FORT BELVIOR, Va. - Developed in the 1940s, antibiotics are powerful medicines used in the treatment and prevention of bacterial infection to save lives. Yet every year approximately two million people in the United States are treated for a bacterial infection that is resistant to antibiotics. These antibiotic-resistant infections result in an average of more than 20,000 deaths annually.
In addition to posing a threat to the civilian population, antibiotic-resistant bacteria, such as plague or smallpox, are highly contagious and could be used as a biological weapon to thwart our military forces. Thus, the arms race against antibiotic-resistant bacteria necessitates a new generation of antimicrobial therapies that must destroy microbes while considering the secondary effects associated with their use.
Research funded by the Defense Threat Reduction Agency’s Chemical and Biological Technologies Department, managed by DTRA’s Dr. Ilya Elashvili and conducted by Dr. James Collins from the Massachusetts Institute of Technology, developed a delivery system for the next generation of antimicrobial therapies.
Their work, recently published in Nano Letters entitled “Engineered Phagemids for Nonlytic, Targeted Antibacterial Therapies,” holds promise as a synthetic biology platform that can be rapidly adapted to deliver targeted antimicrobial therapies to counter multi-drug resistant bacteria.
Traditional antimicrobial therapies employ broad-spectrum antibiotics that indiscriminately kill bacteria, often breaking down the cell membrane through a process called lysis. Such techniques create harmful secondary effects including the release of toxic intracellular intermediates and destroying the natural microbial flora. Antimicrobial peptides (AMPs) are an attractive alternative to broad-spectrum antibiotics because they kill bacteria without inducing lysis.
To maximize this non-lytic effect, AMPs must be expressed in situ, thereby overwhelming the microbe’s intercellular machinery and rapidly shutting down the cell. Researchers have often used bacteriophages for delivery of synthetic biology constructs in situ, and though effective, they are too unreliable to be widely used as a delivery system for alternative therapies.
Dr. Collins and his team improve on AMP and bacteriophage therapy through the use of phagemids plasmids that contain the f1 origin that can be used as a cloning vector, enabling their packaging and delivery by mature M13 bacteriophage particles. A quirk in M13 biology enables expression of virus particles that lack the ability to reproduce, while retaining the ability to infect cells with their DNA. Once delivered, a phagemid plasmid can replicate inside the targeted bacteria, ensuring high expression levels of AMPs.
Additionally, the lack of viral particle reproduction with phagemids prevents resistance to infection in the target cell. This is a critical improvement over traditional bacteriophage therapies where transfected vectors were often unstable and easily rejected by the host. By harvesting and applying M13 phagemid particles containing antimicrobial expressing phagemid plasmids, a target cell population can be converted into an antimicrobial factor system that induces rapid, nonlytic cell death.
Collins and his team compared the ability of a phagemid expression system to the traditional bacteriophage therapy to induce cell death. They transfected E. coli bacteria with phagemid or conventional bacteriophage vectors containing single AMP expression systems known to kill, but not lyse the bacteria.
The survival of cells infected by phagemid particles was reduced by at least an order of magnitude when compared to the survival of cells infected by the conventional bacteriophage vector. Critically, cells infected with the phagemid remained dead throughout the course of the experiment whereas cells infected by the bacteriophage vector began to recover after four hours.
By combining the expression of multiple AMPs on a single phagemid, the researchers enhanced the induction of cell death by another order of magnitude. As an additional benefit, they demonstrated that cells infected by phagemid particles can also be re-infected for additional treatment, unlike conventional bacteriophage therapy where transfected cells gain resistance.
After testing their system in vitro, the authors tested their system in vivo through mice. Mice were infected with E. coli and either treated with AMP expressing phagemid particles or left untreated as a control. The results show that 80 percent of mice treated with AMP expressing phagemid particles survived infection, compared to a 27 percent survival rate in untreated mice. The authors suggest that application of their phagemid platform, in addition to inducing expression of AMPs, may also induce a pro-inflammatory response that further enhances the fight against infection.
Dr. Collins and his team envision phagemids as a platform for delivering the next generation of antimicrobial therapies that can be easily modified to include expression of alternative AMPs, origins of replication and signals to counter new challenges in antimicrobial therapy and target infections. Their work will help realize the full potential of antimicrobial peptides and bacteriophage therapy, protecting the warfighter against antibiotic-resistant microbes.
|Date Posted:||08.26.2015 11:31|
|Location:||FORT BELVOIR, VA, US|
This work, Harnessing Synthetic Biology to Combat Bacterial Pathogens, is free of known copyright restrictions under U.S. copyright law.