CAMBRIDGE, MA -- Tuberculosis, the world's deadliest infectious disease, 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 sulfur-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.
After labeling the glycan, the researchers were able to visualize 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. Chest X-rays and molecular diagnostics are very accurate but are not always available in developing nations where TB rates are high. In those countries, TB is often diagnosed by culturing microbes from a sputum sample, but that test has a high false negative rate, and it can be difficult for some patients, especially children, to provide a sputum sample. This test also requires many weeks for the bacteria to grow, delaying diagnosis.
"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 analyzed. 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.
MIT graduate student Stephanie Smelyansky is the lead author of the paper, which appears this week in the Proceedings of the National Academy of Sciences. Other authors include Chi-Wang Ma, an MIT postdoc; Victoria Marando PhD '23; Gregory Babunovic, a postdoc at the Harvard T.H. Chan School of Public Health; So Young Lee, an MIT graduate student; and Bryan Bryson, an associate professor of biological engineering at MIT.
Labeling glycans
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 labeled 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 sulfur atom sandwiched between two carbon atoms. This chemical group presented an opportunity to use a small-molecule tag that had been previously developed for labeling 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.
"This is the first approach that really selectively allows us to visualize one glycan in particular," Smelyansky says.
Better diagnostics
The researchers also showed that after labeling ManLAM in M. tuberculosis cells, they could track the cells as they infected immune cells called macrophages. Some tuberculosis researchers had hypothesized that the bacterial cells shed ManLAM once inside a host cell, and that those free glycans then interact with the host immune system. However, the MIT team found that the glycan appears to remain in the bacterial cell walls for at least the first few days of infection.
"The bacteria still have their cell walls attached to them. So it may be that some glycan is being released, but the majority of it is retained on the bacterial cell surface, which has never been shown before," Smelyansky says.
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.
"Having a handle to follow the bacteria is really valuable, and it will allow you to visualize processes, both in cells and in animal models, that were previously invisible," Kiessling says.
She also hopes to use this approach to create new diagnostics for tuberculosis. 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.
