Phase 3 (2007 Deadline)
Novel Triple Acting Chimeric Antimicrobials for Eradication of Multidrug-Resistant
Strains of Staphylococcus aureus
David Donovan, USDA Agricultural Research Service, Beltsville, MD
Sheikh Riazuddin, University of the Punjab, Lahore
Pakistani Funding (HEC): $ 80,300
US Funding (State): $ 123,200
Project Dates on US Side: 2008-2010
(Note: This project was not managed by the National Academies. Although it was selected for funding as a result of the Phase 3 grants competition, funds were provided directly by the Department of State to Dr. Donovan’s laboratory at USDA through an interagency agreement. Therefore, no details on progress are available at the Academies.)
Project Overview
Multidrug-resistant Staphylococcus aureus (MRSA) is a serious pathogen in clinics worldwide. Nosocomial and community-acquired MRSA are increasing in both US and Pakistani hospitals at alarming rates. S. aureus also causes mastitis, a serious disease of livestock resulting in both reduced milk production and quality and often premature culling of infected animals. Mastitis costs the US dairy industry $1.7 billion in annual losses, and although Pakistan is the fifth largest milk-producing country in the world, a high disease incidence is cited as one of the reasons why the production per animal is reduced, such that the country is economically burdened with the need to import Rs.1.1 billion worth of dry milk and milk products annually. Although antibiotic treatment of humans and livestock is effective against most pathogens, this is not true for S. aureus, which in addition to multi-drug resistant strains is known to create biofilms, a sessile form of bacterial colony that is highly resistant to antibiotic treatment, but sensitive to enzyme antimicrobials (for example, lysostaphin). Due to the high rate of antibiotic resistance and biofilms associated with staphylococcal infections, novel antimicrobial enzymes to combat this pathogen are sorely needed in both countries.
The goal of this project is to create a staphylococcal antimicrobial enzyme that is refractory to resistance development. Peptidoglycan (PG) hydrolases degrade the major structural component of bacterial cell walls. When applied externally, these enzymes lyse Gram-positive pathogens with near species-specificity and are reported to be stable for months when purified and stored at 4°C or lyophilized. This project will create a repository of mastitis-causing S. aureus pathogens and bacteriophage of Pakistani origin. With these tools the partners on this project intend to create unique chimeric PG hydrolase(s) consisting of three lytic domains, each targeting a unique bond of the staphylococcal cell wall. Mastitis-causing S. aureus isolates will be provided by co-investigator Dr. G. Muhammad of the University of Agriculture, Faisalabad. After in vitro testing of the parental lysins and triple fusion constructs, these enzyme antimicrobials will be poised for future work in animal model testing. A long term goal is to test these same constructs in human clinical paradigms (after success in the appropriate animal models). This investigation should produce mastitis treatments and eventually lead to human S. aureus treatments as well, developments that would benefit both the US and Pakistani people.
The specific objectives of this project are as follows:
- Isolate and characterize staphylococci and staphylococcal bacteriophage from the Pakistani farm environment.
- Isolate and characterize bacteriophage lytic enzymes (endolysins) of Pakistani origin.
- Train four young researchers from the Pakistani labs in these technologies to further strengthen local molecular research and antimicrobial capabilities.
- With the training received in Objective 3, and the endolysin genes characterized in Objectives 1 and 2, the Pakistani researchers will create and characterize novel triple domain antimicrobials and test their efficacy against known Pakistani mastitis pathogens. Once tested in Pakistan, the triple fusions would be sent to the United States for testing against US pathogens.
Major Results
- This project uses cell wall degrading enzymes to klll bacteria. These enzymes are used by bacteriophage to allow nascent phage to escape during the phage lytic cycle. It had been shown previously that these phage endolysins are modular enzymes and that there could be two enzymatic activities in the same enzyme. The research showed that for one such enzyme, the staphylococcal phage K endolysin, LysK, that just the first enzymatic domain ( N-terminal endopeptidase) was responsible for most of the antimicrobial (lytic) activity.
- The study showed that LysK works together with lysostaphin (a staphylococcal bacteriocin that also degrades the S. aureus cell wall) in a manner that was better than just additive (synergistic).
- The project team next determined where the LysK enzyme cuts the cell wall, helping to explain the synergy we observed. The LysK enzyme cleaves peptidoglycan, the major structural component of the cell wall with 1) an endopeptidase activity, cleaving between the D-alanine of the stem peptide and the first glycine of the pentaglycine cross-bridge peptide, and 2) an N-acetylmuramoyl-L-alanine amidase activity cleaving between the N-acetylmuramic acid of the sugar strand and L-alanine of the stem peptide.
- In the knowledge that LysK having two cut sites and Lysostaphin (one cut site) when applied together cut at three unique sites in the staphylococcal cell wall peptidoglycan, the research reasoned that a fusion would harbor three unique enzymatic activities. We engineered fusions of these enzymes and demonstrated that the triple-acting fusions do maintain all three lytic activities, are highly active at lysing S. aureus and because it is rare for a bacterium to evade three simultaneous lytic activities, the triple-acting antimicrobial is highly refractory to resistance development, S. aureus rarely forms resistant strains when exposed to this triple-acting antimicrobial.
- The research showed that our triple-acting staphylolytic fusions (97%) are as good as mupirocin (98%) at reducing S. aureus nasal colonization in a rat model.
- The study demonstrated that we could redirect a streptococcal endolysin to kill staphylococci, by fusing the streptolytic enzyme domain to a staphylococcal cell wall binding domain.
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