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

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.

This project will characterise the role of the chaperonins (Cpn60) in Mycobacterial infection, using a zebrafish embryo infection model. The Mycobacterial chaperonins lack the canonical double ring structure that is required for activity in most chaperonins, but show a phosphorylation-dependent switch into this form. Earlier work showed loss of the non-essential cpn60.1 gene from M. tuberculosis abolishes granuloma formation in animal infection models. The purified protein, or a 30 residue peptide derived from it, activates human monocytes. We wished to establish a quick, robust system for analysing the role of Cpn60.1 in more detail and have now shown that loss of Cpn60.1 from M. marinum leads to loss of infection in zebrafish embryos which can be complemented with the wild-type gene. We have mapped the two serines involved in the phosphorylation-mediated switch to the oligomeric state. If both these sites are mutated to phosphomimetic aspartic acid residues, Cpn60.1 now complements loss of essential GroEL in E. coli. This gives us the opportunity to dissect the role of this protein in detail. We will mutate the protein to abolish its chaperonin function, and also express it in different oligomeric states. We will then assay the impact of these changes on the bacterium, looking specifically for evidence of altered structure of cell wall components (known to be key in pathogenesis); altered protein secretion; or alteration in expression of other genes. We will use complementation analysis in the fish embryo model to assess the roles of chaperonin activity, the switch to oligomerisation, and the stimulatory peptide, in pathogenesis. This will enable us to determine whether it is chaperone activity or modulation of the immune response, or a combination of the two, that determines its role in infection. By complementing the deletion mutant with the chaperonins from other pathogenic Mycobacteia we will examine their possible role in determining host range.

Who might benefit from this research?

This project addresses several of the responsive mode priorities discussed in the BBSRC strategic plan, in particular animal health, combatting antimicrobial resistance, and 3Rs in animal research. The relevant stakeholders in the short term will include academics in related research fields including those with an interest in global health issues, industrial partners interested in developing novel anti-Mycobacterial treatments, regulators engaged with the issue of "gain of function" experiments, and members of the general public with an interest in issues around microbiology and infection.

How might they benefit?

Academics may benefit from the discoveries that we make during the course of the project, to the extent that they are relevant to their own research interests. We see the project as bringing together two disparate disciplines that only slightly overlap: chaperone biology, and Mycobacterial pathogenesis. Demonstrating links between the two will be of benefit to both but more importantly should stimulate others in both fields to consider the importance of chaperones in pathogenesis more widely. We will use standard routes of publication in high impact refereed journals, seminars, and presentations at national and international conferences to raise awareness of our research. We have recently posted a paper on BioRxiv before publication (https://www.biorxiv.org/content/early/2017/03/15/117093) and we were impressed by the rapid exposure that this produced, so this route will also be used.

Industrial partners may benefit from the development of Cpn60.1 (or other related findings from this project) as novel targets or potential vaccine candidates, and we have good links with several companies, particularly with GSK through the other TB groups in the IMI who are part of the Marie Curie-funded CooperaTB project. Our business development partners in the College of Life and Environmental Sciences are aware of the project and are tasked with actively engaging with industrial partners to build collaborative partnerships to develop this kind of research, with University of Birmingham Enterprise Ltd providing expertise in IP and enterprise acceleration.

Part of the proposal involves developing potential gain of function changes in a close relative of human and animal pathogens. These types of experiments, particularly where they involve GM, fall under the remit of HSE in the UK, who are constantly discussing how best to regulate and monitor them. PL has been involved in these discussions through his membership of the Scientific Advisory Committee on Gene Manipulation, and this particular project can be used as an exemplar of how to assess risk vs benefit and how to regulate accordingly.

We have found in the IMI through engaging in activities such as SoapBox Science and Cafe Scientifique that there is significant and well-informed interest among members of the public in research, particularly where they can see clear or potential health benefits. We are committed to sharing both the excitement and frustrations of research with the public, to engage with them with the work we do and to raise the profile of basic research in general. Our university also provides a training course on using social media to increase impact of research across different sectors and Dr Kumar will attend this course and be responsible for highlighting the research as effectively as possible on social media.

More details on the ways we will seek to maximise the impact of our research are discussed in the "Pathways to Impact" statement.