Plant-expressed bacteriophage lysins control pathogenic strains of Clostridium perfringens

The anaerobic spore-forming bacterium Clostridium perfringens is a source of one of the most common food-borne illnesses in the United States and Europe. The costs associated with disease management are high and interventions are limited; therefore, effective and safe antimicrobials are needed to control food contamination by C. perfringens. A viable solution to this problem could be bacteriophage lysins used as food additives or food processing aids. Such antimicrobials could be produced cost-effectively and in ample supply in green plants. By using edible plant species as production hosts the need for expensive product purification can be reduced or obviated. We describe the first successful expression in plants of C. perfringens-specific bacteriophage lysins. We demonstrate that six lysins belonging to two different families (N-acetylmuramoyl-L-alanine amidase and glycosyl hydrolase 25) are active against a panel of enteropathogenic C. perfringens strains under salinity and acidity conditions relevant to food preparation environments. We also demonstrate that plant-expressed lysins prevent multiplication of C. perfringens on cooked meat matrices far better than nisin, the only currently approved bacteriocin food preservative to control this pathogen.


ZP173.
A small portion of frozen leaf tissue was homogenized with chilled mortar and pestle in liquid nitrogen. The powder was mixed with cold extraction buffer (50 mM NaH2PO4/Na2HPO4, 2 mM DTT, pH 5.0) at a ratio of 1 g of plant material to 5 ml of buffer and kept on ice for 10-15 min.
Cell debris were removed by centrifugation at 3220 g, at 4 C for 20 min. Pellets were discarded and the supernatant was filtered by passing the solution through membrane filters (pore sizes 5 µm and 0.22 µm). The pH of solution was adjusted to 5 and formed precipitate was removed by centrifugation at 3220 g, at 4 C for 5 min. The supernatant was taken as total soluble protein and applied for purification in two steps.
At the first purification step the chromatography column was filled with Butyl sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden) and pre-equilibrated with cold buffer (50 mM NaH2PO4/Na2HPO4, 1.1 M (NH4)2SO4, 2 mM DTT, pH 7.0). Protein solution was loaded onto the column and the Butyl sepharose bounded protein fraction was eluted by washing with elution buffer (50 mM NaH2PO4/Na2HPO4, 0.77 M (NH4)2SO4, 2 mM DTT, pH 7.0). Collected protein fraction was transferred to the diafiltrating concentrator (10 kDa) and centrifuged at 3220 g until the volume of protein solution decreased 8-10 folds. Concentrate was diluted up to a primary volume with buffer containing 50 mM NaH2PO4/Na2HPO4, 2 mM DTT (pH 5.0).
Procedure was repeated till conductivity decreased below 9 mS/cm, and protein solution subjected to the final purification step using SP sepharose FF resin (GEHealthcare Life Sciences, Uppsala, Sweden). Chromatography media was pre-equilibrated with cold buffer (50 mM NaH2PO4/Na2HPO4, 2 mM DTT, pH 5.0). Protein solution was loaded onto the column and SP sepharose bounded protein fraction was eluted by linear gradient of cold washing buffer supplemented with 300 mM of NaCl. Collected ZP173 was freeze-dried.

ZP278.
A small portion of frozen leaf tissue was homogenized with chilled mortar and pestle in liquid nitrogen. The powder was mixed with cold extraction buffer (50 mM NaH2PO4/Na2HPO4, 150 mM NaCl, 5 mM DTT, pH 7.0) at a ratio of 1 g of plant material to 5 ml of buffer and kept on ice for 10-15 min. Cell debris were removed by centrifugation at 3220 g, at 4 C for 20 min. Pellets were discarded and the supernatant was filtered by passing solution through membrane filters (pore sizes 5 µm and 0.22 µm). The pH of solution was adjusted to 6.5 and formed precipitate was removed by centrifugation at 3220 g, at 4 C for 5 min. The supernatant was taken as total soluble protein and applied for purification in two steps.
At the first purification step the chromatography column was filled with Butyl sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden) and pre-equilibrated with cold buffer (50 mM NaH2PO4/Na2HPO4, 1.2 M (NH4)2SO4, 5 mM DTT, pH 6.5). Protein solution was loaded onto the column and the Butyl sepharose bounded protein fraction was eluted by washing with elution buffer (50 mM NaH2PO4/Na2HPO4, 0.78 M (NH4)2SO4, 5 mM DTT, pH 6.5). Collected protein fraction was transferred to the diafiltrating concentrator (10 kDa) and centrifuged at 3220 g until the volume of protein solution decreased 8-10 folds. Concentrate was diluted up to a primary volume with buffer containing 50 mM NaH2PO4/Na2HPO4, 5 mM DTT (pH 7.0).
Procedure was repeated till conductivity decreased below 9 mS/cm, and protein solution was subjected to the final purification step using DEAE sepharose FF resin (GEHealthcare Life Sciences, Uppsala, Sweden). Chromatography media was pre-equilibrated with cold buffer (50 mM NaH2PO4/Na2HPO4, 5 mM DTT, pH 7.0). Protein solution was loaded onto the column and DEAE sepharose bounded protein fraction was eluted by linear gradient of cold washing buffer additionally containing 250 mM of NaCl. Collected ZP278 was freeze-dried.

CP25L.
A small portion of frozen leaf tissue was homogenized with chilled mortar and pestle in liquid nitrogen. Prepared powder was mixed with cold extraction buffer (50 mM NaH2PO4/Na2HPO4, 100 mM NaCl, 2 mM DTT, pH 7.5) at a ratio of 1 g of plant material to 5 ml of buffer. The crude extract was kept on ice for 10-15 min. Cell debris were removed by centrifugation at 3220 g, at 4 C for 20 min. Pellets were discarded and the supernatant was filtered by passing solution through membrane filters (pore sizes 5 µm and 0.22 µm). The pH of solution was adjusted to 6.5 and formed precipitate was removed by centrifugation at 3220 g, at 4 C for 5 min. The supernatant was taken as total soluble protein and applied for purification in two steps.
At the first purification step the chromatography column was filled with Butyl sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden) and pre-equilibrated with cold buffer (50 mM NaH2PO4/Na2HPO4, 0.85 M (NH4)2SO4, 2 mM DTT, pH 6.5). Protein solution was loaded onto the column and the Butyl sepharose bounded protein fraction was eluted by washing with elution buffer (50 mM NaH2PO4/Na2HPO4, 0.6 M (NH4)2SO4, 2 mM DTT, pH 6.5). Collected protein fraction was transferred to the diafiltrating concentrator (10 kDa) and centrifuged at 3220 g until the volume of protein solution decreased 6-8 folds. Concentrate was diluted up to a primary volume with buffer containing 20 mM NaH2PO4/Na2HPO4, 2 mM DTT (pH 8).
Procedure was repeated till conductivity decreased below 5 mS/cm and protein solution was subjected to the final purification step using Q sepharose FF resin (GEHealthcare Life Sciences, Uppsala, Sweden). Chromatography media was pre-equilibrated with cold buffer (20 mM NaH2PO4/Na2HPO4, 2 mM DTT, pH 8.0). Protein solution was loaded onto the column and Q sepharose bounded protein fraction was eluted by linear gradient of cold washing buffer supplemented with 125 mM of NaCl. Collected CP25L was freeze-dried.

PlyCP26F.
A small portion of frozen leaf tissue was homogenized with chilled mortar and pestle in liquid nitrogen. Prepared powder was mixed with cold extraction buffer (50 mM NaH2PO4/Na2HPO4, 150 mM NaCl, 2 mM DTT, pH 7.5) at a ratio of 1 g of plant material to 5 ml of buffer. The crude extract kept on ice for 10-15 min. Cell debris were removed by centrifugation at 3220 g, at 4 C for 20 min. Pellets were discarded and the supernatant was filtered by passing solution through membrane filters (pore sizes 5 µm and 0.22 µm). The pH of solution was adjusted to 7.0 and formed precipitate was removed by centrifugation at 3220 g, at 4 C for 5 min. The supernatant was taken as total soluble protein and applied for purification in two steps.
At the first purification step the chromatography column was filled with Butyl sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden) and pre-equilibrated with cold buffer (50 mM NaH2PO4/Na2HPO4, 1.2 M (NH4)2SO4, 2 mM DTT, pH 7.0). Protein solution was loaded onto the column and the Butyl sepharose bounded protein fraction was eluted by washing with elution buffer (50 mM NaH2PO4/Na2HPO4, 0.84 M (NH4)2SO4, 2 mM DTT, pH 7.0). Collected protein fraction was transferred to the diafiltrating concentrator (10 kDa) and centrifuged at 3220 g until the volume of protein solution decreased 8-10 folds. Concentrate was diluted up to a primary volume with buffer containing 50 mM NaH2PO4/Na2HPO4, 2 mM DTT (pH 6).
Procedure was repeated till conductivity decreased below 7 mS/cm and protein solution subjected to the final purification step using SP sepharose FF resin (GEHealthcare Life Sciences, Uppsala, Sweden). Chromatography media was pre-equilibrated with cold buffer (50 mM NaH2PO4/Na2HPO4, 2 mM DTT, pH 6.0). Protein solution was loaded onto the column and SP sepharose bounded protein fraction was eluted with elution buffer (50 mM NaH2PO4/Na2HPO4, 1.0 M NaCl, 2 mM DTT, pH 6.0). Collected PlyCP26F was freeze-dried.

PlyCP39O.
A small portion of frozen leaf tissue was homogenized with chilled mortar and pestle in liquid nitrogen. Prepared powder was mixed with cold extraction buffer (50 mM NaH2PO4/Na2HPO4, 200 mM NaCl, 5 mM DTT, pH 7.5) at a ratio of 1 g of plant material to 5 ml of buffer. The crude extract was kept on ice for 10-15 min. Cell debris were removed by centrifugation at 3220 g, at 4 C for 20 min. Pellets were discarded and the supernatant was filtered by passing solution through membrane filters (pore sizes 5 µm and 0.22 µm). The pH of solution was adjusted to 7.0 and formed precipitate was removed by centrifugation at 3220 g, at 4 C for 5 min. The supernatant was taken as total soluble protein and applied for purification in two steps.
At the first purification step the chromatography column was filled with Butyl sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden) and pre-equilibrated with cold buffer (50 mM NaH2PO4/Na2HPO4, 1.2 M (NH4)2SO4, 5 mM DTT, pH 7.0). Protein solution was loaded onto the column and the Butyl sepharose bounded protein fraction was eluted by washing with elution buffer (50 mM NaH2PO4/Na2HPO4, 0.78 M (NH4)2SO4, 5 mM DTT, pH 7.0). Collected protein fraction was transferred to the diafiltrating concentrator (10 kDa) and centrifuged at 3220 g until the volume of protein solution decreased 6-8 folds. Concentrate was diluted up to a primary volume with buffer containing 50 mM NaH2PO4/Na2HPO4, 5 mM DTT (pH 6).
Procedure was repeated till conductivity decreased below 17 mS/cm and protein solution was subjected to the final purification step using SP sepharose FF resin (GEHealthcare Life Sciences, Uppsala, Sweden). Chromatography media was pre-equilibrated with cold buffer (50 mM NaH2PO4/Na2HPO4, 120 mM NaCl, 5 mM DTT, pH 7.0). Protein solution was loaded onto the column and SP sepharose bounded protein fraction was eluted by linear gradient of cold buffer containing 350 mM of NaCl. Collected PlyCP39O was freeze-dried.

psm.
A small portion of frozen leaf tissue was homogenized with chilled mortar and pestle in liquid nitrogen. Prepared powder was mixed with cold extraction buffer (50 mM NaH2PO4/Na2HPO4, 150 mM NaCl, 2 mM DTT, pH 5.0) at a ratio of 1 g of plant material to 5 ml of buffer. The crude extract was kept on ice for 10-15 min. Cell debris were removed by centrifugation at 3220 g, at 4 C for 20 min. Pellets were discarded and the supernatant was filtered by passing solution through membrane filters (pore sizes 5 µm and 0.22 µm). The pH of solution was adjusted to 6.0 and formed precipitate was removed by centrifugation at 3220 g, at 4 C for 5 min. The supernatant was taken as total soluble protein and applied for purification in two steps.
At the first purification step the chromatography column was filled with Butyl sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden) and pre-equilibrated with cold buffer (50 mM NaH2PO4/Na2HPO4, 1.2 M (NH4)2SO4, 2 mM DTT, pH 6.0). Protein solution was loaded onto the column and the Butyl sepharose bounded protein fraction was eluted by washing with elution buffer (50 mM NaH2PO4/Na2HPO4, 0.78 M (NH4)2SO4, 2 mM DTT, pH 6.0). Collected protein fraction was transferred to the diafiltrating concentrator (10 kDa) and centrifuged at 3220 g until the volume of protein solution decreased 8-10 folds. Concentrate was diluted up to a primary volume with buffer containing 50 mM NaH2PO4/Na2HPO4, 2 mM DTT (pH 8).
Procedure was repeated till conductivity decreased below 8 mS/cm and protein solution was subjected to the final purification step using Q sepharose FF resin (GEHealthcare Life Sciences, Uppsala, Sweden). Chromatography media was pre-equilibrated with cold buffer (50 mM NaH2PO4/Na2HPO4, 2 mM DTT, pH 8.0). Protein solution was loaded onto the column and Q sepharose bounded protein fraction was eluted by linear gradient of cold washing buffer supplemented with 250 mM of NaCl. Collected psm was freeze-dried. Sequence verification of the protein termini. Specialized mass spectrometry technique termed in-source decay (ISD) was used. This technique makes use of N-terminal and C-terminal fragment ions, which are generated due to highly elevated laser energy levels during ionization. These fragment ions can be used to derive the terminal amino acid sequences of proteins. ISD spectra do not directly cover the first amino acids of the N-and C-terminus and hence, do often not allow the unambiguous identification/confirmation of the respective amino acids as well as the exact localization of possible modifications. To solve this issue, a technique termed as T3-sequencing is used. The T3 approach is based on the analysis of selected ISD fragments by LIFT. Since ISD fragment ions are generated within the ion source, they can further fragment inside the mass analyzer. LIFT specifically selects an ISD fragment ion and acquires a fragment (MS/MS) spectrum of it. This fragment spectrum usually allows the direct identification of the first amino acids and their modifications.

Results
When combined, the methods described above provide sufficient data to ensure that all plant produced lysins are intact, and no truncation of N-and C-terminal ends is detected.  Fig 5A and B presents T3-sequencing analysis of ZP278. All lysins were analyzed using the same methods.  Figure S3. MALDI-TOF mass spectrum acquired from ZP278. The acquired MALDI-TOF mass spectrum displayed mass signals for the single and the multiple charged molecular ion of ZP278. Further mass signals that could belong to truncated or modified ZP278 were not detected, indicating that only one proteoform was present. The determined molecular mass displayed a deviation of +56.0 Da compared with the theoretical value, which points towards the presence of an acetylation.

Supplementary
Supplementary Figure S4. In-source decay analysis of ZP178. ISD analysis of ZP278 delivered a fragment spectrum with many ISD fragment signals. Sequence information for the N-and C-terminus of ZP278 was obtained for the a-, c-, y-and z+2-type ion series. These ion series indicate that neither the N-nor Cterminus were truncated and that the N-terminus was acetylated. Further modifications were not detected.   Figure S6. Activity of purified lysins against a mix of 5 food-related C. perfringens strains in cooked turkey meat. C. perfringens strains NCTC8235, NCTC8239, NCTC9851, NCTC8449 and NCTC8797 were grown to OD600 appr. 0.23 in TSB anaerobically. Each strain was diluted to OD600 =0.005 and mixed in equal amounts to get 1 ml of bacterial culture