War on antibiotic resistance

A compound that acts on a protein with a key role may prevent the development of antibiotic resistance 

By Gary Stix

Appeared in Issue 24 of Scientific-American Israel, August September 2006

Floyd A. Romesberg received his PhD from Cornell University in 1994. His research topic was lithium salts of di-alkyl amides. Chemists involved in synthesis usually use these compounds to displace protons from various substances. Romsberg, who was the son of a chemist, spent his days observing how these substances reacted and what their reaction rate was. "It's not that the project was that interesting," says Romesberg, "in fact it was quite boring."
Immediately after completing his degree, he changed direction and went straight to a post-doctorate in a different field at the University of California, Berkeley. There he extracted from the mouth of his future supervisor - the renowned biochemist Peter G. Schultz - a promise that he could say goodbye to physical chemistry and devote himself to immunology. Rumsberg does not regret initially deciding to investigate something so basic and boring, and says that if he had turned to biology immediately, it would probably have been a wrong step. The complexity of biology means that too many research student projects end up with only superficial results. "I was lucky and at Cornell I could work on a small system that could be fully characterized. It was something solvable that allowed me to deal with fundamental questions," he admits. "I always tend to reduce things to the basic questions at the chemical molecular level and I think this tendency has served me faithfully."
During his work on antibodies in Schulz's laboratory, Rumsberg's curiosity about the molecular processes underlying evolution was awakened. In his own laboratory at the Scripps Research Institute in La Hoya, California, there are today 19 researchers (12 research students and 7 postdoctoral researchers). They are organized into separate teams each tackling a different question related to evolution. One group uses powerful lasers to study the evolution of antibodies. Another group hopes to determine how DNA will function in the presence of an unnatural nucleotide, or an artificial letter in the DNA alphabet, that researchers add to the genetic code. The research with the most immediate practical importance tries to check how evolution sometimes goes into high gear. This is a basic process that allows bacteria to mutate quickly. Information that makes it possible to block the process may provide an innovative approach to overcoming bacterial resistance to antibiotics.
distress signal
Genetic mutations usually originate from errors that occur during cell replication. Mutations are usually harmful, so during evolution cells have evolved so that as few mutations as possible occur. The cells are equipped with built-in proofreading machines and repair equipment that ensures minimization of errors when copying the DNA. And yet it sometimes happens that the cell itself initiates a process of genetic mutation - a process that is essentially accelerated evolution.
Since 1970, scientists have known a process that occurs in bacteria, which they call the "SOS reaction". This process actually utilizes the mutation as a way of self-defense. When bacteria are in a state of extreme stress, they first try to take various measures to repair the damage. They then activate genes whose protein products create a flood of mutations, mutations that occur at a rate 10,000 times greater than that occurring during normal cell replication. In fact, the cell undergoes a rapid identity change. The bacterium Escherichia coli (Escherichia coli), for example, reacts to the damage caused to it by the antibiotic ciprofloxacin (known for short Cipro) and by other types of antibiotics by sending a distress signal, SOS. Some of the mutations prevent ciprofloxacin from binding to its target, a protein called gyrase, which is necessary for DNA replication. If the bacterium did not protect the gyrase in this way, the antibiotic would bind to it, inhibiting the normal replication of the bacterial cell and causing DNA breaks that would lead to its death.
When he read about the SOS response, Romesberg hypothesized that turning off the system, that is, stopping the super-evolution, could prevent the cascade of mutations that enable the development of antibiotic resistance in the E. coli bacterium. In experiments published in June 2005 in the online journal PLoS Biology, Rumsberg and his colleagues, Ryan T. Sears, Judy K. Chin and their co-workers at the University of Wisconsin-Madison, found that ciprofloxacin induced an SOS response and excess mutations in E. coli through signaling that caused the truncation of a protein called LexA. This protein suppresses the SOS response, and when it is cut, it allows three enzymes, DNA polymerases, to start producing mutations, and thus resistance develops rapidly.
The researchers created a strain of E. coli in which LexA could not be cut and found that the SOS response did not materialize. Mice infected with this strain of E. coli and treated with ciprofloxacin did not develop antibiotic resistance. The group of researchers obtained similar results with another type of antibiotic, rifampicin. They are now testing whether blocking the truncation of LexA in E. coli may prevent resistance to other types of antibiotics. They are also testing whether LexA may undermine the effectiveness of drug treatment in other bacteria. But ciprofloxacin is an important drug on its own. Some strains of the bacterium that causes epidemic dysentery, Shigella dysenteriae, have developed resistance to all types of antibiotics, except for ciprofloxacin. Dysentery can cause the death of tens of thousands of people in developing countries.
When these results first began to be received in his laboratory in 2002, Romesberg immediately saw the possibility of developing a drug, a small molecule given orally along with the antibiotic. The drug could act like a switch that stops LexA cutting. Romsberg and two partners did not encounter many difficulties in raising 15 million dollars to found a company called Achaogen, ("Achao" means "against chaos", the "Gene" was added by one of the partners, Ned David, because companies that include this suffix in the oil tend to prosper).
Venture capital investors have become cautious about investing in start-up companies in their early stages. But they were open about an innovative approach to antibiotic resistance. So far, most of the solutions to this problem have been related to new types of antibiotics and the company is indeed testing several courses of action today. But, as always, the resourcefulness of a bacterium can turn a lot of hard work into an exercise in wasted time. Stuart B. Levy, an antibiotic resistance expert at Taft University, commented that Romesberg's work provided new insight. But he added that its impact could be limited. "We are always looking for new approaches, especially those that work against resistance," he says. "These findings suggest such an approach, but it focuses on chromosome mutations as a mechanism for creating resistance, a mechanism that is limited in scope." Levy adds that other types of resistance may emerge and directly attack the antibiotics. The bacteria can gain resistance by transferring genes from one strain of bacteria to another or even by transferring genes within the same strain.
In any case, Romesberg and his researchers decided to focus on substances of the fluoroquinolone type, since resistance to these substances develops only through mutations of the chromosomes (the SOS reaction) and because, according to the predictions, these substances are supposed to be the most sold type of antibiotics by the year 2011.
An unexpected, and also unwanted, side reaction to the article published in PLoS Biology was received when the intelligent design community embraced the results of the experiments as confirmation of their unacceptable worldview. The flood of mutations in Romesberg's experiments is not random, the members of the community claimed, but rather the result of a calculated design by the bacterium itself: "Life controls its destiny. "Living beings are not passive participants in the interplay between random events and environmental pressures," wrote the author with the pen name Mike Gann on the website idthink.net. And he says: "The fact that evolution is subject to some kind of self-control is nothing more than a piece of the teleological mosaic (planning of nature). But this is a significant piece, from the point of view that the ability to adapt, at least to these two types of antibiotics, is under control." Romsberg refrains from participating in this debate, but he dismisses any basis for these claims. The SOS response, he says, "says nothing about religion. It talks about the degree of success and creativity of evolution, but there is no magic in it, it is a completely mechanical process."
As he chose a seemingly routine topic for his doctoral research project, Rosmberg says he decided to investigate antibiotic resistance because the steps on the way to developing a drug were clear and relatively simple: "If we win, we can deal with the next level of complexity in a better way." The next level is cancer, a disease in which drug resistance is also a worrying problem. Both his research team and the company he founded plan to create a complementary treatment to chemotherapy that can prevent mutations that lead to resistance to cancer drugs.
Romesberg's gradual path, which began with the displacement of protons using basic salts and progressed to the challenging cancer, may take longer than a direct search for drugs. However, measured scientific progress may lead to success with greater certainty.
 Retaining the power of the antibiotic
Flash mutations in Escherichia coli bacteria may thwart the effectiveness of the antibiotic ciprofloxacin (Cipro), which is becoming more and more accepted by doctors.

How the cipro works
Cipro antibiotics are harmful to bacteria, usually by binding to an enzyme called gyrase and preventing it from working properly.

How does resistance arise?
Resistance begins when the A. coli bacterium reacts by producing single-stranded DNA. Molecules of another protein, RecA, arrange themselves in a chain and adhere to the single-stranded DNA. RecA encourages the cutting of the control protein LexA. This change releases a whole set of genes that were previously blocked, and these genes induce mutations in other sites. Some of the mutations inhibit the binding of Cipro to gyrase, thus preventing the drug from working.

Possible solution
Drugs that bind to LexA and prevent its truncation, such as the hypothetical compound X marked here, will be able to pause the sequence of events described above and thus overcome resistance and restore the antibiotic's effectiveness.

More on:

Revenge of the Microbes: How Bacterial Resistance Is Undermining the Antibiotic Miracle. Abigail A. Salyers and Dixie D. Whitt. American Society for Microbiology Press, 2005
Inhibition of Mutation and Combating the Evolution of Antibiotic Resistance. Ryan T. Cirz et al. in PLoS Biology, Vol. 3, no. 6, pages 1024–1033; June 2005
Induction and Inhibition of Ciprofloxacin Resistance-Conferring Mutations in Hypermutator Bacteria. Ryan T. Cirz and Floyd E. Romesberg in Antimicrobial Agents and Chemotherapy, Vol. 50, no. 1, pages 220–225; January 2006