recently constructed database ( Vázquez et al, 2021b ). In the future, protein structure-related studies could be performed to elucidate the molecular mechanism of bacteriophage lytic enzyme P9ly more accurately from a structural perspective.…”

“…We compared the protein sequence between Ts2631 and P9ly, especially their N-terminal part, but no significant similarity was found. Additionally, several studies ( Thandar et al, 2016 ; Sykilinda et al, 2018 ; Vázquez et al, 2021a , b ) have revealed that antimicrobial peptide (AMP)-like regions in the C-terminal of phage lysins and/or their alpha-helical structure exposed outside of the protein globule showed superb antimicrobial activity and could be engineered for improved bactericidal activity. However, we also did not find similarity between P9ly and these reported C-terminal AMPs, including PlyF307 ( Thandar et al, 2016 ), AcLys ( Sykilinda et al, 2018 ), and Pae87 ( Vázquez et al, 2021a ).…”