Defining the molecular basis of host-pathogen interaction in bovine TB

牛结核病(BTB)的可持续控制是英国农业面临的主要挑战。BTB是由一种名为牛分枝杆菌的细菌引起的，这种细菌可以感染一系列野生和家养动物，也可以感染人类。因此，控制牛结核病对公共卫生以及动物健康和福利都很重要。然而，英国目前的结核病控制措施并不奏效。新感染牛结核病的牛群数量每九年翻一番；2013年，英格兰进行了超过620万头牛结核病检测，导致超过2.6万头牛被宰杀，纳税人损失了近1亿GB。迫切需要新的方法来阻止感染的传播。牛分枝杆菌(MBV)是人类结核病病原体结核分枝杆菌(Mtb)的近亲。虽然结核分枝杆菌用来引起人类疾病的一些机制有很好的特点，但人们对MBV用来导致牛疾病的策略知之甚少。Mtb和MBV基因组DNA测序表明，它们在遗传上有99.9%以上的同源性。虽然MBV和结核分枝杆菌都可以感染人类，但MBV很少在人与人之间传播，而是通过受污染的乳制品传播。另一方面，结核分枝杆菌只能在牛身上引起一种轻微的疾病。MBV和Mtb对宿主的不同偏好表明，这两种分枝杆菌已经进化出不同的策略来在各自的宿主上感染和致病。通过先前的研究和我们自己的工作，已经在Mtb和MBV之间确定了一系列离散的分子变异；其中包括这些细菌之间蛋白质(称为MPB70和MPB83)和脂质(如‘Sulfolipid’)的表达差异。然而，这些蛋白质和脂质在MBV对牛的毒力中的作用尚未确定；在这项研究中，我们将探索这样的假设，即这些分子差异是MBV导致牛疾病的关键原因之一。我们将通过首先产生基因工程MBV毒株来检验我们的假设，这些MBV毒株在这些脂类和蛋白质的表达方面与Mtb相似。然后，这些毒株将与它们的亲本Mtb和MBV毒株一起在细胞感染模型中进行测试。这些模型将侧重于实验室培养的牛巨噬细胞，牛免疫系统的细胞是第一个遇到并吞噬入侵的MBV细胞的细胞。我们有初步数据表明，这些细胞对感染MBV和Mtb的反应不同，并将使用‘类Mtb’MBV基因工程毒株来阐明MBV用来建立牛感染的机制。为了实现这一目标，我们将开发一系列工具和新方法，例如使用荧光标记的MBV和基因敲除技术来研究受感染的巨噬细胞。这些工具将帮助我们监控巨噬细胞机制的关键细胞内组件，并为更广泛的牛免疫学研究社区提供工具。我们的发现将为MBV用来引起牛疾病的分子成分提供新的线索，这些信息将有助于长期开发针对这种对英国农业具有破坏性的疾病的疫苗和诊断方法。

The sustainable control of Bovine tuberculosis (bTB) is a major challenge for UK agriculture. bTB is caused by a bacterium, Mycobacterium bovis, that can infect a range of wild and domesticated animals, as well as man. Control of bTB is therefore important for public health as well as animal health and welfare. However, current bTB control measures have been ineffective in the UK. The number of herds newly infected with bTB has doubled every nine years; in 2013 over 6.2 million bTB tests were performed in England leading to the slaughter of over 26,000 cattle at a cost to the taxpayer of almost £100Million. New approaches to halt the spread of infection are desperately needed. Mycobacterium bovis (Mbv) is a close relative of Mycobacterium tuberculosis (Mtb), the causative agent of TB in humans. While some of the mechanisms used by Mtb to cause disease in humans are well characterised, not much is known about the strategies used by Mbv to cause disease in cattle. DNA sequencing of the genomes of Mtb and Mbv have revealed that they share more than 99.9% genetic identity. While both Mbv and Mtb can infect humans, Mbv rarely transmits between humans and is instead spread by contaminated dairy products. On the other hand, Mtb can only cause a mild form of the disease in cattle. The distinct host preference of Mbv and Mtb indicates that the two mycobacterial species have evolved distinct strategies to infect and cause disease in their respective hosts. Through previous studies and our own work, a range of discrete molecular variations have been identified between Mtb and Mbv; these include differences in the expression of proteins, termed MPB70 and MPB83, and lipids, such as 'sulfolipid', between these bacteria. The role of these proteins and lipids in the virulence of Mbv for cattle has however not been defined; in this study we will explore the hypothesis that these molecular differences are among the key reasons why Mbv causes disease in cattle. We will test our hypothesis by first generating genetically-engineered Mbv strains that are 'Mtb-like' with regards to the expression of these lipids and proteins. These strains will then be tested along with their parental Mtb and Mbv strains in cellular infection models. These models will focus on laboratory grown cell cultures of bovine macrophages, cells of the bovine immune system that are the first to encounter and engulf invading Mbv cells. We have initial data to show that these cells respond differently to infection with Mbv and Mtb, and will use the genetically engineered strains of 'Mtb-like' Mbv strains to elucidate the mechanisms used by Mbv to establish infection in cattle. To achieve this we will develop an array of tools and new methodologies, such as the use of fluorescently-labelled Mbv and genetic-knockdown techniques, to study the infected macrophages. These tools will help us monitor critical intracellular components of the macrophage machinery, as well as providing tools for the wider bovine immunology research community. Our findings will shed new light on the molecular components that Mbv uses to cause disease in cattle, information that will help in the long term development of vaccines and diagnostics against this devastating disease for UK agriculture.