Source: AGRICULTURAL RESEARCH SERVICE submitted to NRP
ENGINEERING BACTERIOPHAGE ENDOLYSINS: ANTIMICROBIALS FOR MASTITIS PATHOGENS THAT ARE REFRACTORY TO RESISTANCE DEVELOPMENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0211127
Grant No.
2007-35204-18395
Cumulative Award Amt.
$348,703.00
Proposal No.
2007-01735
Multistate No.
(N/A)
Project Start Date
Aug 15, 2007
Project End Date
Aug 14, 2011
Grant Year
2007
Program Code
[44.0A]- (N/A)
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
RM 331, BLDG 003, BARC-W
BELTSVILLE,MD 20705-2351
Performing Department
(N/A)
Non Technical Summary
Mastitis is a global problem effecting 1/3 of all dairy cattle and costing the US dairy industry ~$2 Billion annually. Broad range antibiotics used to treat mastitis are less than 50% effective, and often encourage resistant strain development. The goal of this proposal is to create an antimicrobial agent that is refractory to resistance development and targets Staphylococcus aureus with high specificity. The efficacy of this fusion antimicrobial will be tested in a mouse model of mastitis and screened for resistant strain development.
Animal Health Component
(N/A)
Research Effort Categories
Basic
20%
Applied
(N/A)
Developmental
80%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113450110020%
3113840110020%
3114010104030%
3114010110030%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3840 - Laboratory animals; 4010 - Bacteria; 3450 - Milk;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology;
Goals / Objectives
Specific aims. The goal of this project is to create a fusion antimicrobial that is staphylococcal specific and refractory to resistance development. Some peptidoglycan pepetidoglycan hydrolases when applied externally degrade the major structural component of Gram-positive bacterial cell walls with near specie-specificity. The hypothesis is that three fused lytic domains each targeting a unique bond of the staphylococcal peptidoglycan will be highly refractory to resistance development. These fusions would not place selective pressure on non-staphylococci. The first objective is to characterize the triple hydrolase fusion protein (e.g. LysK-Aaa-Lysostaphin), to verify three unique S. aureus pepetidoglycan degrading (exolytic) activities and stable formulations. Included in this goal will be the need to a) verify the three lytic domains are active in the triple fusion via site-directed mutagenesis, b)optimize expression, purification and stabilization of endotoxin-free fusion proteins, and c)prepare formulations for in vivo use in the S. aureus mouse model of mastitis. The second goal is to investigate staphylococcal surface structures, growth phase and the role of a milk environment on the susceptibility of staphylococci to the lytic activity of the parental peptidoglycan hydrolases and resultant fusion(s). This goal will include efforts to a)determine whether the production of capsular polysaccharide, cell wall ribitol teichoic acid, alanylation of wall teichoic acid, poly-N-acetyl glucosamine, surface proteins, or proteases affect the susceptibility of staphylococci to peptidoglycan hydrolases, b) compare the ability of the peptidoglycan hydrolase fusion to disrupt staphylococcal biofilms and c)evaluate the effects of milk and growth phase on bactericidal activity of fusion hydrolase. The final goal of this project is to determine the efficacy of intramammary application of chimeric hydrolases for eradication of S. aureus mastitis. For this goal we will use a mouse model of mastitis to a) evaluate preparations of the chimeric hydrolases in a rodent model of mastitis, b) determine whether mice treated with the fusion proteins develop antibodies to the protein and c)investigate whether S. aureus resistant to the chimeric peptidoglycan hydrolases arise in vitro or in vivo. If resistance does develop, analyze the strains for alterations in peptidoglycan structure, surface polymer content, and protease production.
Project Methods
Peptidoglycan hydrolases degrade the major structural component of cell walls and can lyse Gram-positive bacteria with near specie-specificity when exposed externally. This project proposes to create a chimeric hydrolase consisting of three staphylococcal lytic activities using standard molecular biological techniques, each targeting a unique cell wall (peptidoglycan) bond. These triple activity fusion proteins should be highly refractory to resistance development due to: a) no identified bacterial host can resist the lytic action of its bacteriophage hydrolase; b) peptidoglycan hydrolase domains can maintain their specie-specificity when fused; and c) S. aureus is unlikely to develop three simultaneous compensatory mutations. The specific aims are to: 1) Construct a fusion protein with three cell wall degrading activities and investigate methods for protein stabilization and in vivo delivery, 2) Evaluate whether in vitro culture conditions affect S. aureus susceptibility to the fusion hydrolase (e.g. biofilms; expression of surface antigens induced by milk). 3) Test the chimeric hydrolase in a rodent model of mastitis for eradication of S. aureus. If resistant strains are identified, they will be characterized in an effort to understand the phenotype.

Progress 08/15/07 to 08/14/11

Outputs
Target Audience:The target audience is scientists and industry that might want to license the patents produced from this work. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One Postdoctoral Fellow and numerou volunteer summer students were trained via this grant award. How have the results been disseminated to communities of interest?Aside from the journal publicationsabove, and those in press, there were also numerous posters presented at the Evergreen International Phage meetings in 2009 and 2011, as well as the International Society for Viruses of Microbes meetings in Paris 2010. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Each of the goals was accomplished. Triple fusion lytic enzymes were produced that harbor three unique, synergistic lytic domains that are active in the final fusion construct (by mass spectrometry of peptidoglycan digestion products). These fusions were tested in a mouse model of mastitis and the optimal triple fusion construct identified. A panel of lytic enzymes was also tested on various staphylococcal cell wall mutants for the effect of various cell wall structures on endolysin lytic activity (e.g. capsular polysaccharide, cell wall ribitol teichoic acid, alanylation of wall teichoic acid, poly-N-acetyl glucosamine, surface proteins, or proteases) and ability to degrade staphylococcal biofilms. Manuscripts describing these results are in preparation or in press.

Publications

  • Type: Journal Articles Status: Published Year Published: 2011 Citation: Rodr�guez L, Mart�nez B, Zhou Y, Rodr�guez A, Donovan D M and Garc�a P. Lytic activity of the virion-associated peptidoglycan hydrolase HydH5 of Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88. BMC Microbiology 2011; 11:138
  • Type: Journal Articles Status: Published Year Published: 2008 Citation: Donovan, D.M., Foster-Frey, J.A., Garrett, W.M., Blomberg, L. Resolving database sequence discrepancies for the Staphylococcus aureus bacteriophage phi11 amidase. 2008. Journal of Basic Microbiology, 48, 4852.
  • Type: Journal Articles Status: Published Year Published: 2008 Citation: Becker SC, Foster-Frey J, Donovan DM. The phage K lytic enzyme LysK and lysostaphin act synergistically to kill MRSA. FEMS Microbiol Lett. 2008 Oct;287(2):185-91. Epub 2008 Aug 21.
  • Type: Journal Articles Status: Published Year Published: 2008 Citation: Donovan DM, Foster-Frey J. LambdaSa2 prophage endolysin requires Cpl-7-binding domains and amidase-5 domain for antimicrobial lysis of streptococci. 2008. FEMS Microbiol Lett. Oct;287(1):22-33.
  • Type: Journal Articles Status: Published Year Published: 2009 Citation: Becker S.C., Dong S., Baker J.R., Foster-Frey J., Pritchard D.G., Donovan D.M. LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells. FEMS Microbiol Lett. 2009 May;294(1):52-60.
  • Type: Journal Articles Status: Published Year Published: 2009 Citation: Becker SC, Foster-Frey, J. , Stodola A.J., Anacker, D., Donovan, D.M. Differentially conserved staphylococcal SH3b_5 cell wall binding domains confer increased staphylolytic and streptolytic activity to a streptococcal prophage endolysin domain. Gene. 2009 Aug 15;443(1-2):32-41. Epub 2009 May 5.
  • Type: Journal Articles Status: Published Year Published: 2010 Citation: Filatova, L.Y., Becker, S.C., Donovan, D.M., Gladilin, A.K., Klyachko, N.L. 2010. LysK, the enzyme lysing Staphylococcus aureus cells: specific kinetic features and approaches towards stabilization. Biochimie. May;92(5):507-13. Epub 2010 Feb 6.


Progress 08/15/09 to 08/14/10

Outputs
OUTPUTS: Activities: Despite the ability to eradicate static biofilms, and a formulation that maintains activity for more than 3 months at -80C, the LysK-lysostaphin triple fusion did not show any staphylolytic activity in either bovine or mouse milk. Thus we expanded our search for novel phage endolysins. Deletion analysis was performed on several lysins (including the Haemo, Twort, and 2638A lysins) in order to identify the amino acid domains required for lytic activity. We developed a checkerboard plate lysis assay and were able to demonstrate synergy between Lysostaphin and a staphylolytic fusion protein {streptococcal LambdaSA2 endolysin endopeptidase domain fused to a staphylococcal SH3b domain}. In partial fulfillment of aim 2, nine staphylococcal lysins and several triple fusion proteins were tested for activity against mastitis causing staphylococci in sterile homogenized cow milk. The phi11 endolysin and a closely related lysin from the H5 phage, were both found to exhibit activity in milk, causing >2 log reductions of bacterial cfus within 4 hours. Several enzymes (LysK, Haemo, Twort) are able to eradicate or reduce biofilms of S. aureus SA113 (a known strong biofilm forming strain) in a 96 well static biofilm assay. Initial data indicate that S. aureus strain Newman develops resistance to triple fusion constructs derived from LysK and Lysostaphin at a significantly reduced rate (10X) compared to the parental LysK (100X) and Lysostaphin (1000X). LambdaSA2-SH3b fusion proteins were tested in a mouse model of bovine mastitis. Intramammary infusions of a combination these proteins into mammary glands infected with a staphylococcal mastitis strain at 102 to 104 cfu/gland reduced bacterial numbers by approximately 3 log (after 24 hours). Efforts to create Lysostaphin-LambdaSA2-SH3b fusions are underway. Products: There were four invention disclosures that are accepted for provisional patent applications supported by NRI funds, describing the use of four staphylococcal lysins and deletion constructs (Haemo, Twort, 2638A, phi13). Deletion analyses were conducted in collaboration with Dr. Igor Abaev, SCRAMB, Obolensk, Moscow Region, Russia. Events: Data generated through support by NRI funds were presented at following events: Beltsville Area Research Center Poster Day, April 2010, Beltsville, MD; Crosstalk: Across Cells, Across Campuses, January 2010, University of Maryland, College Park, MD. David Donovan was an invited speaker at following events and locations: National Foundation on Infectious Disease (NFID) annual meeting, Bethesda, MD; Virginia-MD Regional College of Veterinary Medicine. Department of Veterinary Medicine, Sep 23, 2010; Dr. Donovan is a member of the Organizing Committee for the 18th International Evergreen Phage Conference to be held August 2011, has been coordinating the organization of a new Intn'l Society for the Viruses of Microbes and continues to manage an international e-mail bulletin board that supports ~250 phage biologists. Participants: Mathias Schmelcher, Postdoctoral Fellow arrived Jan 2009. Target Audiences: Nothing significant. Project modifications: Nothing significant. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Outcomes/Impact: Mastitis is a global problem effecting dairy production and animal well-being in every country. Broad range antibiotics used to treat mastitis are less than 50% effective, and often encourage resistant strain development. This proposal is engaged in designing an antimicrobial agent that is refractory to resistance development and targets Staphylococcus aureus with high specificity. A first step in this process is to identify novel enzymes and domains with high lytic activity for staphylococci, and those that act synergistically are ideal candidates for ascertaining that we have unique enzymatic activities. Changes in knowledge: Our efforts to determine cut sites of enzymes within the peptidoglycan as well as our deletion analysis identified several unique enzymatic domains (glycosidase, amidase, endopeptidases) that are promising to exhibit high lytic activity and reduced resistance formation (due to different target bonds in the peptidoglycan) when combined in fusion constructs. Using our novel plate lysis checkerboard assay, we discovered the synergistic action between fusion proteins harboring a stem peptide specific streptococcal endopeptidase and Lysostaphin, which is an interpeptide bridge specific endopeptidase. Activity assays in cow milk yielded an additional pair of closely related enzymes, the phi11 and H5 lysin (in addition to Lysostaphin and the LambdaSA2-SH3b fusions that were reported to be active in milk in the last annual report), showing potential for application as an anti-mastitis agent in mammary glands. Furthermore, we identified several enzymes and triple fusion constructs with ability to disrupt staphylococcal biofilms. The results of our resistance development studies suggest that constructs harboring multiple unique enzymatically active domains are indeed more refractory to resistance formation than parental lysins. Using a mouse model of bovine mastitis, we demonstrated that the LambdaSA2-SH3b fusion proteins are able to reduce staphylococcal numbers in infected mammary glands, and preliminary data suggest that one of the fusion constructs acts synergistically with Lysostaphin to kill S. aureus in vivo. Our results yielded patent applications and publications in international journals. Change in actions: none Change in conditions: none

Publications

  • David Donovan. Peptidoglycan hydrolase fusion to protein transduction domains kill intracellular staphylococci. Crosstalk: Across Cells, Across Campuses: The first UMB/UMCP Research Symposia, Jan 12, 2010.
  • Publications: JOURNAL ARTICLES: Filatova LY, Becker SC, Donovan DM, Gladilin AK, Klyachko NL (2010). LysK, the enzyme lysing Staphylococcus aureus cells: specific kinetic features and approaches towards stabilization. Biochimie. 92(5):507-13.
  • ABSTRACTS: Schmelcher M, Becker SC, Pohl CS, Foster-Frey J, Donovan DM (2010). Bacteriophage Endolysins for Control of Mastitis Pathogens. Beltsville Area Research Center Poster Day, April 2010, Beltsville, MD.
  • Schmelcher M, Becker SC, Foster-Frey J, Donovan DM (2010). Peptidoglycan Hydrolases for Control of Mastitis Pathogens. Crosstalk: Across Cells, Across Campuses, January 2010, University of Maryland, College Park, MD
  • David Donovan. A Multi-lytic Domain Peptidoglycan Hydrolase Fusion Protein as a Targeted Topical Antimicrobial for Gram-positive Pathogens. International Conference on Virus of Microbes, June 2010, Paris, France Homan Mohammadi. Double and triple fusion PG hydrolases as novel antimicrobials. UMD Balto Co. Research Day Oct 2009
  • BOOKS: Becker SC, Foster-Frey J, Powell A, Mohammadi H, Kerr DE, Donovan, DM (2010). Lysostaphin: molecular changes that preserve staphylolytic activity. Science and Technology against Microbial Pathogens. Research, Development and Evaluation.
  • Weese S, Mao J, Donovan DM (2010). Staphylococcus aureus (Chapter). Genomes of Food- and Water-Borne Pathogens. Eds. Pina M. Fratamico, Yanhong Liu and Sophia Kathariou, ASM Press, Washington, D.C.
  • Dave Donovan. Peptidoglycan hydrolase fusion to protein transduction domains kill intracellular staphylococci. Presented to UK (Ireland) Representatives 3/29/2010 ARS, Oct 30, 2009
  • Dave Donovan. Peptidoglycan hydrolase fusion to protein transduction domains kill intracellular staphylococci. Presented to David Lowry, Head, Technology Lead for Novartis Animal Health US, Inc, 2010, ARS
  • Dave Donovan. Peptidoglycan hydrolase fusion to protein transduction domains kill intracellular staphylococci. Presented to Abdul Kareem, Univ . Baghdad, August 2010, ARS
  • Emily Cooper. Addition of HIV Trans-activator of Transcription (TAT) Domain to Endolysin Triple Fusion Protein may enable Intracellular Killing of Disease causing Microbes UMD Balto Co. 2009 Graduate student day.
  • Steve Becker. Engineering Antimicrobials Refractory to Resistance Crosstalk: Across Cells, Across Campuses: The first UMB/UMCP Research Symposia Jan 12, 2010


Progress 08/15/08 to 08/14/09

Outputs
OUTPUTS: Activities: In partial fulfillment of Aim 1 to optimize the triple lytic fusion constructs, we examined the relative staphylococcal binding of several SH3b cell wall binding domains, through the use of GFP-SH3b fusions to intact cells. The goal to identify the optimal SH3b cell wall binding domain for use in the final triple lytic domain fusion were not realized. There is not a strong difference between the SH3b domains we examined. We know from preliminary work in our lab that the LysK CHAP endopeptidase and LambdaSa2 endopeptidase domains are the essential domains in these enzymes for lysis of staphylococci. New results indicate that fusion of the streptococcal LambdaSa2 prophage endolysin endopeptidase domain to the cell wall binding domain of either LysK or lysostaphin increases the staphylolytic activity more than 10 fold, making this fusion a highly staphylolytic enzyme for use in our future constructs. Our efforts to identify novel endolysin sequences has resulted in a study to collate the SH3b containing staphylococcal peptidoglycan hydrolases in public datasets. Nine unique lysins (one representative of each of five groups collated by homology and four stand-alone proteins) were recombinantly produced, purified, and characterized for their lytic activity. All nine enzymes are stable in aqueous solution and were shown to exhibit staphylolytic activity in vitro. In partial fulfillment of Aim 2, numerous candidate endolysins and fusion constructs have been tested for their efficacy in a milk environment and on a variety of staphylococcal strains with cell wall mutations. The aforementioned LambdaSa2 endopeptidase - staphylococcal SH3b fusion constructs showed activity in sterile, homogenized whole milk, reducing staphylococcal cell counts by up to 3 log units within 3 hours. Two summer students were mentored by Dr. Donovan and Dr. Schmelcher and determined that most staphylococcal cell wall mutant strains show little difference in susceptibility to a variety of lysins and fusion constructs. The triple fusion constructs made with LambdaSa2 and lysostaphin are still being analyzed. In partial fulfillment of Aim 3, we have also initiated studies to identify resistance development to our fusion constructs and parental staphylococcal lysins via both plate lysis and liquid culture approaches. These studies are still a work in progress. Products: There was one provisional patent application that was supported in part by NRI funds in FY 09. USDA D.N. 0034.09 "Fusion of Peptidoglycan Hydrolase Enzymes to a Protein Transduction Domain Allows Eradication of both Extracellular and Intracellular Gram Positive Pathogens" : Provisional Patent Application filed May 23, 2009, PTO Provisional S.N. 61/216,779. Events: Dr. Donovan served as an Organizing Committee member and Session Chair for the 17th Evergreen International Phage Meeting, held August 2009, Olympia, WA. PARTICIPANTS: Mathias Schmelcher, Postdoctoral Fellow arrived in January 2009. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Mastitis is a global problem effecting dairy production and animal well-being in every country. Broad range antibiotics used to treat mastitis are less than 50% effective, and often encourage resistant strain development. This proposal is engaged in designing an antimicrobial agent that is refractory to resistance development and targets Staphylococcus aureus with high specificity. A first step in this process is to identify novel enzymes with high lytic activity for staphylococci. Change in Knowledge: Our studies have resulted in numerous novel staphylolytic candidate enzymes having been identified and tested in vitro and in a milk environment. In addition to the prior lysins (phi11 endolysin, lysostaphin, LysK) we also have examined the endolysins for phi80alpha, phi68, phi2638a, phi Twort, LambdaSa2 and phi SH2. We have identified enzymatic domains that, when used as modules within fusion constructs, exhibit high staphylolytic activity in whole milk. Furthermore, we investigated the effect of numerous mutant strains with altered cell envelop properties for their susceptibility to our parental enzymes and fusion constructs. Our results yielded patent applications and publications in international journals. Change in conditions: Dr. Donovan initiated an international e-mail bulletin board Phage-list in 2008 for a faster exchange of information between phage-interested parties. He managed that list in 2009. The IT portion is administered through contacts at the Imperial College of London and University of Miami. Phage-List mailing list, Phage-List@imperial.ac.uk, https://mailman.ic.ac.uk/mailman/listinfo/phage-list

Publications

  • These works were supported in part by NRI funding: 1. Becker S.C., Dong S., Baker J.R., Foster-Frey J., Pritchard D.G., Donovan D.M. LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells. FEMS Microbiol Lett. 2009 May;294(1):52-60.
  • 2. Becker SC, Foster-Frey, J. , Stodola A.J., Anacker, D., Donovan, D.M. Differentially conserved staphylococcal SH3b_5 cell wall binding domains confer increased staphylolytic and streptolytic activity to a streptococcal prophage endolysin domain. Gene. 2009 Aug 15;443(1-2):32-41. Epub 2009 May 5.
  • 3. David M Donovan 2009. PEPTIDOGLYCAN HYDROLASE FUSIONS: MASTITIS ANTIMICROBIALS THAT ARE REFRACTORY TO RESISTANCE DEVELOPMENT. Elanco, Inc., Indianapolis, Indiana.
  • 4. David M. Donovan. 2009. PEPTIDOGLYCAN HYDROLASE FUSIONS: MASTITIS ANTIMICROBIALS THAT ARE REFRACTORY TO RESISTANCE DEVELOPMENT. Neogen, Inc,. Lexington, KY
  • 5. Steve Becker, Juli Foster-Frey, Ryan Willard, Richard Lease, Raul Almeida, Ian Marriott, Shengli Dong, John R. Baker, David Pritchard, David Donovan. 2009. PEPTIDOGLYCAN HYDROLASE FUSIONS TO PROTEIN TRANSDUCTION DOMAINS KILL INTRACELLULAR STAPHYLOCOCCI. Evergreen Phage Meeting, Olympia WA, August 9-14, 2009. page 5-6.
  • 3. Donovan, D.M., Becker, S.C., Dong, S., Baker, J.R., Foster-Frey, J., and Pritchard, D.G. Peptidoglycan hydrolase enzyme fusions for treating multi-drug resistant pathogens. 2009 Biotech International 21(2): 6-10.
  • ABSTRACTS: Dr. Donovan was an invited speaker: 1. Steve Becker, Juli Foster-Frey, Ryan Willard, Richard Lease, Raul Almeida, Ian Marriott, Shengli Dong, David Pritchard, David Donovan. 2009. ANTIMICROBIALS FOR STAPHYLOCOCCAL PATHOGENS THAT ARE REFRACTORY TO RESISTANCE DEVELOPMENT. International Symposium on Antimicrobial Peptides. St. Malo, France ; page 43
  • 2. David M. Donovan. 2009. DESIGNING A BETTER ANTIMICROBIAL THROUGH PEPTIDOGLYCAN HYDROLASE ENGINEERING. ARS, USDA. Plant Pathogens : Laboratory seminar.
  • Other Presentations: 1. Mathias Schmelcher, Stephen C. Becker, Juli Foster-Frey, David M. Donovan. 2009. PEPTIDOGLYCAN HYDROLASES FOR CONTROL OF MASTITIS PATHOGENS., Evergreen Phage Meeting, Olympia WA, August 9-14, 2009. page 4-3.
  • 2. Stephen C. Becker, Calvin Pohl, Homan Mohammadi, Mathias Schmelcher, Julie Foster-Frey, Richard Lease, Shengli Dong, John R. Baker, David G. Pritchard, and David M. Donovan. 2009. ENGINEERING ANTIMICROBIALS REFRACTORY TO RESISTANCE. Evergreen Phage Meeting, Olympia WA, August 9-14, 2009. page 9-2.
  • NEWSLETTERS: 1. Dr. Donovans work was showcased in Science Daily web newsletter (Aug. 31, 2009). VIRUS ENZYMES COULD PROMOTER HUMAN, ANIMAL HEALTH. http://www.sciencedaily.com/releases/2009/08/090830102451.htm
  • 2. Dr. Donovans work was showcased in the ARS newsletter: VIRUS ENZYMES COULD PROMOTE HUMAN, ANIMAL HEALTH. ARS Newsmakers, Friday August 14, 2009, and on the ARS News and Events web site. http://www.ars.usda.gov/is/pr/2009/090814.htm
  • MEDIA RELEASES: 1. Dr. Donovan (2009) was interviewed on air by Jim LaPorte, Lexington, Nebraska for Farm Broadcaster, KRNV (Rural Voice of Nebraska) radio talk show.


Progress 08/15/07 to 08/14/08

Outputs
OUTPUTS: Activities: A deletion analysis of the staphylococcal phage K endolysin, LysK, was performed to identify the optimal domains for use in constructing triple fusions. A second candidate enzyme reported to have staphylolytic activity was the LambdaSa2 streptococcal prophage endolysin. A deletion analysis was performed on this enzyme to again identify the optimal domains for use in constructing triple fusions. We also have numerous LysK-lysostaphin triple domain containing constructs that were optimized for use in this project. There were two summer students mentored by Dr. Donovan in 2008. Also, in-house technical support and Postdoctoral Fellows were utilized in a part time capacity to meet program goals despite the lack of a full time Fellow hired through award funding. Despite the absence of the intended Postdoctoral Fellow, we were able to partially fulfill the goals of Aim 1 by creating and verifying the triple lytic activity of a LysK-lysostaphin fusion by electron spray ionization mass spectrometry via a collaboration with Dr. David Pritchard, Univ. Alabama, Birmingham. Events: Dr. Donovan also was co-organizer of the 2008 Edinburgh International Phage meeting, Edinburgh, UK. Where he was an invited speaker and presented his work : Stephen C. Becker, David M. Donovan, Juli Foster-Frey. "SH3b cell wall binding domains can ehanace antistaphylococcal activity of endolysin lytic domains" Edinburgh International Phage Meeting, 2008. Dr. Donovan also served as an Organizing Committee member on the 17th Evergreen International Phage Meeting, to be held August 2009, Olympia, WA. David M. Donovan, Stephen C. Becker, Juli Foster-Frey.2008. Making better antimicrobials through bacteriophage endolysin engineering. Departmental Seminar, Department of Infectious Diseases, University of GA, Athens GA. Products: There were three provisional patent applications that were supported in part by the NRI funding. 1. USDA D.N. 0065.08 "Lys K Endolysin Is Synergistic with Lysostaphin Against MRSA": Provisional Patent Application filed May 23, 2008, PTO Provisional S.N. 61/128,707 2. USDA D.N. 0064.08 "LambdaSa2 Endolysin Truncation": Provisional Patent Application filed May 23, 2008, Provisional S.N. 61/128,713 3. USDA D.N. 0113.08 "Triple acting antimicrobials that are refractory to resistance development": Provisional Patent Application filed July 24, 2008, PTO Provisional S.N. 61/135,810 Dissemination: August 2008, Dr. Donovan initiated an international e-mail bulletin board Phage-list. The IT portion is administered through contacts at the Imperial College of London and University of Miami. Phage-List mailing list, Phage-List@imperial.ac.uk, https://mailman.ic.ac.uk/mailman/listinfo/phage-list PARTICIPANTS: Postdoctoral Fellow has been identified, Mathias Schmelcher. Did not arrive in 2007-08. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: The LysK-AAA-Lysostaphin triple fusion was deemed too instable, and thus impossible to reliably create and purify. LysK-lysostaphin triple fusions are much more amenable to purification and are being optimized along with efforts to identify essential domains and new endolysins for use in the fusion constructs, as described in the original application.

Impacts
Mastitis is a global problem effecting dairy production and animal well-being in every country. Broad range antibiotics used to treat mastitis are less than 50% effective, and often encourage resistant strain development. This proposal is engaged in designing an antimicrobial agent that is refractory to resistance development and targets Staphylococcus aureus with high specificity. A first step in this process is to identify novel enzymes with high lytic activity for staphylococci, and those that act synergistically are ideal candidates for ascertaining that we have unique enzymatic activities. We have demonstrated and published on the fact that LysK and lysostaphin act synergistically in lysing staphylococci. We have also identified that the LysK CHAP endopeptidase and LambdaSa2 endopeptidase domains are the essential domains in these enzymes for lysis of staphylococci (see publication list). Preliminary data suggests that fusion of the LambdaSa2 endolysin endopeptidase domain to the cell wall binding domain of either LysK or lysostaphin increases the staphylolytic activity more than 10 fold. We are currently creating and testing constructs that utilize these modified enzymes. We have also performed a preliminary analysis of the SH3b containing staphylococcal peptidoglycan hydrolases in public datasets, as a means to identify novel enzymes for use in creating triple fusions. We also adopted a novel method to perform turbidity reduction assays. This method uses a plate reader and rapid (20 second interval) absorbance measurements. This has much improved the reproducibility of our experiments and the reliability of our results.

Publications

  • JOURNAL ARTICLES: Donovan, D.M., Foster-Frey, J. LambdaSa2 prophage endolysin requires Cpl-7-binding domains and amidase-5 domain for antimicrobial lysis of streptococci. In Press. FEMS Microbiol Lett. Epub 2008 Jul 31.
  • Becker SC, Foster-Frey J, Donovan DM. The phage K lytic enzyme LysK and lysostaphin act synergistically to kill MRSA. In Press FEMS Microbiol Lett. Epub 2008 Aug 21.
  • ABSTRACTS: Stephen C. Becker, David M. Donovan. 2008. Engineering MRSA antimicrobials that are refractory to resistance development. Beltsville Area Research Center Poster Day, Beltsville, MD.
  • Stephen C. Becker, David. M. Donovan. 2008. Engineering antimicrobials that are refractory to resistance development. Edinburgh International Phage Meeting.