Tuberculosis lives and thrives in the lungs. When the bacteria that cause the disease are coughed into the air, they are thrust into a comparatively hostile environment, with drastic changes to their surrounding pH and chemistry. How these bacteria survive their airborne journey is key to their persistence, but very little is known about how they protect themselves as they waft from one host to the next.
Now MIT researchers and their collaborators have discovered a family of genes that becomes essential for survival specifically when the pathogen is exposed to the air, likely protecting the bacterium during its flight.
Many of these genes were previously considered to be nonessential, as they didn’t seem to have any effect on the bacteria’s role in causing disease when injected into a host. The new work suggests that these genes are indeed essential, though for transmission rather than proliferation.
“There is a blind spot that we have toward airborne transmission, in terms of how a pathogen can survive these sudden changes as it circulates in the air,” says Lydia Bourouiba, who is the head of the Fluid Dynamics of Disease Transmission Laboratory, an associate professor of civil and environmental engineering and mechanical engineering, and a core faculty member in the Instiute for Medical Engineering and Science at MIT. “Now we have a sense, through these genes, of what tools tuberculosis uses to protect itself.”
The team’s results, appearing this week in the Proceedings of the National Academy of Sciences, could provide new targets for tuberculosis therapies that simultaneously treat infection and prevent transmission.
“If a drug were to target the product of these same genes, it could effectively treat an individual, and even before that person is cured, it could keep the infection from spreading to others,” says Carl Nathan, chair of the Department of Microbiology and Immunology and R.A. Rees Pritchett Professor of Microbiology at Weill Cornell Medicine.
Nathan and Bourouiba are co-senior authors of the study, which includes MIT co-authors and mentees of Bourouiba in the Fluids and Health Network: co-lead author postdoc Xiaoyi Hu, postdoc Eric Shen, and student mentees Robin Jahn and Luc Geurts. The study also includes collaborators from Weill Cornell Medicine, the University of California at San Diego, Rockefeller University, Hackensack Meridian Health, and the University of Washington.
Pathogen’s perspective
Tuberculosis is a respiratory disease caused by Mycobacterium tuberculosis, a bacterium that most commonly affects the lungs and is transmitted through droplets that an infected individual expels into the air, often through coughing or sneezing. Tuberculosis is the single leading cause of death from infection, except during the major global pandemics caused by viruses.
“In the last 100 years, we have had the 1918 influenza, the 1981 HIV AIDS epidemic, and the 2019 SARS Cov2 pandemic,” Nathan notes. “Each of those viruses has killed an enormous number of people. And as they have settled down, we are left with a ‘permanent pandemic’ of tuberculosis.”
Much of the research on tuberculosis centers on its pathophysiology — the mechanisms by which the bacteria take over and infect a host — as well as ways to diagnose and treat the disease. For their new study, Nathan and Bourouiba focused on transmission of tuberculosis, from the perspective of the bacterium itself, to investigate what defenses it might rely on to help it survive its airborne transmission.
“This is one of the first attempts to look at tuberculosis from the airborne perspective, in terms of what is happening to the organism, at the level of being protected from these sudden changes and very harsh biophysical conditions,” Bourouiba says.
Critical defense
At MIT, Bourouiba studies the physics of fluids and the ways in which droplet dynamics can spread particles and pathogens. She teamed up with Nathan, who studies tuberculosis, and the genes that the bacteria rely on throughout their life cycle.
To get a handle on how tuberculosis can survive in the air, the team aimed to mimic the conditions that the bacterium experiences during transmission. The researchers first looked to develop a fluid that is similar in viscosity and droplet sizes to what a patient would cough or sneeze out into the air. Bourouiba notes that much of the experimental work that has been done on tuberculosis in the past has been based on a liquid solution that scientists use to grow the bacteria. But the team found that this liquid has a chemical composition that is very different from the fluid that tuberculosis patients actually cough and sneeze into the air.
Additionally, Bourouiba notes that fluid commonly sampled from tuberculosis patients is based on sputum that a patient spits out, for instance for a diagnostic test. “The fluid is thick and gooey and it’s what most of the tuberculosis world considers to represent what is happening in the body,” she says. “But it’s extraordinarily inefficient in spreading to others because it’s too sticky to break into inhalable droplets.”
Through Bourouiba’s work with fluid and droplet physics, the team determined the more realistic viscosity and likely size distribution of tuberculosis-carrying microdroplets that would be transmitted through the air. The team also characterized the droplet compositions, based on analyses of patient samples of infected lung tissues. They then created a more realistic fluid, with a composition, viscosity, surface tension and droplet size that is similar to what would be released into the air from exhalations.
Then, the researchers deposited different fluid mixtures onto plates in tiny individual droplets and measured in detail how they evaporate and what internal structure they leave behind. They observed that the new fluid tended to shield the bacteria at the center of the droplet as the droplet evaporated, compared to conventional fluids where bacteria tended to be more exposed to the air. The more realistic fluid was also capable of retaining more water.
Additionally, the team infused each droplet with bacteria containing genes with various knockdowns, to see whether the absence of certain genes would affect the bacteria’s survival as the droplets evaporated.
In this way, the team assessed the activity of over 4,000 tuberculosis genes and discovered a family of several hundred genes that seemed to become important specifically as the bacteria adapted to airborne conditions. Many of these genes are involved in repairing damage to oxidized proteins, such as proteins that have been exposed to air. Other activated genes have to do with destroying damaged proteins that are beyond repair.
“What we turned up was a candidate list that’s very long,” Nathan says. “There are hundreds of genes, some more prominently implicated than others, that may be critically involved in helping tuberculosis survive its transmission phase.”
The team acknowledges the experiments are not a complete analog of the bacteria’s biophysical transmission. In reality, tuberculosis is carried in droplets that fly through the air, evaporating as they go. In order to carry out their genetic analyses, the team had to work with droplets sitting on a plate. Under these constraints, they mimicked the droplet transmission as best they could, by setting the plates in an extremely dry chamber to accelerate the droplets’ evaporation, analogous to what they would experience in flight.
Going forward, the researchers have started experimenting with platforms that allow them to study the droplets in flight, in a range of conditions. They plan to focus on the new family of genes in even more realistic experiments, to confirm whether the genes do indeed shield Mycobacterium tuberculosis as it is transmitted through the air, potentially opening the way to weakening its airborne defenses.
“The idea of waiting to find someone with tuberculosis, then treating and curing them, is a totally inefficient way to stop the pandemic,” Nathan says. “Most people who exhale tuberculosis do not yet have a diagnosis. So we have to interrupt its transmission. And how do you do that, if you don’t know anything about the process itself? We have some ideas now.”
This work was supported, in part, by the National Institutes of Health, the Abby and Howard P. Milstein Program in Chemical Biology and Translational Medicine, and the Potts Memorial Foundation, the National Science Foundation Center for Analysis and Prediction of Pandemic Expansion (APPEX), Inditex, NASA Translational Research Institute for Space Health , and Analog Devices, Inc.
