Co-ordinated CHAOS

In the nineteenth century, Charles Darwin laid the groundwork for our current understanding of natural selection. The same rules governing natural selection over millions of years also apply in our current society, albeit on substantially shorter timescales. Our indiscriminate use of antibiotics in agriculture and healthcare has created scary new varieties of bacteria. These bacteria have evolved to be impervious to many types of antibiotics that were initially designed to kill them. With some strains even resistant to what we consider as “last-resort” antibiotics, the implications of this problem are enormous.

Now, researchers from the Chatterjee Lab at the University of Colorado, Boulder have developed a new way to interfere with the emergence of antibiotic resistance in pathogenic bacteria. The approach works by messing with the normal expression of multiple genes. Called CHAOS (for Controlled Hindrance of Adaptation of OrganismS), it overwhelms the inherent ability of bacteria to adapt to environmental changes.

Peter Otoupal, first author of the study published in Communications Biology discovered this ground-breaking method by exploiting a commonly underappreciated aspect of evolution.

If an organism’s genetic information changes as a result of a mutation in its DNA, for instance, and that mutation makes the organism more successful at reproduction than its competitors, then more offspring will carry the “improved” DNA to the next generation. However, predicting which new DNA variants may be beneficial for an organism is not as simple as it may seem.

The benefits of a mutation exist solely relative to its current environment. If the environment changes, then that mutation may not be beneficial anymore. Moreover, to really understand the effects of a mutation in a gene, we need to understand the context of that gene’s interactions with the dozens or hundreds of other genes with which it coexists. Worse yet, you may be dealing with several simultaneous mutations all having occurred at once!

In the case of antibiotic resistance, when we bombard bacteria with deadly antibiotics, we abruptly and drastically alter their environment. Under normal conditions, certain DNA mutations may render bacteria less competitive by making them grow slower. These would be unfavorable in natural selection. But the same mutations in this new, harsher environment may produce sluggish bacteria that are also impervious to these drugs—these would be immediately chosen by natural selection.

All these reasons make it pretty hard to predict the benefits of any given DNA alteration. But bacteria are really good at playing around with their DNA—over the course of generations, they make many small changes, some of which have surprisingly large effects.

One of the reasons that small DNA changes can have a large effect on how fit bacteria are is due to a phenomenon called “epistasis.” Certain genes are interconnected in a special way, such that changing them simultaneously has a greater effect on the overall fitness than what you would expect from both individual changes by themselves.

Say a mutation in one gene changes your eye color from green to blue, and a mutation in a different gene changes your eyebrow shape from flat to arched. But if you make both mutations simultaneously, suddenly your face is upside down. That suggests the two genes are in an “epistatic relationship”.

During some preliminary experiments in which bacteria were exposed to different perturbations, the researchers were inspired by an early observation: that bacteria not only evolve by mutating their genes, but also by changing how much their genes are “turned on,” or expressed.

“Instead of nature doing it, how about we do it ourselves?” said Chatterjee.  “How about if we synthetically introduce gene expression perturbations?”

Chatterjee hypothesized that they could use epistasis for their own devices. What if they made small changes that exacerbated each other in ways that were detrimental to the bacteria? Her lab set out to harness the power of epistasis to force bacterial evolution unproductively, thereby serving our species’ interests instead of bacteria’s.

To perturb the normal expression levels of target genes in the bacterium Escherichia coli, Otoupal used the DNA editing technology CRISPR in a non-conventional manner. The DNA editing precision of CRISPR stems from its ability to guide DNA-cutting molecules to certain DNA sequence within the genome. Once there, it makes a cut which then produces the desired DNA changes as designed by researchers.

Otoupal, however, used a modified deactivated version of one CRISPR’s components, called dCas9, that is not capable of producing any cuts, but it still retains its ability to be recruited to whatever DNA sequence you need it to go to. To lower gene expression levels, he could recruit dCas9 to the middle of a target gene to serve as a roadblock to the cellular machinery responsible for activating such gene.

To do the opposite experiment, or increase gene expression levels, he fused dCas9 with a gene-activator protein. This recruited gene expression machinery to the start of the gene and made expression of that gene go up.

Using these dCas9 variants, Otoupal then tested the idea that he could restrain bacterial evolution using epistasis. He decided to perturb two different groups of genes. First, he decreased the expression of key genes that are essential for bacterial metabolism. He suspected this would hamper bacterial growth. In another, parallel experiment, Otoupal increased the expression of genes whose job is to help bacteria to adapt faster.

As expected, when these two groups of genes were perturbed, the bacteria’s fitness decreased and increased, respectively. What was surprising, however, was that when Otoupal started combining perturbations–disturbing the expression of multiple genes simultaneously–fitness dropped sharply to levels that could only be explained by epistatic interactions. Combining the two types of mutations—slowing the bacteria’s ability to metabolize while also forcing them to adapt quickly—impaired the bacteria more than either of the two types of changes by themselves.

Otoupal and Chatterjee showed that if you prevent bacteria from responding to environmental changes at the speed that they are best equipped to react, they get sick. He could do this by making several small changes—but these small changes make a bigger impact than you might expect because they all exacerbate each other.

Otoupal and colleagues also successfully tested their CHAOS approach in clinical isolates of E. coli resistant to carbapenem, a drug considered a last-resort resource for certain infections.

“The other thing that we do in our lab is work towards producing new therapies. For us it is very important that we can translate an idea into the clinic.” added Chatterjee. Perhaps in the not-so-distant future, we may be able to get CRISPR pills in the hospital or in your local pharmacy to fight bacterial infections.

By Daniel Ramirez