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Characterization of MSlys, the endolysin of Streptococcus pneumoniae phage MS1.

ABSTRACT: Despite the use of pneumococcal conjugate vaccines, the number of infections related to Streptococcus pneumoniae continues to be alarming. Herein, we identified, characterized the MSlys endolysin encoded in the phage MS1. We further tested its antimicrobial efficacy against planktonic and biofilm cells, assessing the culturability of cells and biofilm structure by scanning electron microscopy, and confocal laser scanning microscopy. The modular MSlys endolysin consists of an amidase catalytic domain and a choline-binding domain. MSlys is active against isolates of children with otitis media, and conditions close to those found in the middle ear. Treatment with MSlys (2?h, 4 ?M) reduced planktonic cultures by 3.5 log10 CFU/mL, and 24- and 48-h-old biofilms by 1.5 and 1.8 log10 CFU/mL, respectively. Imaging of the biofilms showed thinner and damaged structures compared to control samples. The recombinantly expressed MSlys may be a suitable candidate for treating pneumococcal infections, including otitis media.

SUBMITTER: Silva MD

PROVIDER: S-EPMC7648177 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Characterization of MSlys, the endolysin of <i>Streptococcus pneumoniae</i> phage MS1.

Silva Maria Daniela MD Oliveira Hugo H Faustino Alberta A Sillankorva Sanna S

Biotechnology reports (Amsterdam, Netherlands) 20201024

Despite the use of pneumococcal conjugate vaccines, the number of infections related to <i>Streptococcus pneumoniae</i> continues to be alarming. Herein, we identified, characterized the MSlys endolysin encoded in the phage MS1. We further tested its antimicrobial efficacy against planktonic and biofilm cells, assessing the culturability of cells and biofilm structure by scanning electron microscopy, and confocal laser scanning microscopy. The modular MSlys endolysin consists of an amidase catal ...[more]

PMID: 33204659

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Project description:Bacteriophage Mu is a paradigm coliphage studied mainly because of its use of transposition for genome replication. However, in extensive nonsense mutant screens, only one lysis gene has been identified, the endolysin gp22. This is surprising because in Gram-negative hosts, lysis by Caudovirales phages has been shown to require proteins which disrupt all three layers of the cell envelope. Usually this involves a holin, an endolysin, and a spanin targeting the cytoplasmic membrane, peptidoglycan (PG), and outer membrane (OM), respectively, with the holin determining the timing of lysis initiation. Here, we demonstrate that gp22 is a signal-anchor-release (SAR) endolysin and identify gp23 and gp23.1 as two-component spanin subunits. However, we find that Mu lacks a holin and instead encodes a membrane-tethered cytoplasmic protein, gp25, which is required for the release of the SAR endolysin. Mutational analysis showed that this dependence on gp25 is conferred by lysine residues at positions 6 and 7 of the short cytoplasmic domain of gp22. gp25, which we designate as a releasin, also facilitates the release of SAR endolysins from other phages. Moreover, the entire length of gp25, including its N-terminal transmembrane domain, belongs to a protein family, DUF2730, found in many Mu-like phages, including those with cytoplasmic endolysins. These results are discussed in terms of models for the evolution and mechanism of releasin function and a rationale for Mu lysis without holin control. IMPORTANCE Host cell lysis is the terminal event of the bacteriophage infection cycle. In Gram-negative hosts, lysis requires proteins that disrupt each of the three cell envelope components, only one of which has been identified in Mu: the endolysin gp22. We show that gp22 can be characterized as a SAR endolysin, a muralytic enzyme that activates upon release from the membrane to degrade the cell wall. Furthermore, we identify genes 23 and 23.1 as spanin subunits used for outer membrane disruption. Significantly, we demonstrate that Mu is the first known Caudovirales phage to lack a holin, a protein that disrupts the inner membrane and is traditionally known to release endolysins. In its stead, we report the discovery of a lysis protein, termed the releasin, which Mu uses for SAR endolysin release. This is an example of a system where the dynamic membrane localization of one protein is controlled by a secondary protein.

Dataset Information

Characterization of MSlys, the endolysin of Streptococcus pneumoniae phage MS1.

Publications

Characterization of MSlys, the endolysin of <i>Streptococcus pneumoniae</i> phage MS1.

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