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

结核病是由结核分枝杆菌引起的。该病是所有传染病中的头号杀手，95%以上的死亡发生在低收入和中等收入国家。虽然有疫苗，但效果很差。结核病通常是可以治疗的，尽管治疗时间长且昂贵，但是一些结核病菌株对现有药物具有高度耐药性，感染这些菌株的人的存活率很低。到2030年终止目前的结核病流行是联合国可持续发展目标之一，但如果不能找到新的治疗方法，这一目标将无法实现。由密切相关的牛分枝杆菌和海洋分枝杆菌引起的类似疾病分别在牛（和其他哺乳动物）和鱼类中发现，并可造成农业和渔业损失。这种细菌如此有效的原因之一是它有几种对抗免疫系统的策略。这些包括有一个难以被免疫系统发现的细胞壁，以及能够在杀死大多数其他细菌的免疫系统细胞内生存。它还会导致一种叫做肉芽肿的结构的形成，这种结构是由免疫系统的细胞组成的，在引发疾病或感染其他人之前，它可以在肉芽肿中潜伏多年。了解感染过程和肉芽肿的形成是更好地了解疾病和设计治疗或预防新方法的关键。结核分枝杆菌和几乎所有细菌一样，表达一种叫做“伴侣蛋白”的蛋白质。这是一种大型的复杂蛋白质，可以帮助其他蛋白质形成它们的最终形状，这是它们正常运作所必需的。伴侣蛋白是所有细胞生长和存活所必需的。然而，结核杆菌及其近亲是不寻常的，因为它产生两种不同的伴侣蛋白，这两种伴侣蛋白似乎都没有形成其他细菌伴侣蛋白通常看到的大复合体。其中一个仍然是必要的，但另一个不是。这些蛋白质也承担了额外的作用。它们可以导致体内细胞分泌细胞因子：这些分子可以刺激炎症，帮助免疫系统清除感染，但也参与肉芽肿的形成。因此，科学家们通过删除非必需的伴侣蛋白基因，并使用产生的菌株感染小鼠和豚鼠，来观察这些伴侣蛋白是否在导致肉芽肿形成的过程中起重要作用。研究发现，尽管细菌仍在生长，但它们不再引起肉芽肿的形成，而且通常感染时对免疫系统的刺激也大大减少了。这是一个重要的发现，但不幸的是，结核分枝杆菌很难研究，因为它生长非常缓慢，动物感染研究既缓慢又昂贵。因此，我们决定看看同样的事情是否也适用于近亲（但生长速度更快）的海洋分枝杆菌。为了做到这一点，我们制造了一种缺乏非必需伴侣蛋白的突变菌株，并用它感染斑马鱼的胚胎，在那里它通常会导致肉芽肿形成。事实上，突变生物体不再引起肉芽肿的形成。如果我们把基因放回去，但现在以更高的水平表达，我们发现肉芽肿现在形成了，感染照常发生。现在我们有了这个实验，我们计划更详细地研究这种蛋白质的作用。我们已经知道蛋白质的哪些部分对其伴侣功能很重要，包括它形成大型复合物的能力，我们也有一些关于哪些部分对刺激免疫系统很重要的数据。通过突变这些区域或表达致病分枝杆菌的伴侣蛋白，我们可以测试我们关于蛋白质如何在感染中起作用的想法。这将帮助我们确定这种蛋白质是否可以成为未来新的结核病治疗的潜在靶标，包括它是否可以成为一种新疫苗的良好靶标。

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.