The multidomain architecture of a bacteriophage endolysin enables intramolecular synergism and regulation of bacterial lysis

Abstract

Endolysins are peptidoglycan hydrolases produced at the end of the bacteriophage (phage) replication cycle to lyse the host cell. Endolysins in Gram-positive phages come in a variety of multimodular forms that combine different catalytic and cell wall binding domains. However, the reason why phages adopt endolysins with such complex multidomain architecture is not well understood. In this study, we used the Streptococcus dysgalactiae phage endolysin PlySK1249 as a model to investigate the role of multidomain architecture in phage-induced bacterial lysis and lysis regulation. PlySK1249 consists of an amidase (Ami) domain that lyses bacterial cells, a nonbacteriolytic endopeptidase (CHAP) domain that acts as a dechaining enzyme, and a central LysM cell wall binding domain. We observed that the Ami and CHAP domains synergized for peptidoglycan digestion and bacteriolysis in the native enzyme or when expressed individually and reunified. The CHAP endopeptidase resolved complex polymers of stem-peptides to dimers and helped the Ami domain to digest peptidoglycan to completion. We also found that PlySK1249 was subject to proteolytic cleavage by host cell wall proteases both in vitro and after phage induction. Cleavage disconnected the different domains by hydrolyzing their linker regions, thus hindering their bacteriolytic cooperation and possibly modulating the lytic activity of the enzyme. PlySK1249 cleavage by cell-wall-associated proteases may represent another example of phage adaptation toward the use of existing bacterial regulation mechanism for their own advantage. In addition, understanding more thoroughly the multidomain interplay of PlySK1249 broadens our knowledge on the ideal architecture of therapeutic antibacterial endolysins. Endolysins are peptidoglycan hydrolases produced at the end of the bacteriophage (phage) replication cycle to lyse the host cell. Endolysins in Gram-positive phages come in a variety of multimodular forms that combine different catalytic and cell wall binding domains. However, the reason why phages adopt endolysins with such complex multidomain architecture is not well understood. In this study, we used the Streptococcus dysgalactiae phage endolysin PlySK1249 as a model to investigate the role of multidomain architecture in phage-induced bacterial lysis and lysis regulation. PlySK1249 consists of an amidase (Ami) domain that lyses bacterial cells, a nonbacteriolytic endopeptidase (CHAP) domain that acts as a dechaining enzyme, and a central LysM cell wall binding domain. We observed that the Ami and CHAP domains synergized for peptidoglycan digestion and bacteriolysis in the native enzyme or when expressed individually and reunified. The CHAP endopeptidase resolved complex polymers of stem-peptides to dimers and helped the Ami domain to digest peptidoglycan to completion. We also found that PlySK1249 was subject to proteolytic cleavage by host cell wall proteases both in vitro and after phage induction. Cleavage disconnected the different domains by hydrolyzing their linker regions, thus hindering their bacteriolytic cooperation and possibly modulating the lytic activity of the enzyme. PlySK1249 cleavage by cell-wall-associated proteases may represent another example of phage adaptation toward the use of existing bacterial regulation mechanism for their own advantage. In addition, understanding more thoroughly the multidomain interplay of PlySK1249 broadens our knowledge on the ideal architecture of therapeutic antibacterial endolysins. Phage endolysins represent a fascinating family of peptidoglycan hydrolases that are critical for bacterial lysis and release of phage progeny at the end of the phage life cycle (1Young R. Phage lysis: Three steps, three choices, one outcome.J. Microbiol. 2014; 52: 243-258Crossref PubMed Scopus (158) Google Scholar). They have gained recent interest in biomedical development as potential antibacterial agents (2Nelson D.C. Schmelcher M. Rodriguez-Rubio L. Klumpp J. Pritchard D.G. Dong S. Donovan D.M. Endolysins as antimicrobials.Adv. Virus Res. 2012; 83: 299-365Crossref PubMed Scopus (207) Google Scholar, 3Fischetti V.A. Bacteriophage lysins as effective antibacterials.Curr. Opin. Microbiol. 2008; 11: 393-400Crossref PubMed Scopus (289) Google Scholar, 4Fischetti V.A. Bacteriophage endolysins: A novel anti-infective to control gram-positive pathogens.Int. J. Med. Microbiol. 2010; 300: 357-362Crossref PubMed Scopus (265) Google Scholar). Phage endolysins target the bacterial peptidoglycan, an essential structure made of a complex meshwork of N-acetylglucosamine (GlcNAc)–N-acetylmuramic acid (MurNAc) glycan strands cross-linked by short stem peptides attached to MurNAc residues (5Schleifer K.H. Kandler O. Peptidoglycan types of bacterial cell walls and their taxonomic implications.Bacteriol. 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G. the for and Microbiol. 2014; Google Scholar, Bacteriophage of and Opin. Microbiol. 2005; PubMed Scopus Google Scholar). They are at the or of the and are in the central G. A. J. a for binding to Microbiol. 2008; PubMed Scopus Google Scholar, K. S. domains of bacteriophage murein hydrolases and binding to bacterial cell wall Microbiol. PubMed Scopus Google Scholar). However, CBDs are not for bacterial lysis has the lytic activity of endolysins of as for endolysin (7Horgan M. O'Flynn G. Garry J. Cooney J. Coffey A. Fitzgerald G.F. Ross R.P. McAuliffe O. Phage lysin LysK can be truncated to its CHAP domain and retain lytic activity against live antibiotic-resistant staphylococci.Appl. Environ. Microbiol. 2009; 75: 872-874Crossref PubMed Scopus (88) Google Scholar, D.M. J. Dong S. S. Pritchard D.G. The cell lysis activity of the Streptococcus agalactiae bacteriophage B30 endolysin on the Environ. Microbiol. PubMed Scopus Google Scholar, Dong S. Baker J.R. J. Pritchard D.G. Donovan D.M. LysK CHAP endopeptidase domain is for lysis of live staphylococcal Microbiol. 2009; PubMed Scopus Google Scholar). nonbacteriolytic CDs or CBDs may have or in the endolysin In this we to the functional of the CDs and the CBD of the Streptococcus dysgalactiae phage endolysin PlySK1249 J. M. G. In vitro of a novel phage lysin, and of its antibacterial activity in a model of Streptococcus agalactiae PubMed Scopus Google and their in the of phage-induced bacterial endolysin has a complex structure with a central LysM an amidase (Ami) and a (CHAP) domain. are to the between the glycan and the of the stem peptides L. of acid amidase with cell wall Biol. Chem. Full Text PDF PubMed Google CHAP domains are with amidase activities domains from bacterial and phage a family of Full Text Full Text PDF PubMed Scopus Google Scholar). We that the Ami domain was a bacteriolytic CHAP was a nonbacteriolytic endopeptidase that for bacterial The CHAP domain also as a dechaining enzyme and functional and with dechaining in or of as dechaining Bacteriol. PubMed Google Scholar, S. J. The CHAP domain of functions as an endopeptidase that acts at to Streptococcus cell Microbiol. 2009; PubMed Scopus Google Scholar). We that PlySK1249 was subject to proteolytic cleavage by cell wall proteases both and after phage in Cleavage the CDs by hydrolyzing their linker regions, thus hindering their bacteriolytic cooperation and possibly modulating the lytic activity of the enzyme. for proteolytic regulation of A. M. J. J. G. of is of peptidoglycan and Bacteriol. 2005; 187: PubMed Scopus Google Scholar, G. G. J. of is by Bacteriol. PubMed Google Scholar, C. M. L. M. S. analysis of the of Bacteriol. PubMed Scopus Google PlySK1249 cleavage represent a mechanism of regulation of In addition, understanding more thoroughly the interplay of PlySK1249 may be in the of novel therapeutic the of of the various truncated forms of PlySK1249 in the by a at the The and the on to for for for for the CHAP domain not be to the LysM domain. The of the two CDs and the CBD to bacterial lysis was with the of the enzyme. The tested against an of different Gram-positive bacteria PlySK1249 was more lytic against Streptococcus against more such as and from a the PlySK1249 truncated forms the enzyme, with more Ami and lytic activity when for and not We tested the of a activity between the Ami and CHAP catalytic domains by the of and cell when used or against S. dysgalactiae and The the of lytic activity of the observed in PlySK1249 a in lysis in in and was not lytic at However, of to lysis a lysis at as with the cooperation was the of the domain was the of their and was also observed between Ami and not with the cell in A at the activity and of of S. dysgalactiae that composed of of chains from to However, of with in with the of chains only to bacteria, the of activity was by in a Gram-positive bacterial with PlySK1249 or as by the of a of PlySK1249 and with in with of lysis after the of domains to the activity of the enzyme, we at their peptidoglycan cleavage or activity was for PlySK1249 and its truncated S. dysgalactiae cell with the of the activities to In a release was after peptidoglycan by and the constructs, an amidase endopeptidase between amidase and endopeptidase activities for the Ami and CHAP catalytic peptidoglycan was and for the of the stem peptide and polymers of digestion with two with short by and as stem peptides dimers by also of and was digestion with or Ami the two and by an of in the and and analysis that these an from to that in and and for Ami and that the LysM CBD was not for the The digestion with not two be observed at and not observed with the Ami domain in the PlySK1249 digestion and that the is to peptidoglycan to a can be to its endopeptidase of the peptide not stem peptides from the glycan is the glycan with the digestion of the PlySK1249 enzyme The that the digestion of the native enzyme is composed of dimers also that to to glycan strands can are to a structure and thus bacterial this why the CHAP domain is not lytic on its the digestion in an activity for the Ami domain and an endopeptidase activity for the CHAP domain to the structure of the The complex of PlySK1249 may be a of as observed of multidomain by cell-wall-associated proteases A. M. J. J. G. of is of peptidoglycan and Bacteriol. 2005; 187: PubMed Scopus Google Scholar, G. G. J. of is by Bacteriol. PubMed Google Scholar, C. M. L. M. S. analysis of the of Bacteriol. PubMed Scopus Google Scholar). For this reason, we tested the of PlySK1249 to cleavage by and by cleavage of PlySK1249 was observed to be to the two the different enzyme the domains a be observed after of the endolysin with of acid of the of two peptides to the CHAP and Ami domains in the and the Ami domain only in the also by observed when the or the to also with the cleavage of the and PlySK1249 was composed of different to the and CBD domains and linker of S. dysgalactiae cell-wall-associated also and with the native enzyme or the and to the of in the cell wall was not to the by with an was used as for two for PlySK1249 at and in the control with the endolysin as well as in the wall not was also observed for the with an at to the of the in the of the enzyme, the Ami domain was not observed these are to the one observed and that PlySK1249 was between its two by both and We used a to the host cell-wall-associated proteases that are for PlySK1249 was used for a of and tested on the proteolytic was observed for at to A was by a to and the was tested for proteolytic activity on the different and by and a of different proteases be and of observed only in the two proteolytic and that PlySK1249 can be by a of host a of we tested cleavage also be phage in we not the prophage PlySK1249 from its Streptococcus we of Streptococcus agalactiae for is and a prophage that is with C. prophage an endolysin to PlySK1249 and including the domain architecture and with the PlySK1249 endolysin of S. agalactiae with or as after and on an The was different a of from to and by for the of prophage A of in the of the to a in the of the prophage and A at the peptides that in the different that was in the in its also in cleavage the of the enzyme in A from in and in the and Ami catalytic also observed in the of the at two different cleavage in the on both of the that cleavage of endolysin in as observed with its PlySK1249 in was also when with the of the phage for only was observed the of the endolysin cleavage The of proteolytic cleavage on the activity and of PlySK1249 was of PlySK1249 with for an in a lytic activity on S. dysgalactiae a of only was observed after with an after proteolytic cleavage also in the of the domain from the catalytic we the of and a of S. dysgalactiae to the lytic activity of the amidase we the of the different by the of the produced by bacterial lysis LysM of the Ami domain when tested against three different from for Ami with for and The of PlySK1249 was to that of that the was of the and In addition, of Ami was LysM a of In the study, we at understanding the and of the PlySK1249 functional as well as their potential in endolysin lysis regulation. the enzyme and truncated constructs, we found that the three domains to the lytic activity of the enzyme. CHAP was not have a was in the activity of domains was the of the activities of when with Ami as in the of native We the functions of the three domains and that the nonbacteriolytic CHAP domain a dechaining activity that also to peptidoglycan CHAP was an endopeptidase that resolved complex polymers of stem-peptides to dimers and helped the Ami domain to digest peptidoglycan to completion. polymers to dimers to the of CHAP bacteriolytic as stem-peptides glycan chains cross-linked and a cell wall In digestion to have the of glycan chains have in The CHAP dechaining activity is of cell wall by in also as dechaining as well as the of Streptococcus is composed of a LysM and a CHAP domain that cell of as dechaining Bacteriol. PubMed Google Scholar, S. J. The CHAP domain of functions as an endopeptidase that acts at to Streptococcus cell Microbiol. 2009; PubMed Scopus Google Scholar). domains may different in different functional of endolysin has been the enzymatic of helped to the of the nonbacteriolytic CHAP to the endolysin activity and wall in of cell the CHAP domain is a endopeptidase the Ami domain. domain cooperation be by the of host cell-wall-associated We proteolytic cleavage in the both CDs to the central LysM CBD when PlySK1249 was with cell-wall-associated proteases or cell lysis by prophage induction. A of different be as potential three and and two endopeptidases and J.A. and acid in acid 1999; PubMed Scopus Google the two are more to be in the proteolytic cleavage is the potential mechanism the of domain domain modulating the lytic activity of the enzyme. A was observed for the of and of also by cell wall proteases A. M. J. J. G. of is of peptidoglycan and Bacteriol. 2005; 187: PubMed Scopus Google Scholar, G. G. J. of is by Bacteriol. PubMed Google Scholar, C. M. L. M. S. analysis of the of Bacteriol. PubMed Scopus Google Scholar). In the of proteolytic cleavage in a enzymatic and cell wall binding activity A. M. J. J. G. of is of peptidoglycan and Bacteriol. 2005; 187: PubMed Scopus Google Scholar, G. G. J. of is by Bacteriol. PubMed Google Scholar, A. G. G. J. of is an with an of LysM domains for J. 2005; PubMed Scopus Google Scholar). In the a for the multimodular architecture of possibly be to multimodular both catalytic domains observed to to bacterial the CBD is to proteolytic of the lysin by host cell-wall-associated is also possibly bacteria and from the multimodular structure of PlySK1249 a to several functions in a as cleavage or the to different Ami for lysis and CHAP for have both and the of the of between and host Endolysins and are to have and bacteria can the activity of in the for the of cell wall It that the phage endolysin the adaptation of phage endolysins toward the use of existing bacterial regulation mechanism for their own advantage. the of on the ideal architecture of therapeutic antibacterial endolysins. this is relevant for the of domains that not lytic activity not be when with the lytic of domains. the of the not be and in to enzyme therapeutic bacterial used in this are Gram-positive bacteria at in and on with of and at in with or on The to the when for and for or from in the in and at The J. M. G. In vitro of a novel phage lysin, and of its antibacterial activity in a model of Streptococcus agalactiae PubMed Scopus Google was used as a for of encoding for truncated forms of the PlySK1249 endolysin and and and in was the the and by for recombinant by as J. M. G. In vitro of a novel phage lysin, and of its antibacterial activity in a model of Streptococcus agalactiae PubMed Scopus Google and on to to the and their of bacterial lysis was for the PlySK1249 endolysin and its various truncated by the in of a bacterial as J. M. G. In vitro of a novel phage lysin, and of its antibacterial activity in a model of Streptococcus agalactiae PubMed Scopus Google Scholar). In to the potential between or Ami and the truncated at different from to In vitro as J. M. G. In vitro of a novel phage lysin, and of its antibacterial activity in a model of Streptococcus agalactiae PubMed Scopus Google Scholar). S. dysgalactiae with the various endolysin at a of for for the PlySK1249 and and for for the control and the CHAP domain. For on and with a on an For cell with for at and with The was for at and and at and the in of the in for that as by J. and with for PubMed Scopus Google Scholar). with a at an of with a Peptidoglycan of S. dysgalactiae was as H. D. M. of Streptococcus cell walls with its peptidoglycan hydrolase stem peptides activity.J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). of an was to of The an of to and in an for to The was at and in to a of The bacterial was of and with for to The at to with and with the the bacterial was in and at The the a three of at with a between The was a and at for at and for at The was in the of and the was for at with and after and For the steps, was to the was for at to an for at and the was with in and at with for The was at for was in to by the bacterial peptidoglycan was with and in of and by The bacterial peptidoglycan was to a of For enzymatic of the peptidoglycan was with of the endolysin at The was at with The the was for at to the at for and the the walls was a Peptidoglycan was as analysis was a and with a with as by Schmelcher M. Y. D.C. D.C. Dong S. Pritchard D.G. J. Donovan D.M. bacteriophage endolysins peptidoglycan cleavage from infection.J. PubMed Scopus Google Scholar). peptidoglycan was used as a and or used for in The peptidoglycan was at for and by the of of the was to the and another of was to The was at for and at for The was by for In was was at for and by for was at in to the glycan chains from the peptides by The was and on for at for at the the stem-peptides and peptide was a and at the peptides by and in the an of an an and an was used as H. D. M. of Streptococcus cell walls with its peptidoglycan hydrolase stem peptides activity.J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). of a at a A of to in acid at a of of the at with the Peptidoglycan was as the end of the at to the enzymatic They a and a Peptidoglycan with an and by in on a a with a Peptidoglycan a on a at a of A was with an a to from to in Full at In by the in the for and for analysis in the at The for was of the and from analysis for with and used for peptide by de and a For the a of S. dysgalactiae was from the and used to by from S. the de was used to peptidoglycan The and de as enzyme was used as the enzyme of and of used as and a of and a of wall from the S. dysgalactiae as J.A. of Staphylococcus aureus A in a PubMed Scopus Google Scholar). PlySK1249 and its truncated at by with of the endolysin also by at for different at peptidoglycan was to the at a of The was by the with and for at The on a to of interest from the and the acid by by as A. H. J. M. digestion for of and PubMed Scopus Google and peptide as on a with to the S. dysgalactiae in the of S. dysgalactiae at was used as the enzyme with two was with a of and a of of was in as a of and of as was as H. T. J. of from to and S. A. PubMed Scopus Google and for with a of by a with a of to by For of the cell wall was used at from to For was to of cell wall and for at The by at for in and with of the for was used to in the different as A was with to on a The was in with one of tested for proteolytic activity the as by by with a J.R. A. M. for 2009; PubMed Scopus Google Scholar). by an and with as was used to and peptide from and to For prophage S. dysgalactiae was at in a to an of was at a of to prophage a the was at and to a to after on a A of from to from the and peptides in by by and peptide on a with to the Streptococcus in the of S. at was used as the enzyme with two was with a of and a of of was in as a of and of as was used to and peptide from was to the of the CBD in the of the endolysins a of with of S. dysgalactiae was in and in of was to the cell and for at of was and at in the of the and of the enzyme at different the was by the of the lysis that after at The have been to the the with the The that have of interest with the of this The to for with the of for the and for with the We are to and for and for the O. and the O. and C. M. the O. and M. the G. R. the the critical and helped the and was by a from the for in and was also by the

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