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Abstract
Diffocins are high-molecular-weight phage tail-like bacteriocins (PTLBs) that some Clostridium difficile strains produce in response to SOS induction. Similar to the related R-type pyocins from Pseudomonas aeruginosa, R-type diffocins act as molecular puncture devices that specifically penetrate the cell envelope of other C. difficile strains to dissipate the membrane potential and kill the attacked bacterium. Thus, R-type diffocins constitute potential therapeutic agents to counter C. difficile-associated infections. PTLBs consist of rigid and contractile protein complexes. They are composed of a baseplate, receptor-binding tail fibers and an inner needle-like tube surrounded by a contractile sheath. In the mature particle, the sheath and tube structure form a complex network comprising up to 200 copies of a sheath and a tube protein each. Here, we report the crystal structures together with small angle X-ray scattering data of the sheath and tube proteins CD1363 (39 kDa) and CD1364 (16 kDa) from C. difficile strain CD630 in a monomeric pre-assembly form at 1.9 and 1.5 Å resolution, respectively. The tube protein CD1364 displays a compact fold and shares highest structural similarity with a tube protein from Bacillus subtilis but is remarkably different from that of the R-type pyocin from P. aeruginosa. The structure of the R-type diffocin sheath protein, on the other hand, is highly conserved. It contains two domains, whereas related members such as bacteriophage tail sheath proteins comprise up to four, indicating that R-type PTLBs may represent the minimal protein required for formation of a complete sheath structure. Comparison of CD1363 and CD1364 with structures of PTLBs and related assemblies suggests that several conformational changes are required to form complete assemblies. In the sheath, rearrangement of the flexible N- and C-terminus enables extensive interactions between the other subunits, whereas for the tube, such contacts are primarily established by mobile α-helices. Together, our results combined with information from structures of homologous assemblies allow constructing a preliminary model of the sheath and tube assembly from R-type diffocin.
Highlights
In order to infect bacteria, bacteriophages use an attachment organelle known as the “tail,” which recognizes the host cell and attaches the phage’s capsid to it
We provide a model of the assembled sheath and tube structures of the R-type diffocin contractile apparatus, which may serve as templates for future cryo electron microscopy studies
The correct molecular masses of the purified proteins were confirmed at the Department Chemical Biology at the Helmholtz Center for Infection Research, using a maXis HD ultra-high resolution (UHR) quadrupole time of flight (Q-TOF) mass spectrometer equipped with a Apollo II electrospray source (Bruker Daltoniks, Bremen, Germany) and an Ultimate 3000RS autosampler together with a binary high gradient pump (Dionex/Thermo Fisher Scientific, Waltham, MA, United States)
Summary
In order to infect bacteria, bacteriophages use an attachment organelle known as the “tail,” which recognizes the host cell and attaches the phage’s capsid to it. Bacterial genomes often contain gene clusters encoding for structural elements that are evolutionary related to bacteriophage tail structures with regard to morphology, size, and the mechanism of action but without containing a phage capsid This includes, e.g., the T6SS (Leiman et al, 2009), needle-like particles such as the Afp from Serratia entomophila (Hurst et al, 2004) or the PVC (Yang et al, 2006), as well as PTLBs such as the “pyocins” from Pseudomonas aeruginosa or the “monocins” from Listeria monocytogenes (Nakayama et al, 2000; Lee et al, 2016). A rather recently identified group of PTLBs are the R-type “diffocins” from Clostridium difficile, which constitute the only Grampositive R-type PTLBs known to date (Gebhart et al, 2012; Scholl, 2017)
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