Progress 03/15/16 to 03/14/19
Outputs
Target Audience:The target audience includes the pork industry and pork farmers who will benefit from new therapeutics to treat porcine infection. The audience also includes consumers who will also benefit from safe, healthy livestock. Finally, students and postdocs who have worked on this project have received mentorship, education, and training. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The postdoc funded by this project has received training on equipment necessary to complete the project. She has also been responsible for training and mentoring incoming graduate, undergraduate, and high school students, all of which have helped with the project. Additionally, the postdoc attended a workshop to learn new techniques and instrumentation that will be essential for full characterization of the S. suis endolysins. How have the results been disseminated to communities of interest?The results have been presented in posters at the Evergreen International Phage Biology meeting, the Agriculture and Food Research Initiative Animal Health and Well-being Program Annual Project Director Meeting, and the Virology of Microbes meeting. Further, once complete, results will be submitted for peer review and published in leading scientific journals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
A bioinformatic search was conducted to identify endolysins potentially active against Streptococcus suis. Our initial investigation resulted in nine putative candidates, but upon further examination, we narrowed this list to five endolysins with distinct architectures. These five endolysins (named PlySp0, PlySs0, PlySs9, PlySsM, and PlySs3) were synthesized by GeneArt (ThermoFisher Scientific). Additionally, we synthesized PlySs2, a known endolysin with lytic activity against S. suis. Each gene was cloned into pBAD24 expression vector and transformed into E. coli DH5α, whereupon the sequence was verified by Macrogen. The plasmids were then transformed into E. coli BL21 (DE3) pLysS and expression was induced with arabinose. All endolysins were successfully expressed in this system and were also able to be purified by nickel column chromatography. Each endolysin was then tested for lytic activity against a panel of ten S. suis strains. Using the turbidity reduction assay, only one endolysin (PlySs9) other than PlySs2 was shown to possess activity against S. suis in the native, un-engineered form. Further characterization was performed to determine PlySs9's optimal lytic activity in a range of pH, salt concentrations, and temperatures. This endolysin was shown to display activity against not only all S. suis strains tested, but multiple other species of streptococci, as well as against Staphylococcus aureus. Additionally, PlySs9 exhibited activity against biofilms formed by both S. suis or S. aureus. Identification of catalytic residues in PlySs9 was determined by site-directed mutagenesis, and preliminary results indicate that PlySs9 contains two active catalytic domains (which are annotated as an Amidase3 domain and a CHAP domain). Toward our goal of creating a triple-acting enzyme, PlySs9 will be the chassis upon which an extra domain will be added. Fusions between PlySs9 and the catalytic domains from the four other endolysins have been constructed, resulting in five new potential triple acting enzymes. Two were successfully expressed and purified (PlySp0Glucosaminidase+PlySs9 and PlySs0CHAP+PlySs9) and will need to be tested in comparison to the parental PlySs9 to determine if the addition of a third catalytic domain has resulted in increased lytic activity. Since the project ended early (i.e. the remainder of the project will be transferred to another institution), the final enzyme to put forward for in vivo studies has not yet been determined. Specific accomplishments include: Endolysins were synthesized, cloned into expression plasmid, transformed into cloning strain of E. coli, sequence verified, and transformed into expression strain of E. coli. All endolysins were successfully expressed and purified via affinity chromatography. Endolysins were tested for activity via turbidity reduction assay. Optimal lytic activity was determined in a range of pH, salt concentrations, and temperatures. Activity was observed against multiple species and against bacteria in biofilms. Catalytic residues of PlySs9 were identified. Construction of triple catalytic domain endolysins resulted in two new enzymes that could successfully be expressed and purified and will need to be tested for activity.
Publications
- Type:
Journal Articles
Status:
Other
Year Published:
2019
Citation:
A novel Streptococcus suis endolysin with broad host specificity (proposed title) - Viruses (Journal)
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Progress 03/15/16 to 03/14/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The postdoc funded by this project has received training on equipment necessary to complete the project. She has also been responsible for training and mentoring incoming graduate, undergraduate, and high school students, all of which have helped with the project. Additionally, the postdoc attended a workshop in April to learn new techniques and instrumentation that will be essential for full characterization of the S. suis endolysins. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, the multiple catalytic abilities of PlySs9 and triple catalytic domain enzymes will be confirmed by mass spectrometry and/or biochemical assay. Furthermore, the lytic activity of the triple catalytic domain constructs will be tested in comparison to the parental enzymes. If found to be more active than the parental enzymes, these fusions will be our lead candidates for all following experiments. The optimal conditions for lytic activity for these new constructs will be determined (pH, salt concentration, cation dependence, temperature, dose) and activity against our panel of target bacteria will be confirmed. Of the fusions are not more active than the single or double catalytic domain constructs, then these enzymes will be benchmarked against each other, it will be determined if they display synergy, and ultimately a final candidate or candidates will be selected for scale up expression and purification for the animal studies. Importantly, for all final candidates, resistance development studies will be performed.
Impacts
What was accomplished under these goals?
Technical summary: A bioinformatic search was conducted to identify endolysins potentially active against Streptococcus suis. Our initial investigation resulted in nine putative candidates, but upon further examination, we narrowed this list to five endolysins with distinct architectures. These five endolysins were synthesized by GeneArt (ThermoFisher Scientific). Additionally, we synthesizeda known endolysin with lytic activity against S. suis. Each gene was cloned into pBAD24 expression vector and transformed into E. coli DH5α, whereupon the sequence was verified by Macrogen. The plasmids were then transformed into E. coli BL21 (DE3) pLysS and expression was induced with arabinose. All endolysins were successfully expressed in this system and were also able to be purified by nickel column chromatography. Each endolysin was then tested for lytic activity against a panel of ten S. suis strains. Using the turbidity reduction assay, only one new endolysin and the knownendolyin were shown to possess activity against S. suis in the native, un-engineered form. Further characterization was performed to determinethe new endolysinoptimal lytic activity in a range of pH, salt concentrations, and temperatures. This endolysin was shown to display activity against not only all S. suis strains tested, but multiple other species of streptococci, as well as against Staphylococcus aureus. Additionally,it exhibited activity against biofilms formed by both S. suis or S. aureus. Identification of catalytic residues in PlySs9 was determined by site-directed mutagenesis, and preliminary results indicate thatit contains two catalytic domains. Toward our goal of creating a triple-acting enzyme,the new endolysinwill be the chassis upon which an extra domain will be added. Fusions between the new endolysin and the catalytic domains from the four other endolysins have been constructed, resulting in five new potential triple acting enzymes. Two were successfully expressed and purified and will need to be tested in comparison to the parental PlySs9 to determine if the addition of a third catalytic domain has resulted in increased lytic activity. Accomplishments Endolysins were synthesized, cloned into expression plasmid, transformed into cloning strain of E. coli, sequence verified, and transformed into expression strain of E. coli. All endolysins were successfully expressed and purified via affinity chromatography. Endolysins were tested for activity via turbidity reduction assay. Optimal lytic activity was determined in a range of pH, salt concentrations, and temperatures. Activity was observed against multiple species and against bacteria in biofilms. Catalytic residues of PlySs9 were identified. Construction of triple catalytic domain endolysins resulted in two new enzymes that could successfully be expressed and purified and will need to be tested for activity.
Publications
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