Chemists at Massachusetts Institute of Technology (MIT) have found a way to identify a complex sugar molecule in the cell walls of Mycobacterium tuberculosis.
Tuberculosis is estimated to infect around 10 million people each year, and kills more than 1 million annually. Once established in the lungs, the bacteria’s thick cell wall helps it to fight off the host immune system. Much of that cell wall is made from complex sugar molecules known as glycans, but it’s not well-understood how those glycans help to defend the bacteria. One reason for that is that there hasn’t been an easy way to label them inside cells.
MIT chemists have now overcome that obstacle, demonstrating that they can label a glycan called ManLAM using an organic molecule that reacts with specific sulphur-containing sugars. These sugars are found in only three bacterial species, the most notorious and prevalent of which is Mycobacterium tuberculosis, the microbe that causes TB. The findings have been published in Proceedings of the National Academy of Sciences.
After labelling the glycan, the researchers were able to visualise where it is located within the bacterial cell wall, and to study what happens to it throughout the first few days of tuberculosis infection of host immune cells.
The researchers now hope to use this approach to develop a diagnostic that could detect TB-associated glycans, either in culture or in a urine sample, which could offer a cheaper and faster alternative to existing diagnostics.
“There aren’t a lot of good diagnostic options, and there are some patient populations, including children, who have a hard time giving samples that can be analysed. There’s a lot of impetus to develop very simple, fast tests,” says Laura Kiessling, the Novartis Professor of Chemistry at MIT and the senior author of the study.
Glycans are found on the surfaces of most cells, where they perform critical functions such as mediating communication between cells. In bacteria, glycans help the microbes to enter host cells, and they also appear to communicate with the host immune system, in some cases blocking the immune response.
“Mycobacterium tuberculosis has a really elaborate cell envelope compared to other bacteria, and it’s a rich structure that’s composed of a lot of different glycans,” Smelyansky says. “Something that’s often underappreciated is the fact that these glycans can also interact with our host cells. When our immune cells recognize these glycans, instead of sending out a danger signal, it can send the opposite message, that there’s no danger.”
Glycans are notoriously difficult to tag with any kind of probe, because unlike proteins or DNA, they don’t have distinctive sequences or chemical reactivities that can be targeted. And unlike proteins, they are not genetically encoded, so cells can’t be genetically engineered to produce sugars labelled with fluorescent tags such as green fluorescent protein.
One of the key glycans in M. tuberculosis, known as ManLAM, contains a rare sugar known as MTX, which is unusual in that it has a thioether — a sulphur atom sandwiched between two carbon atoms. This chemical group presented an opportunity to use a small-molecule tag that had been previously developed for labelling methionine, an amino acid that contains a similar group.
The researchers showed that they could use this tag, known as an oxaziridine, to label ManLAM in M. tuberculosis. The researchers linked the oxaziridine to a fluorescent probe and showed that in M. tuberculosis, this tag showed up in the outer layer of the cell wall. When the researchers exposed the label to Mycobacterium smegmatis, a related bacterium that does not cause disease and does not have the sugar MTX, they saw no fluorescent signal.
The image above shows macrophages infected with Mycobacterium tuberculosis fixed and imaged at four hours and 72 h postinfection. (Scale bar, 4 μm.) LAM labelling with oxaziridines persists for up to 72 h. Images are representative of at least two independent experiments.
The researchers now plan to use this approach to study what happens to the bacteria following treatment with different antibiotics, or immune stimulation of the macrophages. It could also be used to study in more detail how the bacterial cell wall is assembled, and how ManLAM helps bacteria get into macrophages and other cells.
There is currently a diagnostic in development that uses antibodies to detect ManLAM in a urine sample. However, this test only works well in patients with very active cases of TB, especially people who are immunosuppressed because of HIV or other conditions. Using their small-molecule sensor instead of antibodies, the MIT team hopes to develop a more sensitive test that could detect ManLAM in the urine even when only small quantities are present.
The research was funded by the National Institute of Allergy and Infectious Disease, the National Institutes of Health, the National Science Foundation, and the Croucher Fellowship.
- Smelyansky SR, Ma CW, Marando VM, et al. Exploiting thioether reactivity to label mycobacterial glycans. Proc Natl Acad Sci U S A. 2025;122(19):e2422185122. doi:10.1073/pnas.2422185122