Cracking tuberculosis’ shield

The rise of drug- and multidrug-resistant strains of Mycobacterium tuberculosis, the bacterium responsible for tuberculosis, has exposed the limits of current treatments and made the search for new strategies more urgent than ever.

A key reason for the bacterium’s resilience lies in its unique cell envelope, a complex outer layer that protects it from antibiotics and the host’s immune system. A key component of this shield is a group of molecules called phosphatidyl-myo-inositol mannosides (PIMs). These molecules help maintain the envelope’s structure, regulate what gets in and out of the cell, and influence how the bacteria interact with the host.

Now, a study published in Nature Communications and involving researchers from ITQB NOVA has revealed insights into how PimE, a protein responsible for the synthesis of PIMs, actually works. The work complements another recently published study involving ITQB NOVA that also focused on the mycobacteria’s cell envelope, highlighting the growing efforts in understanding this vital aspect of tuberculosis pathogenicity.

PIMs are made up of fat molecules linked to one to six sugar units. The enzyme the researchers focused on adds the fifth sugar molecule to the growing PIM structure. While this step is critical for creating a functional cell envelope, scientists had long struggled to understand how exactly PimE works.

Using cryo-electron microscopy (cryo-EM), the researchers captured three-dimensional images of PimE from Mycobacterium abscessus, a bacterium related to the one that causes tuberculosis, Mycobacterium tuberculosis. For the first time, they visualized the enzyme both when it is “naked” and when bound to its products. The images revealed a unique binding pocket that accommodates both the sugar donor, polyprenyl phosphate-mannose (PPM), and the receiver PIM molecule.

By combining lab experiments, tests in live bacteria, and advanced computer simulations, the team was able to identify the precise parts of the enzyme involved in binding the reagents and carrying out the reaction. “Because the substrates and products of PimE’s reaction aren’t commercially available, we had to develop methods to synthesize and purify these molecules ourselves”, explains Helena Santos, who led the Cell Physiology & NMR Lab at ITQB NOVA until 2022, co-author of the study. “The work performed at our lab was instrumental in achieving these results”, she adds.

Although the study used M. abscessus, the findings apply to M. tuberculosis, where PimE plays a similar role, as shown by this study. By revealing how this enzyme functions, the research opens up new possibilities for drug development against tuberculosis, a disease that affects over 10 million people and kills more than 1.3 million each year, as well as other infections.

Researchers Nuno Borges, Rodrigo N. Nobre, Ana M. Esteves and Cristina G. Timóteo from ITQB NOVA are also part of this team. The study results from the collaboration with University of Warwick (UK), Academia Sinica (Taiwan) and Columbia University Irving Medical Center, University of Chicago and University of Massachusetts (USA).