Abstract
Bacteriophages produce endolysins, which lyse the bacterial host cell to release newly produced virions. The timing of lysis is regulated and is thought to involve the activation of a molecular switch. We present a crystal structure of the activated endolysin CTP1L that targets Clostridium tyrobutyricum, consisting of a complex between the full-length protein and an N-terminally truncated C-terminal cell wall binding domain (CBD). The truncated CBD is produced through an internal translation start site within the endolysin gene. Mutants affecting the internal translation site change the oligomeric state of the endolysin and reduce lytic activity. The activity can be modulated by reconstitution of the full-length endolysin-CBD complex with free CBD. The same oligomerization mechanism applies to the CD27L endolysin that targets Clostridium difficile and the CS74L endolysin that targets Clostridium sporogenes. When the CTP1L endolysin gene is introduced into the commensal bacterium Lactococcus lactis, the truncated CBD is also produced, showing that the alternative start codon can be used in other bacterial species. The identification of a translational switch affecting oligomerization presented here has implications for the design of effective endolysins for the treatment of bacterial infections.
Highlights
Bacteriophages are viruses that infect bacterial cells, and they can hijack the bacterial metabolism to produce new phages
When the CTP1L endolysin gene is introduced into the commensal bacterium Lactococcus lactis, the truncated cell wall binding domain (CBD) is produced, showing that the alternative start codon can be used in other bacterial species
The Crystal Structure of Endolysin CTP1L Contains a Truncated C-terminal Domain—CTP1L protein that is expressed in E. coli using the native bacteriophage oligonucleotide sequence reveals the presence of two species of different molecular weight on an SDS-polyacrylamide gel when purified [16]
Summary
Protein Expression, and Purification—The bacteriophage nucleotide sequences of the full-length endolysins for CTP1L, CD27L, and CS74L were inserted into pET15b (Novagen), containing an N-terminal His tag and a thrombin cleavage site [13,14,15]. The refined model was used to phase an anomalous difference density map using Phaser [27], using a cut-off for the Z score to find sites at 5.0 In this way, all sulfur atoms on the cysteine and methionine residues were identified, as well as a peak in the dimer interface between the N terminus of the truncated CBD and Val-195 of the full-length CTP1L endolysin. Masses as well as ratios of protein species were determined from at least three independent measurements, and errors given correspond to the S.D. Analysis of Cell Binding and Lysis—Assessments of lytic and binding activity were performed using Ni-NTA-purified protein as described previously, using buffer or Ni-NTA-purified GFP linker protein, respectively, as negative controls [5]. Cells were viewed with a ϫ100 magnification oil immersion lens
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