Phthisis. Scrofula. Consumption. The White Plague.
Tuberculosis has been around long enough to earn many labels. Back in 18th and 19th century Europe it was characterized as “a romantic disease,” given its high prevalence among artists and writers, but recent research has shown that the earliest trace of TB can be found in human remains from nearly 9,000 years ago. Yet, despite its long history and largely successful efforts at wiping the disease out in most Western countries, tuberculosis is still very much a problem in the developing world.
Caused by Mycobacterium tuberculosis, the infectious disease that primarily infects the lungs is responsible for nearly 9 million cases and roughly 2 million fatalities worldwide every year, and continued drug resistance to current antibiotic therapies is fueling the need for new drugs. The drugs that are most commonly used, rifampin and isoniazid, have been waning in effectiveness against the bacterium. As a result, combinatorial therapy has been in place for nearly 50 years, but the bacterium has resisted this as well.
Robert Koch first discovered the bacterium in 1882, and won the Nobel Prize for the discovery in 1905. Eventually, the main challenge in producing antibiotics against this bacterium was also recognized: the organism has an unusually thick, waxy cell wall that is hard to penetrate. Getting any drugs through this barrier, while possible, is tough.
Given TB’s virulence, researchers often use a close relative, Mycobacterium smegmatis, to study it in the lab. The two types of bacteria share many qualities, including the peculiar cell wall, but M. smegmatis has low pathogenicity, which makes it relatively safe. Several HMS scientists, however, have been working with both M. smegmatis and M. tuberculosis to better understand tuberculosis in the hope of developing novel ways of treating the disease.
E. Coli Clues
Current TB research at HMS stems from work conducted in the bacterial model organism Escherichia coli. Throughout his career, Alfred Goldberg, HMS professor of cell biology, has been studying proteases—enzymes that help break proteins down into smaller molecules or amino acids. This work has led to breakthroughs in cancer treatment. In 1987, he discovered an E. coli protease called ClpP, an enzyme that decades later has been shown to be an essential component of the tuberculosis bacterium. Currently, Tatos Akopian and Olga Kandror, HMS instructors of cell biology and members of the Goldberg lab, are exploring the role of ClpP in Mycobacterium tuberculosis. In this case, it so happens that a version of ClpP is conserved in M. tuberculosis.
Specifically, the researchers found that ClpP has two parts, ClpP1 and ClpP2, which form a joint complex. Both of these parts are essential to the normal functioning of the bacterial ClpP complex. Without either one, the complex, and ultimately the bacteria, can't function properly.
"Because the organism needs this complex for survival, ClpP would be a terrific target for the development of new antibiotics," said Kandror. "In addition, humans don’t have the ClpP protease in their cytoplasm, so manipulating the complex shouldn't affect human cells."
As with Goldberg, Jonathan Beckwith, HMS professor of microbiology and immunobiology, also looked to identify qualities in M. tuberculosis that he first observed in E. coli. In this case, a former graduate student initiated a project to see if the tuberculosis bacterium and E. coli use disulfide bonds, molecules that bind proteins together, in similar ways.
“Disulfide bonds are important for folding in some proteins,” explained Beckwith, “particularly in proteins that often determine bacterial virulence.”
Although proteins in both E. coli and M. tuberculosis have disulfide bonds, the bonds are formed differently in each of the organisms. M. tuberculosis uses an enzyme called vitamin K epoxide reductase (VKOR). This enzyme, important for blood coagulation pathways in humans, is the target of the drug warfarin (commercially known as Coumadin). “We found that when we added warfarin to M. tuberculosis, the function of VKOR in the bacterium was inhibited, and so the growth of the bacterium is inhibited as well,” said Beckwith.
Eye on the Target
These results, combined with a readily available drug, make VKOR another desirable target for killing the bacterium. With support from Harvard Catalyst, Beckwith has begun collaborating with Bruce Furie, HMS professor of medicine at Beth Israel Deaconess Medical Center, and with Eric Rubin, professor of immunology and infectious diseases at Harvard School of Public Health.
“We are currently working on developing assays that will screen for inhibitors of VKOR,” said Beckwith. “Bruce is helping make sure that it doesn’t result in blood thinning in humans, and Eric is helping with measuring effectiveness in the TB bacterium.”
Rubin, in turn, is collaborating not only with Beckwith but also with Goldberg. “My lab is working on very early stages of drug discovery,” he said. “We have isolated ClpP complexes as a possible target, but we are also looking into other options.”
One such option is simply to look at known killers of bacteria and replicate their mechanisms. “A good drug target,” said Rubin, “would be one that is hypersensitive—such that when you inhibit the target or manipulate the target in some way, the bacterium simply dies.”
Previous studies have shown that when antibiotics are applied to bacteria, instead of weakening or killing the organism, some of the drugs are simply pumped back out by the bacterium. Using this knowledge, members of Rubin’s lab are working to better understand the nature of this "pump."
“If we know what triggers the pump system, we may also be able to learn how to block it,” said Rubin.
Rubin and his lab members are also studying variation within a given population of M. tuberculosis. “Just like people are different and are either more prone to disease or have better immunity, it seems that the TB bacteria also have genetic differences that protect the population when antibiotics are applied,” said Rubin. Finding compounds that are not affected by the variability in individual bacteria might lead to developing much more effective drugs.
While the goal of many researchers is to find new targets for anti-tuberculosis therapies, the issue of antibiotic resistance is always a problem. "The bacteria will find a way to beat antibiotics, particularly if they are used extensively," said Beckwith, a veteran microbiologist. "Clinicians and public health officials can take steps to reduce such overuse and avoid the further spread of the disease on a community level."
Next week: How clinicians and public health experts are managing the disease on a global scale.