New weapons against multi-drug resistance in tuberculosis

Tuberculosis is an infectious disease caused by a bacterium called Mycobacterium tuberculosis, which commonly infects the lungs. In 2012, the World Health Organization estimated that tuberculosis affected 8.6 million people worldwide, causing death in 1.4 million. However, the fight against the disease is hampered by the fact that treatment requires a long time and that the bacterium often develops muti-drug resistance. Consequently, there is a growing need for alternative medications and drug screening methods, which in turn require a deeper and better understanding of the molecular machinery that the tuberculosis bacterium uses to infect a person’s lung cells. Scientists at EPFL have used a sensitive screening assay to test new compounds that can be used against the bacterium, and have discovered two small molecules that show remarkable promise. Their findings are published in Cell Host & Microbe.

In tuberculosis, Mycobacterium tuberculosis enters and infects the lung’s immune cells, the macrophages, rendering them unable to digest and eradicate the bacterium. The bacterium uses a specialized secretion system to release “virulence proteins" upon infection, which affect the lung’s macrophages.

One of the main virulence proteins that the tuberculosis bacterium uses is called “EsxA”. It helps the bacterium invade the macrophages of the lungs, replicate itself, and break them down, damaging the lung tissue and allowing the bacterium to escape and spread. Targeting the EsxA system offers an effective therapeutic strategy because, instead of aiming to kill the bacterium directly, it would rather help the body’s immune system fight it naturally. If successful, it could minimize the risk of multi-drug resistance, which is the main obstacle in treating tuberculosis.

The team of Stewart Cole at EPFL’s Global Health Institute developed a new drug-screening assay capable of exploring thousands of compounds simultaneously. It allowed the researchers to screen a total of 10,880 different compounds, from a library of a pharmaceutical company working on tuberculosis.

For the assay, the scientists used human lung cells infected with Mycobacterium tuberculosis and then incubated with the candidate compounds. The survival rate of the cells was then quantified using fluorescent staining, selecting the compounds that worked best against EsxA.

Using the screening method, Cole’s team was able to narrow down the compounds from over 10’000 down to only two (BBH7 and BTP15). Both compounds can inhibit the secretion of the virulence protein EsxA even when given at very low doses. In effect, both compounds can reverse the suppressive effects of the tuberculosis bacterium on infected macrophages, which can lead to efficient killing of intracellular bacteria.

This therapeutic approach is different to antibiotics, which act directly on the bacterium, often forcing the multidrug resistance that characterizes tuberculosis. “The novelty of our work is that, rather than trying to kill the bacterium, we’re trying to disarm it so as to prevent tissue damage,” says Stewart Cole. “This approach would be used as part of a larger treatment, possibly combined with conventional drugs.”

Cole’s team is now planning to test different combinations of compounds to see if they can improve the efficiency of BBH7 and BTP15 against the bacterium. In addition, they want to apply their screening method on other bacteria. “The whole screening process we set up is fairly simple and basic, but quite robust,” says lead author Jan Rybniker, who designed the assay. “I am sure that we can use similar screening techniques to target the virulence protein systems of many other multi-drug resistant bacteria.”

This work represents a collaboration between EPFL’s Global Health Institute, the University of Cologne, Vichem Chemie Research Ltd (Hungary) and Semmelweis University (Budapest).

Reference

Rybniker J, Chen JM, Sala C, Hartkoorn RC, Vocat A, Benjak A, Boy-Röttger S, Zhang M, Székely R, Greff Z, Őrfi L, Szabadkai I, Pató J, Kéri G, Cole ST. Anticytolytic Screen Identifies Inhibitors of Mycobacterial Virulence Protein Secretion.Cell Host & Microbe DOI: http://dx.doi.org/10.1016/j.chom.2014.09.008