NEW ANTIBIOTIC ATTACK S DRUG RESISTANT MICROBES
The multitude of
microbes scientists have found populating the human body have good, bad and
mostly mysterious implications for our health. But when something goes wrong,
we defend ourselves with the undiscriminating brute force of traditional
antibiotics, which wipe out everything at once, regardless of the consequences.
Researchers at Rockefeller University and their collaborators
are working on a smarter antibiotic. And in research to be published October 5
in Nature Biotechnology, the team describes a 'programmable'
antibiotic technique that selectively targets the bad bugs, particularly those
harboring antibiotic resistance genes, while leaving other, more innocent
microbes alone.
"In experiments, we succeeded in instructing a bacterial
enzyme, known as Cas9, to target a particular DNA sequence and cut it up,"
says lead researcher Luciano Marraffini, head of the Laboratory of
Bacteriology. "This selective approach leaves the healthy microbial community
intact, and our experiments suggest that by doing so you can keep resistance in
check and so prevent certain types of secondary infections, eliminating two
serious hazards associated with treatment by classical antibiotics."
The new approach could, for instance, reduce the risk of C.
diff, a severe infection of the colon, caused by the Clostridium
difficile bacterium, that is associated with prolonged courses of
harsh antibiotics and is a growing public health concern.
The Cas9 enzyme is part of a defense system that bacteria use to
protect themselves against viruses. The team coopted this bacterial version of
an immune system, known as a CRISPR (clustered regularly interspaced short
palindromic repeats) system and turned it against some of the microbes. CRISPR
systems contain unique genetic sequences called spacers that correspond to
sequences in viruses. CRISPR-associated enzymes, including Cas9, use these
spacer sequences as guides to identify and destroy viral invaders.
The researchers were able to direct Cas9 at targets of their
choosing by engineering spacer sequences to match bacterial genes then
inserting these sequences into a cell along with the Cas9 gene. The cell's own
machinery then turns on the system. Depending on the location of the target in
a bacterial cell, Cas9 may kill the cell or it may eradicate the target gene.
In some cases, a treatment may prevent a cell from acquiring resistance, they
found.
"We previously showed that if Cas9 is programmed with a target
from a bacterial genome, it will kill the bacteria. Building on that work, we
selected guide sequences that enabled us to selectively kill a particular
strain of microbe from within a mixed population," says first author David
Bikard, a former Rockefeller postdoc who is now at the Pasteur Institute in
Paris.
In initial experiments, Bikard and colleagues targeted a strain
of the common skin and respiratory bacteria Staphylococcus aureus that
is resistant to the antibiotic kanamycin. Treatment by Cas9 programmed to
target a part of the resistance gene killed most of the resistant Staph,
but left behind the kanamycin-susceptible Staph.
Targeted bacterial genocide is only one option. Bacteria share
genes, including those conferring drug resistance, in the form of rings of DNA
known as plasmids. In a second series of experiments, researchers turned Cas9
on tetracycline resistance-harboring plasmids in a strain of the potentially
deadly multidrug resistant bacteriaStaphylococcus aureus (MRSA).
Not only did the resistant cells become sensitive to tetracycline after Cas9
destroyed the plasmids, but the arrival of Cas9 in other Staphcells
acted as an immunization, preventing them from taking on resistance-carrying
plasmids.
And, in a final set of experiments, conducted in collaboration
with Vincent Fischetti's Laboratory of Bacterial Pathogenesis and Immunology,
adjunct faculty member Chad Euler confirmed their test tube results on living
skin, by using Cas9 to selectively kill kanamycin-resistant Staph infecting
the shaved backs of mice.
In spite of the promising results, the delivery system needs
improvement. The researchers used bacteria-infecting viruses to inject the
programmed Cas9 enzymes into the bacterial cells, but these viruses only attack
specific types of cells. Scientists need to devise a less discriminating method
of delivery, before the technology can be used to develop a new class of
antibiotics, Marraffini says.
In addition to its potential as a much-needed new weapon against
drug-resistant microbes, the new system could also be used to advance research
on the complex populations of microbes in the body, about which very little is
known. "There are enormous microbial communities in the human body,"
Marraffini says. "Programmable Cas9 enzymes may make it possible to
analyze these populations by eliminating their members, one by one, and
studying the effects."
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