SH3 BINDING DOMAINS FROM PHAGE ENDOLYSINS: HOW TO USE THEM FOR DETECTION OF GRAMPOSITIVE PATHOGENS
| Authors | |
|---|---|
| Year of publication | 2020 |
| Type | Article in Periodical |
| Magazine / Source | Journal of Microbiology, Biotechnology & Food Sciences |
| MU Faculty or unit | |
| Citation | |
| Web | https://www.jmbfs.org/wp-content/uploads/2020/06/jmbfs_1692_Jarabkova.pdf |
| Doi | http://dx.doi.org/10.15414/jmbfs.2020.9.6.1215-1220 |
| Keywords | phage; endolysin; SH3 binding domain; Staphylococcus aureus; Streptococcus agalactiae |
| Description | Phage-encoded enzymes, called endolysins, could be linked to the purpose of specifically detecting and treating Gram-positive pathogens, such as drug-resistant bacteria Staphylococcus aureus or Streptococcus agalactiae. Most endolysins encoded by staphylococcal phages are composed of a C-terminal SH3b type domain, an amidase catalytic domain in the central position and an Nterminal catalytic domain with endopeptidase activity. Furthermore, endolysins from a streptococcal phage, e.g. B30 consist of a dualenzyme active domain (EAD)- muramidase and endopeptidase and one or several cell wall binding domains (CBD), in this case SH3. An nderstanding of CBDs and domain design that has a high degree of host specificity may lead to shifts in the lytic spectrum of a chimeric endolysin. Our research was focused on the in silico analysis of CBDs of endolysins encoded by staphylococcal and streptococcal phages. Gene for specific binding domain (SBD) was designed based on bioinformatics approaches. The coding gene sequence of the SBD domain was subsequently fused with thegene for GFP (green fluorescent protein) and subcloned into the pET21 expression vector. The domain was prepared as a fusion recombinant protein and analyzed for its binding function. This capability was shown by the fusion of SBD with fluorescent tag - GFP, followed by fluorescent microscopy. Binding assays showed that SBD was able to bind to living cells of Staphylococcus aureus and Streptococcus agalactiae. Because of these properties, CBDs may be used as tools in alternative diagnostic methods. The fusions of SBD and EAD may bring the chimeric endolysin for being utilized as novel therapeutic agents. |
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