NEW TECHNOLOGIES FOR COMBATING SUPERBUGS
In recent years, new
strains of bacteria have emerged that resist even the most powerful
antibiotics. Each year, these superbugs, including drug-resistant forms of
tuberculosis and staphylococcus, infect more than 2 million people nationwide,
and kill at least 23,000. Despite the urgent need for new treatments,
scientists have discovered very few new classes of antibiotics in the past
decade
MIT engineers have now
turned a powerful new weapon on these superbugs. Using a gene-editing system
that can disable any target gene, they have shown that they can selectively
kill bacteria carrying harmful genes that confer antibiotic resistance or cause
disease.
Led by Timothy Lu, an
associate professor of biological engineering and electrical engineering and
computer science, the researchers described their findings in the Sept. 21
issue of Nature Biotechnology. Last month, Lu's lab reported a
different approach to combating resistant bacteria by identifying combinations
of genes that work together to make bacteria more susceptible to antibiotics.
Lu hopes that both
technologies will lead to new drugs to help fight the growing crisis posed by
drug-resistant bacteria.
"This is a pretty
crucial moment when there are fewer and fewer new antibiotics available, but
more and more antibiotic resistance evolving," he says. "We've been
interested in finding new ways to combat antibiotic resistance, and these
papers offer two different strategies for doing that."
Cutting out resistance
Most antibiotics work
by interfering with crucial functions such as cell division or protein
synthesis. However, some bacteria, including the formidable MRSA
(methicillin-resistant Staphylococcus aureus) and CRE
(carbapenem-resistant Enterobacteriaceae) organisms, have evolved to become
virtually untreatable with existing drugs.
In the new Nature
Biotechnology study, graduate students Robert Citorik and Mark Mimee
worked with Lu to target specific genes that allow bacteria to survive
antibiotic treatment. The CRISPR genome-editing system presented the perfect
strategy to go after those genes.
CRISPR, originally
discovered by biologists studying the bacterial immune system, involves a set
of proteins that bacteria use to defend themselves against bacteriophages
(viruses that infect bacteria). One of these proteins, a DNA-cutting enzyme
called Cas9, binds to short RNA guide strands that target specific sequences,
telling Cas9 where to make its cuts.
Lu and colleagues
decided to turn bacteria's own weapons against them. They designed their RNA
guide strands to target genes for antibiotic resistance, including the enzyme
NDM-1, which allows bacteria to resist a broad range of beta-lactam
antibiotics, including carbapenems. The genes encoding NDM-1 and other
antibiotic resistance factors are usually carried on plasmids -- circular
strands of DNA separate from the bacterial genome -- making it easier for them
to spread through populations.
When the researchers
turned the CRISPR system against NDM-1, they were able to specifically kill
more than 99 percent of NDM-1-carrying bacteria, while antibiotics to which the
bacteria were resistant did not induce any significant killing. They also
successfully targeted another antibiotic resistance gene encoding SHV-18, a
mutation in the bacterial chromosome providing resistance to quinolone
antibiotics, and a virulence factor in enterohemorrhagic E. coli.
In addition, the
researchers showed that the CRISPR system could be used to selectively remove
specific bacteria from diverse bacterial communities based on their genetic
signatures, thus opening up the potential for "microbiome editing"
beyond antimicrobial applications.
To get the CRISPR
components into bacteria, the researchers created two delivery vehicles --
engineered bacteria that carry CRISPR genes on plasmids, and bacteriophage
particles that bind to the bacteria and inject the genes. Both of these
carriers successfully spread the CRISPR genes through the population of
drug-resistant bacteria. Delivery of the CRISPR system into waxworm larvae
infected with a harmful form of E. coli resulted in increased
survival of the larvae.
The researchers are
now testing this approach in mice, and they envision that eventually the
technology could be adapted to deliver the CRISPR components to treat
infections or remove other unwanted bacteria in human patients.
High-speed genetic
screens
Another tool Lu has
developed to fight antibiotic resistance is a technology called CombiGEM. This
system, described in the Proceedings of the National Academy of
Sciences the week of Aug. 11, allows scientists to rapidly and
systematically search for genetic combinations that sensitize bacteria to
different antibiotics.
To test the system, Lu
and his graduate student, Allen Cheng, created a library of 34,000 pairs of
bacterial genes. All of these genes code for transcription factors, which are
proteins that control the expression of other genes. Each gene pair is
contained on a single piece of DNA that also includes a six-base-pair barcode
for each gene. These barcodes allow the researchers to rapidly identify the
genes in each pair without having to sequence the entire strand of DNA.
"You can take
advantage of really high-throughput sequencing technologies that allow you, in
a single shot, to assess millions of genetic combinations simultaneously and
pick out the ones that are successful," Lu says.
The researchers then
delivered the gene pairs into drug-resistant bacteria and treated them with
different antibiotics. For each antibiotic, they identified gene combinations
that enhanced the killing of target bacteria by 10,000- to 1,000,000-fold. The
researchers are now investigating how these genes exert their effects.
"This platform
allows you to discover the combinations that are really interesting, but it
doesn't necessarily tell you why they work well," Lu says. "This is a
high-throughput technology for uncovering genetic combinations that look really
interesting, and then you have to go downstream and figure out the
mechanisms."
Once scientists
understand how these genes influence antibiotic resistance, they could try to
design new drugs that mimic the effects, Lu says. It is also possible that the
genes themselves could be used as a treatment, if researchers can find a safe
and effective way to deliver them.
CombiGEM also enables
the generation of combinations of three or four genes in a more powerful way
than previously existing methods. "We're excited about the application of
CombiGEM to probe complex multifactorial phenotypes, such as stem cell
differentiation, cancer biology, and synthetic circuits," Lu says.
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