Enterococcal bacteriophage: A survey of the tail associated lysin landscape

Abstract

Bacteriophages are viruses that exclusively infect bacteria which require local degradation of cell barriers. This degradation is accomplished by various lysins located mainly within the phage tail structure. In this paper we surveyed and analysed the genomes of 506 isolated bacteriophage and prophage infecting or harboured within the genomes of the medically important Enterococcus faecalis and faecium. We highlight and characterise the major features of the genomes of phage in the morphological groups podovirus, siphovirus and myovirus, and explore their categorisation according to the new ICTV classifications, with a focus on putative extracellular lysins chiefly within tail modules. Our analysis reveals a range of potential cell-wall targeting enzyme domains that are part of tail, tape measure or other predicted base structures of these phages or prophages. These largely fall into protein domains targeting pentapeptide or glycosidic linkages within peptidoglycan but also potentially the enterococcal polysaccharide antigen (EPA) and wall teichoic acids of these species (i.e., Pectinesterases and Phosphodiesterases). Notably, there is a great variety of domain architectures that reveal the diversity of evolutionary solutions to attack the Enterococcus cell wall. Despite this variety, most phage and prophage possess a putative endopeptidase (70%), reflecting the ubiquity of this cell surface barrier. We also identified a predicted lytic transglycosylase domain belonging to the glycosyl hydrolase (GH) family 23 and present exclusively within tape measure proteins. Our data also reveal distinct features of the genomes of podo-, sipho- and myo-type viruses that most likely relate to their size and complexity. Overall, we lay a foundation for expression of recombinant TAL proteins and engineering of enterococcal and other phage that will be invaluable for researchers in this field.

Keywords: Bacteriophages; Enterococcus; Prophage; Tail-associated lysin.

Fig. 1

Schematic representation of the bacterial peptidoglycan structure showing target locations of Glycosidases (grey), Amidases (yellow) and Endopeptidases (red). MurNAc: N-acetylmuramic acids GlcNAc: N-acetylglucosamine. This figure was created with Biorender.com.

Fig. 2

(A) 100 enterococcal phage genomes were plotted against genome size. The genomes are labelled in accordance with the new ICTV classification as follow: Schiekvirus (dark green), Kochikohdavirus (light green), Andrewesvirinae (dark blue), Saphexavirus (blue), Efquatrovirus (Azure), Phifelvirus (sky), Copernicusvirus (red), Minhovirus (orange), Studiervirinae (brown),. The grey colour indicates unclassified genomes regarding the current ICTV classification and further details are included in the supplementary file1. Phage morphologies are also included according to the ICTV classification. Temperate phages are underlined and labelled with asterisks. (B) 406 intact prophage genomes were plotted against genome size. The genomes are in ascending order in both figures.

Fig. 3

(A) Schematic representation of the identified lysins and their targets in this study. This part was created with Biorender.com. (B) The order of the Functional modules of the enterococcal Phage genomes. Modules and specific genes are coloured as follow: Green= DNA packaging and head, Red= Head alone, Purple= Packaging alone, Blue= Tail, Pink= lysis, Orange= DNA metabolism. Of note, podovirus general genome organisation contains some labelled gene like yellow= NLPC/P60 gene. HNH stands for homing endonuclease. The general scheme of the siphovirus tail module is also drawn Brown=TMP, Dark green= Dit and Dark blue=Tal. The new ICTV classification is also indicated regarding each phage morphology.

Fig. 4

Domain architectures of TAEP proteins based on Pfam. Five DAs are shown with coloured domains. Red= endopeptidase, Blue= lysozyme, Green= CHAP, Yellow= M23 peptidase, Dark blue= amidase, Grey= chaperone of endosialidase. The left side contains the phage or prophage's name and ICTV classification while the right side contains the DA type and its abundance in percentage. The length of the protein is also indicated on the right side.

Fig. 5

(A) Domain architectures of NLPC/P60 containing proteins. Five DA are shown with coloured domains. Green= NLPC/P60, Blue= M23 peptidase, Orange= lysozyme. The domain type and abundance (%) are indicated on the right side while phage or prophage name and the ICTV classification is on the left side. The length of the protein is also indicated. (B) a phylogenetic tree of all identified NLPC/P60 containing proteins show two main groups based on phage genomic classification and morphology: sequences from podoviruses labelled Blue while myoviruses labelled red. The tree was constructed using FastTree and visualised using the ITOL online website.

Fig. 6

(A) Domain architecture of TMP-LT proteins. Based on CDD, 12 DA are shown with coloured domains. Purple= lytic transglycosylase-like domain (LT), Green= tape measure protein domain (TMP), Dark blue= structural maintenance of chromosomes (SMC), Red= Tail protein, Sky blue= Synaptonemal complex protein (SCP-1), Orange= Minor tail protein, Dark Grey=SbcC, Blue= Phage-related protein, Light green= Merozoite Apical Erythrocyte Binding-ligand (MAEBL), Brown= Endonuclease, light grey= Hyaluronan mediated motility receptor N-terminal (HMMR_N). The domain type and abundance (%) are indicated on the right side while phage or prophage name and the ICTV classification is on the left side. The asterisks indicate conserved residues specific for analysed sequences. The length of the protein is also mentioned. (B) Weblogo of the TMP-LT domains showing highly conserved domains including the catalytic residue Glutamic acid (red arrow) and GH23 specific motif (Blue arrows). Sequence logos were created using Weblogo (http://weblogo.berkeley.edu/logo.cgi).

Fig. 7

(A) Domain architecture of a Pectinesterase containing protein from E7663-prophage3 genome based on NCBI domain database which showed Pectinesterase domain. (B) MSA of tail modules showing Pectinesterase protein (yellow), TMP-LT (Blue), TAEP (Orange). HP= Hypothetical proteins. The level of identity is indicated by the grey region between genomes.

Fig. 8

(A) Domains architectures of the GDPD containing proteins based on Pfam. Three DA are shown with coloured domains. Green= GDPD, Blue= membrane and dark blue=baseplate upper protein. The domain type and abundance (%) are indicated on the right side while phage or prophage name and the ICTV classification is on the left side. The length of the protein is also indicated. (B) MSA of tail modules showing GDPD protein (Green), TAEP (Orange), HP= Hypothetical proteins. The level of identity is indicated by the grey region between genomes. C) weblogo of the aligned GDPD domains which shows catalytic residues (Blue arrows) and metal binding residues (Red arrows).

Fig. 9

The general organisation of Tail modules in enterococcal phage genomes. (A) examples of podovirus genomes harbour NLPC/P60 containing protein (pink colour). (B) Group1 of siphovirus genomes contain both TAEP (orange) and TMP-LT (blue) proteins while Group2 (C) contains only TAEP proteins (orange). (D) myovirus genomes harbour TMP-LT and NLPC/P60 proteins. The phage names and the ICTV classification are indicated on the left side. The level of identity is indicated by the grey region between genomes.