)
We have to find ways to remove the bacteria at a faster rate and also improve lung function.German version/Deutsche Version
A tiny droplet
This moment happens somewhere at the end of the lung tree, which branches out ever more finely over a length of 700 meters in an adult to finally end in around 300 million alveoli, which inflate and collapse like tiny balloons when we breathe. The wafer-thin wall of each alveolus is surrounded by very fine blood vessels. Oxygen diffuses through this wall into the blood, which in turn releases carbon dioxide. In a healthy adult lung, the combined breathing surface of all the alveoli is almost as large as half of a tennis court. Tuberculosis is transmitted by spit droplets containing bacteria. The lungs make short work of large droplets: the cilia on the mucous membrane of their upper airways propel them back into the throat at more than 1000 beats per minute, where they are swallowed or coughed up. However, it cannot resist the tiny droplets, which only contain one or two bacteria, so easily. They penetrate into the alveoli, where the contact of a single M. tuberculosis with a single host cell can lead to an infection, unless a macrophage of the immune system kills it in time. A certain degree of protection is probably also provided by the detergent-like surfactant that the alveoli produce in order to reduce their surface tension and thus enable breathing at normal air pressure. This assumption cannot be easily tested on living organisms because surfactant is essential for life.
A soft spot for biophysics
Vivek Thacker knew little of all this when he left his parents’ home in the megacity of Mumbai at the age of less than 18 to study in the tranquil city of Cambridge in England on a Commonwealth Trust scholarship. Although tuberculosis is particularly common in India, it was of little scientific interest to him. Physics seemed to be the ideal subject for him to realize his childhood dream of becoming a basic researcher. However, his first year at Cambridge also brought him closer to biology. The Natural Sciences Tripos course there gave him a sound knowledge of all three natural sciences. “I sensed the wealth of exciting ideas and concepts that biology has to offer.” Nevertheless, he continued to feel drawn to quantitative science and specialized in physics. He spent a year as an exchange student at the Massachusetts Institute of Technology in Cambridge, USA. There he realized that neither particle physics nor cosmology were his thing. “I was good enough to understand the theory, but I would never have been able to contribute original new results to these fields.” Biophysics, on the other hand, appealed to him and its unanswered questions ignited his thirst for research. After completing his master’s degree, he therefore applied for admission to the doctoral program at the Physics Department at the University of Cambridge – with success, so that he was able to start his doctoral thesis there in October 2010 under the aegis of Prof. Ulrich Keyser, in which he dealt with the physical behavior of macromolecules in nanopores and the self-regulated folding of DNA into organic nanopores. In the long run, however, “the toy-like aspects of this work” frustrated him a bit, recalls Thacker. Soon after completing his doctorate, he decided to do “something more biological”. Partly because the use of nanopores was beginning to drive the development of ultra-fast sequencing technologies at the time, Thacker thought he had a good chance of being hired as a postdoctoral researcher in laboratories where new single-cell sequencing methods were being used. However, this was a fallacy. As a physicist with little experience in cell biology, his applications were usually rejected. “Hence, I extended my search to microbiology.” Vivek Thacker found a position with Prof. John McKinney at the Global Health Institute of the École Polytechnique Fédérale in Lausanne. “He was open-minded enough to consider my scientific background for his laboratory and I am grateful to him for this.”
A chip to simulate TB infection
McKinney’s open-mindedness was also due to the fact that he could make good use of a physicist with an interest in biology like Vivek Thacker. One of his main areas of research is devoted to the astonishing tenacity with which some bacteria defy the immune system and antibiotic therapy. He is investigating the reasons for this using M. tuberculosis as a model system. Shortly before he hired Thacker, researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University had succeeded in producing a biochip on which the events at the interface between the pulmonary alveoli and the capillaries surrounding them could be simulated. As a biophysicist, Thacker was the ideal postdoctoral fellow to integrate the lung-on-a-chip (LoC) technology into McKinney’s group. “We were the first lab that was able to use the chip to study tuberculosis.” Doing this under reproducible conditions at biosafety level 3 is a major achievement. Together with researchers from the start-up Emulate, which had since spun out of the Wyss Institute, Thacker adapted their technology platform in such a way that he was able to reproduce the interaction of tuberculosis bacteria with their host cells during respiration in a spatial and temporal resolution that he would never have been able to achieve in animal experiments.
A soft spot for biophysics
Vivek Thacker knew little of all this when he left his parents’ home in the megacity of Mumbai at the age of less than 18 to study in the tranquil city of Cambridge in England on a Commonwealth Trust scholarship. Although tuberculosis is particularly common in India, it was of little scientific interest to him. Physics seemed to be the ideal subject for him to realize his childhood dream of becoming a basic researcher. However, his first year at Cambridge also brought him closer to biology. The Natural Sciences Tripos course there gave him a sound knowledge of all three natural sciences. “I sensed the wealth of exciting ideas and concepts that biology has to offer.” Nevertheless, he continued to feel drawn to quantitative science and specialized in physics. He spent a year as an exchange student at the Massachusetts Institute of Technology in Cambridge, USA. There he realized that neither particle physics nor cosmology were his thing. “I was good enough to understand the theory, but I would never have been able to contribute original new results to these fields.” Biophysics, on the other hand, appealed to him and its unanswered questions ignited his thirst for research. After completing his master’s degree, he therefore applied for admission to the doctoral program at the Physics Department at the University of Cambridge – with success, so that he was able to start his doctoral thesis there in October 2010 under the aegis of Prof. Ulrich Keyser, in which he dealt with the physical behavior of macromolecules in nanopores and the self-regulated folding of DNA into organic nanopores. In the long run, however, “the toy-like aspects of this work” frustrated him a bit, recalls Thacker. Soon after completing his doctorate, he decided to do “something more biological”. Partly because the use of nanopores was beginning to drive the development of ultra-fast sequencing technologies at the time, Thacker thought he had a good chance of being hired as a postdoctoral researcher in laboratories where new single-cell sequencing methods were being used. However, this was a fallacy. As a physicist with little experience in cell biology, his applications were usually rejected. “Hence, I extended my search to microbiology.” Vivek Thacker found a position with Prof. John McKinney at the Global Health Institute of the École Polytechnique Fédérale in Lausanne. “He was open-minded enough to consider my scientific background for his laboratory and I am grateful to him for this.”
Two remarkable insights
An LoC consists of a tiny box with a central and two side chambers. A 40-micrometer thin, porous membrane stretches across the middle chamber, which is densely coated with alveolar epithelial cells at the top and endothelial cells at the bottom. A vacuum pump attached to the side chambers stretches and relaxes the membrane like the skin of a pulmonary alveolus. Air flows above the membrane and a blood-like fluid below it. Thacker then added some macrophages to the air side of the chip, which only patrol there in small numbers also under natural conditions. “Introducing M. tuberculosis via the air stream, corresponding to the natural situation, is challenging for safety reasons.” The infection was therefore introduced artificially via brief contact with a bacteria-containing liquid. Thacker and his team then observed what happens in the LoC system using a microscope pointed at it. Using it to take time-lapse images of living cells, they have already uncovered two remarkable insights. By altering the surfactant production of the alveolar epithelial cells on the chip, they confirmed for the first time the hypothesis of a protective function of surfactant: cells that produced a lot of it usually warded off the growth of TB bacteria, while cells that lacked it (as occurs in smokers, for example) often succumbed to it quickly[i]. On the other hand, they succeeded in documenting in high-resolution photos how TB bacteria can undermine the immune defense in the first moments of infection[ii] .
Strangulation of the nucleus of the immune cell
M. tuberculosis is one of the bacterial species that lead an intracellular lifestyle, as Thacker puts it. “It has a unique ability to hide in host cells and inhabit in tissues permanently.” In the vast majority of cases, TB bacteria persist but do not significantly harm their hosts as a result. The infected cells are usually enclosed by defense cells of the immune system. It is estimated that a significant fraction of the world’s population may have such exposure to M. tuberculosis. However, under certain conditions, a small proportion of (difficult to estimate but <only one in ten) infections eventually develops into the dangerous disease, which gradually destroys the lung tissue and causes those affected to cough it up in bloody shreds. The infectiousness of tuberculosis bacteria is facilitated by their ability to intertwine in dense cords. This has been known for many decades. However, it remained unclear what roles these cords may play in the infection. Their highly-ordered structure fascinated him from the very beginning, says Vivek Thacker. “I wondered why the bacteria put so much energy into building this structure.” One answer: the structure is rigid enough to shackle the nucleus of the invaded cell. Although a single TB bacterium can invade both an epithelial cell and a macrophage, it apparently prefers the latter. While multiplying in it, it turns its own kind into a rope that through compression prevents the immune cell from normal function and producing warning messenger substances. How the structure is held together is not fully understood, but it likely emerges from the stored hydrophobic attraction of the lipids of their outer membranes during assembly. With these findings, Thacker has not only made a significant contribution to explaining the “immune escape” of M. tuberculosis, but also its frequent tolerance to antibiotics. Just as biofilms of other bacterial species in the extracellular space make it more difficult for antibiotics to gain access, so do its cords in the infected cell’s carriage.
Promising prospects in Heidelberg
Despite the successes to date, his lung-on-a-chip has a long road ahead for further development, says Thacker. “We are not yet at the stage of development where we can map the infection process in humans in all its complexity.” One limitation of the LoC model, for example, was that it is flat, but the lungs actually breathe in balloons. “We will need to continue working in tandem with animal models in the immediate future.” The researchers also want to find out what the cords of the TB bacterium do not only to the cell nucleus, but also to the other organelles once they have taken up 30 or 40 percent of the cell volume. All of this has been on Vivek Thacker’s agenda since he became head of his own research group at the Center for Infectiology at Heidelberg University Hospital in October 2023 where he is particularly excited by the opportunities to use advanced imaging to investigate how exactly the TB bacteria are glued together in cords. “If we know this, we can separate them from each other more easily and thus make them more accessible for antibiotics.” There are many TB patients, says Thacker, whose lungs are still severely damaged even after successful antibiotic treatment. “We have to find ways to remove the bacteria at a faster rate and also improve lung function.” He is of course aware that this has been attempted for a long time. “But I am optimistic that our research will help to develop more effective therapies against tuberculosis.” A good reason for the Aventis Foundation to support him on his way to a permanent professorship with its Life Sciences Bridge Award.
Author: Joachim Pietzsch, Wissenswort Photos: © Uwe Dettmar
[1] V.V. Thacker&, N. Dhar, K. Sharma, R. Barrile, K. Karalis, and J.D. McKinney&. A lung- on-chip infection model of early M. tuberculosis infection reveals an essential role for alveolar epithelial cells in controlling bacterial growth. eLife 2020; 9: e59961
[2] R Mishra, M Hannebelle, VP Patil, A Dubois, C Garcia-Mouton, G Kirsch, M Jan, K Sharma, N Guex, J Sordet-Dessimoz, J Perez-Gil, M Prakash, GW Knott, N Dhar, JD McKinney, and VV Thacker. (senior, corresponding author). Mechanopathology of biofilm-like M. tuberculosis cords. Cell 186(23), 5135-5150.e28, 2023.