Reducing and replacing the animal cost of functional genetics in African trypanosomiasis

传染病动物模型的关键应用之一是了解病原体基因如何影响疾病结果。这是至关重要的，例如，在确定毒力因素和耐药性机制，影响我们如何治疗感染者。非洲锥虫是一种血液寄生虫，在撒哈拉以南非洲地区的人们中引起一种致命疾病，也是一种消耗牛的疾病，对肉类和乳制品生产产生巨大的有害影响，给非洲、亚洲和南美洲的发展中经济体造成约40亿美元的损失。密切相关的寄生虫利什曼原虫引起一系列疾病，其症状包括溃疡性病变、口鼻粘膜完全破坏、器官衰竭和死亡，每年约有100万新病例。了解这些寄生虫的毒力需要通过动物模型获得基因突变。传统上，这是通过使用单个突变体感染一组动物，并将疾病进展与感染非突变体寄生虫的动物进行比较来完成的。然而，每一种寄生虫都含有大约1万个基因，每种基因都有许多不同的突变，所以即使只研究很少的基因，也意味着要用大量的实验动物来进行这样的研究。由于技术上的原因，这些研究通常也是用不能概括真实疾病重要方面的寄生虫菌株进行的。此外，由于感染和同一基因的不同突变体之间的寄生虫水平存在差异，这些研究检测变化的敏感性相对较弱。我们已经开发了一种利用当代基因技术的方法，可以在包含数千个单个突变体的复杂混合物中快速测试突变体在感染期间的影响。该方法适用于捕获真实人类疾病生物学的寄生虫菌株，也适用于引起动物疾病的物种。我们的试验数据表明，该方法可以用于动物疾病模型，以在感染过程中可靠地评估突变体的适应性，捕获同一基因突变体之间的变异和动物-动物变异，但所需的动物数量比测试单个基因要少。在这个项目中，我们将证明该方法可以转化为人类疾病中最重要的锥虫，并验证其用于有效测试从实验中产生的基因集，而动物使用最少。我们还将扩大该方法的使用范围，以涵盖导致人类和动物疾病的锥虫基因组中的每个基因，从而有效地消除了在感染期间进行基本适应性测试时使用新的实验动物的需要。在这些感染模型中证明有效性将鼓励实验室广泛采用这些高度并行的方法，从而在取得更好的科学成果的同时大幅减少动物的使用。它还将消除对最常见用途之一的需求，并展示用较小的模型代替大型动物的潜力。

One of the key applications of animal models to infectious disease is in understanding how pathogen genes affect disease outcome. This is critical, for example, in identification of virulence factors and mechanisms of drug resistance, which affect how we treat infected individuals. African trypanosomes are parasites of the blood which cause a fatal disease in people in sub-Saharan Africa and a wasting disease of cattle that has a huge detrimental impact on meat and dairy production, creating losses of ~$4 billion from developing economies in Africa, Asia and South America. Closely related parasites called Leishmania cause a range of diseases whose symptoms include ulcerative lesions, complete destruction of the mucous membranes in the nose and mouth, and organ failure and death, with ~1 million new cases each year. Understanding the virulence of these parasites involves taking gene mutants through animal models. Traditionally, this is done by using an individual mutant to infect a set of animals, and comparing the disease progression to animals infected with non-mutant parasites. However, each parasite species contains ~10,000 genes and there are many different mutations for each, so even only looking at a very few genes means a lot of experimental animals are used for such studies. For technical reasons, these studies are also typically performed with strains of the parasite that do not recapitulate important aspects of the real disease. Moreover, because of variation in parasite levels between infections and different mutants of the same gene, the sensitivity of these studies to detect changes is relatively weak. We have developed a method using contemporary genetic technology that can rapidly test the effect of mutants during infections in a complex mixture containing many 1000s of individual mutants. The method is compatible with parasite strains that capture real human disease biology and also species that cause animal disease. We have pilot data showing the method can be used in animal models of disease to robustly assess mutant fitness over the course of infections, capturing both variation between mutants in the same gene and animal-animal variation, but requiring fewer animals than would testing a single gene. In this project, we will demonstrate that the method can be translated to the most important trypanosome for human disease and validate its use to efficiently test sets of genes arising from experiments with minimal animal usage. We will also expand the use of the method to cover every gene in the genomes of trypanosomes causing human and animal disease, effectively eliminating the need for new experimental animal usage in basic tests of fitness during infection. Demonstration of effectiveness in these infection models will encourage the wide adoption of these highly-parallel methods by labs, leading to substantial reduction in animal usage at the same time as resulting in better scientific outcome. It will also remove the need for one of the most common usages and demonstrate the potential to replace large animals with smaller models.