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.
Highlights
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 [1]
The relative contribution of the two catalytic domain (CD) and the cell-wall-binding domain (CBD) to bacterial lysis was compared with the relative contribution of the parent enzyme
Using the parent enzyme and truncated constructs, we found that the three domains cooperated to increase the overall lytic activity of the enzyme
Summary
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 [1]. Apart from a few exceptions, the PlySK1249 truncated forms were less active than the parent enzyme, with Ami_LysM being generally more active than Ami alone and LysM_CHAP showing no lytic activity even when incubated for 2 h (Fig. 1C and data not shown). To discriminate between amidase and endopeptidase activities for the Ami and CHAP catalytic domains, digested peptidoglycan was further analyzed RP-HPLC and LC-MS for the presence of precursor masses containing the stem peptide motif AQKAAA and polymers of it.
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