Mycobacterial determinants of survival and fitness within the bovine host

牛结核病（BTB）是英国某些地区持续存在的问题。目前的控制策略是及时检测牛群是否存在结核分枝杆菌，并屠宰检测呈阳性的动物。然而，观察到结核分枝杆菌结核在地理区域和流行率上都在蔓延，这表明现有的控制战略没有有效地发挥作用。每年控制计划的费用为1亿英镑，因此，该政策不仅无效，而且是一个重大的经济负担。此外，结核分枝杆菌可在人类中引起结核病，虽然目前这种情况很少见，但结核分枝杆菌的流行率不断上升，有可能发展成为人畜共患（疾病从动物传播给人类）的风险。BTB是由一种叫做牛分枝杆菌的细菌引起的。这与结核分枝杆菌密切相关，结核分枝杆菌是一种通常引起结核病的细菌。牛的结核病与人的结核病有很大的相似之处，与人的结核病一样，疫苗接种是控制结核的另一种策略。预防人类结核病的疫苗是牛分枝杆菌的一种菌株，称为牛分枝杆菌卡介苗，然而这对牛和人都提供了有限的预防疾病的保护。牛分枝杆菌通过气溶胶传播，一旦进入动物体内，它就会进入巨噬细胞。这些细胞，通常致力于杀死和清除细菌病原体，不能杀死细菌。相反，牛分枝杆菌以肉芽肿的形式适应并存活于肺内。然而，我们对允许细菌存活并在宿主中引起疾病的基因知之甚少。我们将敲除牛分枝杆菌基因组中每一个基因的功能（大约4000个），并确定那些在牛巨噬细胞和全牛中存活所需的基因。我们将通过使用一种称为转座子定向插入测序（TraDIS）的技术来实现这一点。该技术已成功应用于其他具有医学和兽医重要性的细菌病原体的研究，但这将是第一次将其用于研究牛分枝杆菌疾病的遗传决定因素。我们将建立突变体文库，其中基因组中每个基因的功能都被敲除。然后我们将把库放入“屏幕”中。通过“筛选”后恢复的突变体通过DNA测序与原始预筛选的突变体池进行比较。无法在筛选中存活的突变体被识别出来，因此我们将识别出在宿主中存活所需的所有基因。那些不能在宿主体内存活的突变体代表了潜在的候选疫苗。我们将能够利用计算分析、文献检索和在不同类型的细菌培养基（体外）上进行的筛选比较相结合来评估必需基因的功能。宿主生存所必需的功能也反映了宿主内部的条件。在该项目结束时，我们将填补宿主病原体相互作用领域BTB知识的关键空白，并确定几个潜在的候选疫苗，供未来开发考虑。

Bovine tuberculosis (BTB) is a persistent problem in certain areas of the UK. The current control strategy is to test herds for the presence of BTB at timely intervals and slaughter test-positive animals. However, the observation that BTB is spreading in both geographical area and prevalence suggests that the existing control strategy is not working effectively. The cost of the control programme is £100 million annually, therefore not only is the policy ineffective, it is also a significant economic burden. In addition, BTB can cause tuberculosis in humans, and while this is currently rare, the escalating prevalence of BTB has the potential to develop into a zoonotic (the passage of disease from animals to humans) risk.BTB is caused by a bacterium called Mycobacterium bovis. This is very closely related to Mycobacterium tuberculosis, a bacterium that commonly causes tuberculosis. Tuberculosis in cattle and humans show great similarities and, like the human disease, vaccination is an alternative strategy to control BTB. The vaccine against human tuberculosis is a strain of Mycobacterium bovis called Mycobacterium bovis BCG, however this provides limited protection against the disease in both cattle and humans. Mycobacterium bovis is transmitted by aerosol, once within the animal it enters into cells called macrophages. These cells, normally dedicated to the killing and removal of bacterial pathogens, are unable to kill the bacteria. Instead, Mycobacterium bovis adapts and survives within the lungs in structures called granulomas. However, we do not know much about the genes that allow the bacteria to survive and cause disease in the host. We will knock-out the function of every single gene in the genome of Mycobacterium bovis (approximately 4000) and determine those genes that are required for survival in bovine macrophages and in the whole cow. We will achieve this by using a technique called Transposon Directed Insertion Seqeuncing (TraDIS). This technique has been applied successfully to the study of other bacterial pathogens of medical and veterinary importance, but this will be the first time it has been used to study the genetic determinants of disease in Mycobacterium bovis. We will make libraries of mutants where the function of every gene in the genome has been knocked-out. We will then put the libraries into "screens". Mutants that are recovered after being through a "screen" are compared to the original pre-screened mutant pool by DNA sequencing. Mutants that are unable to survive the screen are identified and as a result we will identify all the genes necessary for survival in the host. Those mutants that are unable to survive in the host represent potential vaccine candidates. We will be able to assess the function of essential genes using a combination of computational analysis, literature searching and comparisons to screens performed on different types of bacteriological media (in vitro). The functions that are essential for survival in the host also reflects the conditions within the host. At the end of this project we will have filled in key gaps in the knowledge of BTB in the area of host pathogen interactions and have identified several potential vaccine candidates to be considered for future development.