3D model reveals stubborn TB’s defense

JOHNS HOPKINS (US) — A new 3D model of a tuberculosis protein opens the way to drugs that help crack the problem of super-resistant TB bacteria, the model’s developers say.

The protein in question creates unique bonds within the cell wall of bacteria that cause tuberculosis, making them resistant to currently available drugs, the researchers say.

Now that the protein’s structure is documented, the scientists say, drug designers can use it as a target for medicine to weaken the cell wall and kill the deadly resistant TB bacteria.

A 3D model of the outside surface of the enzyme that helps M. tuberculosis bacteria resist common antibiotics. Blue indicates positively charged atoms; red indicates negatively charged atoms. A peptidoglycan (green) is bound inside the enzyme’s active site. (Credit: Mario A. Bianchet/JHU)


“We’ve known for a while that this protein would make a good drug target, but without a structural model, drug discovery is like blindly choosing random objects to fit into a small hole of unknown shape and size,” says L. Mario Amzel, director of the department of biophysics and biophysical chemistry at the Johns Hopkins University School of Medicine.

“The results of our study have removed the blindfold.”

Amzel’s team used X-ray crystallography to scatter radiation off a specially prepared portion of the enzyme that forms those unique molecular bonds within the cell wall of Mycobacterium tuberculosis. They then used information about the direction and intensity of the radiation scattered to build their 3D model of the arrangement of atoms in the enzyme. A paper on the research was published online today in the journal Structure.

Mario A. Bianchet, assistant professor of neurology at Johns Hopkins and a member of the research team, says the challenge of TB is that most of the standard six-month multiple-drug regimen is needed just to get rid of the roughly 1 percent of bacteria that persist after the first week of a patient’s treatment.

If patients stop the expensive treatment too soon, perhaps because they start to feel better, these super-bacteria are left to take over. The World Health Organization reported in 2011 that about 3.4 percent of new TB patients, and 20 percent of previously treated patients, are infected with a multi-drug resistant form. Of the 8.8 million TB cases estimated worldwide in 2010, approximately 650,000 of them were resistant to multiple drugs.

Breaking the chain

“The ‘persisters’ resist, in part, because of unique bonds within their cell walls,” Bianchet says. “Their cell walls form a thick, three-layered boundary between the bacteria and the outside world, including a middle layer of interlocking molecules, called peptidoglycans, that form a network resembling a chain-link fence,” says Bianchet.

Peptidoglycans are long chains of sugar molecules with short protein branches extending from every other sugar on alternating sides of the chain. Specific enzymes bond the protein branches to each other to create a meshwork.

In most species of bacteria, Amzel says, the majority of the bonds between these branches are created between position 4 on one branch and position 3 on an opposing branch. In M. tuberculosis, however, the majority of bonds are created between position 3 on both branches.

The most common antibiotics interfere with the enzyme that creates the 4-3 bonds, which is enough to destabilize the cell wall and kill most TB bacteria. The bacteria that persist have a particularly high level of 3-3 bonds. These 3-3 bonds are created by the enzyme that Amzel and Bianchet studied, one not specifically targeted by any current drugs.

In addition to showing the structure of the enzyme, the team also showed a peptidoglycan molecule inside the action site where the 3-3 bonds are made, giving drug designers even more details about the way the protein works.

“Beyond fighting TB,” Amzel says, “the structure of this enzyme may help us fight other disease-causing bacteria that have similar enzymes, such as Enterococcus faecium and the spore-forming, drug-resistant Clostridium difficile.”

The study was supported by grants from the National Institutes of Health Office of the Director and the National Institute of Allergy and Infectious Diseases.

Source: Johns Hopkins University