The CD27L and CTP1L endolysins targeting Clostridia contain a built-in trigger and release factor.
The bacteriophage ΦCD27 is capable of lysing Clostridium difficile, a pathogenic bacterium that is a major cause for nosocomial infection. A recombinant CD27L endolysin lyses C. difficile in vitro, and represents a promising alternative as a bactericide. To better understand the lysis mechanism, we...
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2014
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oai:doaj.org-article:76e1091774d84f3ca598478c1eec73672021-11-25T05:46:15ZThe CD27L and CTP1L endolysins targeting Clostridia contain a built-in trigger and release factor.1553-73661553-737410.1371/journal.ppat.1004228https://doaj.org/article/76e1091774d84f3ca598478c1eec73672014-07-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/25058163/pdf/?tool=EBIhttps://doaj.org/toc/1553-7366https://doaj.org/toc/1553-7374The bacteriophage ΦCD27 is capable of lysing Clostridium difficile, a pathogenic bacterium that is a major cause for nosocomial infection. A recombinant CD27L endolysin lyses C. difficile in vitro, and represents a promising alternative as a bactericide. To better understand the lysis mechanism, we have determined the crystal structure of an autoproteolytic fragment of the CD27L endolysin. The structure covers the C-terminal domain of the endolysin, and represents a novel fold that is identified in a number of lysins that target Clostridia bacteria. The structure indicates endolysin cleavage occurs at the stem of the linker connecting the catalytic domain with the C-terminal domain. We also solved the crystal structure of the C-terminal domain of a slow cleaving mutant of the CTP1L endolysin that targets C. tyrobutyricum. Two distinct dimerization modes are observed in the crystal structures for both endolysins, despite a sequence identity of only 22% between the domains. The dimers are validated to be present for the full length protein in solution by right angle light scattering, small angle X-ray scattering and cross-linking experiments using the cross-linking amino acid p-benzoyl-L-phenylalanine (pBpa). Mutagenesis on residues contributing to the dimer interfaces indicates that there is a link between the dimerization modes and the autocleavage mechanism. We show that for the CTP1L endolysin, there is a reduction in lysis efficiency that is proportional to the cleavage efficiency. We propose a model for endolysin triggering, where the extended dimer presents the inactive state, and a switch to the side-by-side dimer triggers the cleavage of the C-terminal domain. This leads to the release of the catalytic portion of the endolysin, enabling the efficient digestion of the bacterial cell wall.Matthew DunneHaydyn D T MertensVasiliki GarefalakiCy M JeffriesAndrew ThompsonEdward A LemkeDmitri I SvergunMelinda J MayerArjan NarbadRob MeijersPublic Library of Science (PLoS)articleImmunologic diseases. AllergyRC581-607Biology (General)QH301-705.5ENPLoS Pathogens, Vol 10, Iss 7, p e1004228 (2014) |
| institution |
DOAJ |
| collection |
DOAJ |
| language |
EN |
| topic |
Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5 |
| spellingShingle |
Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5 Matthew Dunne Haydyn D T Mertens Vasiliki Garefalaki Cy M Jeffries Andrew Thompson Edward A Lemke Dmitri I Svergun Melinda J Mayer Arjan Narbad Rob Meijers The CD27L and CTP1L endolysins targeting Clostridia contain a built-in trigger and release factor. |
| description |
The bacteriophage ΦCD27 is capable of lysing Clostridium difficile, a pathogenic bacterium that is a major cause for nosocomial infection. A recombinant CD27L endolysin lyses C. difficile in vitro, and represents a promising alternative as a bactericide. To better understand the lysis mechanism, we have determined the crystal structure of an autoproteolytic fragment of the CD27L endolysin. The structure covers the C-terminal domain of the endolysin, and represents a novel fold that is identified in a number of lysins that target Clostridia bacteria. The structure indicates endolysin cleavage occurs at the stem of the linker connecting the catalytic domain with the C-terminal domain. We also solved the crystal structure of the C-terminal domain of a slow cleaving mutant of the CTP1L endolysin that targets C. tyrobutyricum. Two distinct dimerization modes are observed in the crystal structures for both endolysins, despite a sequence identity of only 22% between the domains. The dimers are validated to be present for the full length protein in solution by right angle light scattering, small angle X-ray scattering and cross-linking experiments using the cross-linking amino acid p-benzoyl-L-phenylalanine (pBpa). Mutagenesis on residues contributing to the dimer interfaces indicates that there is a link between the dimerization modes and the autocleavage mechanism. We show that for the CTP1L endolysin, there is a reduction in lysis efficiency that is proportional to the cleavage efficiency. We propose a model for endolysin triggering, where the extended dimer presents the inactive state, and a switch to the side-by-side dimer triggers the cleavage of the C-terminal domain. This leads to the release of the catalytic portion of the endolysin, enabling the efficient digestion of the bacterial cell wall. |
| format |
article |
| author |
Matthew Dunne Haydyn D T Mertens Vasiliki Garefalaki Cy M Jeffries Andrew Thompson Edward A Lemke Dmitri I Svergun Melinda J Mayer Arjan Narbad Rob Meijers |
| author_facet |
Matthew Dunne Haydyn D T Mertens Vasiliki Garefalaki Cy M Jeffries Andrew Thompson Edward A Lemke Dmitri I Svergun Melinda J Mayer Arjan Narbad Rob Meijers |
| author_sort |
Matthew Dunne |
| title |
The CD27L and CTP1L endolysins targeting Clostridia contain a built-in trigger and release factor. |
| title_short |
The CD27L and CTP1L endolysins targeting Clostridia contain a built-in trigger and release factor. |
| title_full |
The CD27L and CTP1L endolysins targeting Clostridia contain a built-in trigger and release factor. |
| title_fullStr |
The CD27L and CTP1L endolysins targeting Clostridia contain a built-in trigger and release factor. |
| title_full_unstemmed |
The CD27L and CTP1L endolysins targeting Clostridia contain a built-in trigger and release factor. |
| title_sort |
cd27l and ctp1l endolysins targeting clostridia contain a built-in trigger and release factor. |
| publisher |
Public Library of Science (PLoS) |
| publishDate |
2014 |
| url |
https://doaj.org/article/76e1091774d84f3ca598478c1eec7367 |
| work_keys_str_mv |
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| _version_ |
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