A zebrafish model to study the role of chaperonins in Mycobacterial infection

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The bacterium Mycobacterium tuberculosis causes the disease TB. This disease is the number one killer among all infectious diseases, with more than 95% of deaths occurring in lower and middle income countries. Although there is a vaccine, its effectiveness is poor. TB is usually treatable, though the treatment is lengthy and expensive, but some strains of TB are highly resistant to current drugs and survival rates in people infected with these strains are low. Ending the current TB epidemic by 2030 is one of the UN's Sustainable Development Goals, but this target will be missed if new treatments cannot be found. Similar diseases caused by the closely related organisms M. bovis and M. marinum are found in cattle (and other mammals) and fish respectively, and can cause losses in agriculture and fish farming. One of the reasons the bacterium is so effective is that it has several strategies to combat the immune system. These include having a cell wall which is hard for the immune system to spot, and being able to survive inside immune system cells that kill most other bacteria. It also causes the formation of structures called granulomas, made up from cells of the immune system, in which it can shelter for many years before emerging to cause disease or to infect other people. Understanding the process of infection and the formation of granulomas is key to better understanding the disease and devising new ways to treat or prevent it. M. tuberculosis, like nearly all bacteria, expresses proteins called "chaperonins". These are large complex proteins that help other proteins to achieve their final shape, which is required for them to function properly. Chaperonins are essential for all cells to grow and survive. The TB bacterium and its relatives are unusual however in that it makes two different kinds of chaperonin, neither of which appears to form the large complex that is typically seen with chaperonins from other bacteria. One of these is still essential, but the other is not. These proteins have taken on an additional role as well. They can cause cells in the body to secrete cytokines: these are molecules that can stimulate inflammation and help the immune system clear infections, but are also involved in granuloma formation. Scientists therefore looked to see whether these chaperonins might be important in causing granulomas to form, by deleting the gene for the non-essential chaperonin and using the resultant strains to infect mice and guinea pigs. It was found that although the bacteria still grew, they no longer caused granuloma formation, and that stimulation of the immune system normally seen with infection was much reduced. This was an important finding, but unfortunately M. tuberculosis is hard to study as it grows very slowly and the animal infection studies are slow and expensive. We therefore decided to see whether the same thing was true in the closely related (but faster growing) M. marinum. To do this we made a mutant strain that lacked the non-essential chaperonin and used it to infect embryos of zebrafish, where it normally causes granuloma formation. Indeed, the mutant organism no longer caused granuloma formation. If we put the gene back in, but now expressed at a higher level, we found that granulomas were now formed and infection happened as normal. Now that we have this assay, we plan to study the role of this protein in more detail. We already know which parts of the protein are important for its chaperone function, including its ability to form a large complex, and we have some data on which parts are important in stimulating the immune system. By mutating these regions or by expressing chaperonins from disease-causing Mycobacteria we can test our ideas about how the protein works in infection.This will help us determine whether the protein could be a potential target for new TB treatments in the future, including whether it would make a good target for a new vaccine.