Battling superbugs: Two new technologies could enable novel strategies for combating drug-resistant bacteria

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.

Engineered proteins stick like glue -- even in water

Shellfish such as mussels and barnacles secrete very sticky proteins that help them cling to rocks or ship hulls, even underwater. Inspired by these natural adhesives, a team of MIT engineers has designed new materials that could be used to repair ships or help heal wounds and surgical incisions.

To create their new waterproof adhesives, the MIT researchers engineered bacteria to produce a hybrid material that incorporates naturally sticky mussel proteins as well as a bacterial protein found in biofilms -- slimy layers formed by bacteria growing on a surface. When combined, these proteins form even stronger underwater adhesives than those secreted by mussels.

This project, described in the Sept. 21 issue of the journal Nature Nanotechnology, represents a new type of approach that can be exploited to synthesize biological materials with multiple components, using bacteria as tiny factories.

"The ultimate goal for us is to set up a platform where we can start building materials that combine multiple different functional domains together and to see if that gives us better materials performance," says Timothy Lu, an associate professor of biological engineering and electrical engineering and computer science (EECS) and the senior author of the paper.

The paper's lead author is Chao Zhong, a former MIT postdoc who is now at ShanghaiTech University. Other authors are graduate student Thomas Gurry, graduate student Allen Cheng, senior Jordan Downey, postdoc Zhengtao Deng, and Collin Stultz, a professor in EECS.

Complex adhesives

The sticky substance that helps mussels attach to underwater surfaces is made of several proteins known as mussel foot proteins. "A lot of underwater organisms need to be able to stick to things, so they make all sorts of different types of adhesives that you might be able to borrow from," Lu says.

Scientists have previously engineered E. coli bacteria to produce individual mussel foot proteins, but these materials do not capture the complexity of the natural adhesives, Lu says. In the new study, the MIT team wanted to engineer bacteria to produce two different foot proteins, combined with bacterial proteins called curli fibers -- fibrous proteins that can clump together and assemble themselves into much larger and more complex meshes.

Lu's team engineered bacteria so they would produce proteins consisting of curli fibers bonded to either mussel foot protein 3 or mussel foot protein 5. After purifying these proteins from the bacteria, the researchers let them incubate and form dense, fibrous meshes. The resulting material has a regular yet flexible structure that binds strongly to both dry and wet surfaces.

"The result is a powerful wet adhesive with independently functioning adsorptive and cohesive moieties," says Herbert Waite, a professor of chemistry and biochemistry at the University of California at Santa Barbara who was not part of the research team. "The work is very creative, rigorous, and thorough."

The researchers tested the adhesives using atomic force microscopy, a technique that probes the surface of a sample with a tiny tip. They found that the adhesives bound strongly to tips made of three different materials -- silica, gold, and polystyrene. Adhesives assembled from equal amounts of mussel foot protein 3 and mussel foot protein 5 formed stronger adhesives than those with a different ratio, or only one of the two proteins on their own.

These adhesives were also stronger than naturally occurring mussel adhesives, and they are the strongest biologically inspired, protein-based underwater adhesives reported to date, the researchers say.

More adhesive strength

Using this technique, the researchers can produce only small amounts of the adhesive, so they are now trying to improve the process and generate larger quantities. They also plan to experiment with adding some of the other mussel foot proteins. "We're trying to figure out if by adding other mussel foot proteins, we can increase the adhesive strength even more and improve the material's robustness," Lu says.

The team also plans to try to create "living glues" consisting of films of bacteria that could sense damage to a surface and then repair it by secreting an adhesive.

The research was funded by the Office of Naval Research, the National Science Foundation, and the National Institutes of Health.

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The above story is based on materials provided by Massachusetts Institute of Technology . The original article was written by Anne Trafton. Note: Materials may be edited for content and length.