Global assignment of the function of Salmonella genes in livestock

肠道沙门氏菌对公众健康和可持续农业构成威胁。已经描述了超过2460种不同的生物体变体，这些变体可以分为两组；受限制或适应于特定宿主的血清型，可引起全身性疾病（如导致人类伤寒的伤寒沙门氏菌），以及在广泛宿主中引起肠胃炎的血清型（如伤寒沙门氏菌）。伤寒是一种主要由水传播的疾病，由于不卫生的条件而长期存在，估计全世界每年发生2100万人患病和216 510人死亡。由非伤寒沙门氏菌菌株引起的肠胃炎也很常见，仅在美国，估计每年就有140万例病例和600例死亡。这些感染通常与食用来自鸡、猪和牛的食物有关，因为细菌能够在这些动物的肠道内定植。减少产粮动物携带肠炎沙门氏菌的战略预计将降低人类感染的发生率并改善动物健康。虽然已有控制家禽沙门氏菌的疫苗，但它们对不同的血清型提供的保护很弱且不稳定，人们普遍认为，需要有效的疫苗或治疗方法用于其他牲畜物种。此外，并非在动物身上发现的所有肠球菌血清型都对人类构成威胁，需要工具来预测这类菌株的流行潜力。沙门氏菌新疫苗、治疗方法和诊断方法的开发需要了解肠道定植的分子事件。我们的实验室已经确定了鸡、猪和小牛定植所需的一些鼠伤寒沙门氏菌因子，但是迄今为止，只有不到五分之一的沙门氏菌基因的功能得到了检测。犊牛和猪感染鼠伤寒沙门氏菌可导致急性胃肠炎，而鸡携带鼠伤寒沙门氏菌往往无症状。沙门氏菌在一个宿主中致病而在另一个宿主中不致病的原因尚不清楚。此外，尚不清楚为什么一些肠链球菌血清型在特定宿主中引起全身性疾病，而另一些则局限于消化道。我们已经发明了一种新方法，可以在动物感染期间同时筛选数千个个体沙门氏菌突变体，每个突变体都缺乏特定的特征。这种方法（转座子介导的差异杂交，TMDH）可以快速描述几乎所有沙门氏菌基因在感染过程中的作用，并依赖于检测芯片上每个突变的信号，芯片上排列着所有细菌基因。细菌基因随机突变，然后突变体被组装成可以接种到动物身上的大池。大型水池的使用大大减少了所需动物的数量。如果在接种物中有突变体，但在细菌通过动物后恢复的池中没有突变体，则可以推断该突变体中被破坏的特征对定植很重要。TMDH已经在老鼠身上得到了验证，但现在需要在生产食品的动物身上使用。该方法取代了现有的方法，因为它可以告知研究人员在感染期间何时需要或不需要基因，而无需分离和检查突变基因。我们建议用这种方法筛选1万个鼠伤寒沙门氏菌突变体，以了解它们在鸡、猪和牛的肠道中定植的能力。这将提供使用替代啮齿动物或基于细胞的分析无法获得的见解，并依赖于申请人实验室的独特专业知识和设施。TMDH将大大加快沙门氏菌发病机制所需因素的识别，其中一些可能适合作为药物靶点或包含在疫苗中。为牲畜开发的治疗方法也可能适用于人类治疗伤寒。

Bacteria of the species Salmonella enterica are a threat to public health and sustainable agriculture. Over 2460 distinct variants of the organism have been described and these can be divided into two groups; serovars restricted or adapted to a given host that cause systemic illness (e.g. S. Typhi, which causes Typhoid fever in humans) and serovars causing gastroenteritis in a wide range of hosts (e.g. S. Typhimurium). Typhoid fever is a largely water-borne disease perpetuated by unsanitary conditions and 21 million human illnesses and 216,510 deaths are estimated to occur each year worldwide. Gastroenteritis caused by non-typhoidal Salmonella strains is also common, with an estimated 1.4 million cases and 600 deaths per annum in the United States alone. These infections are often associated with the consumption of foods derived from chickens, pigs and cattle owing to the ability of the bacteria to colonise the intestines of such animals. Strategies to reduce the carriage of S. enterica in food-producing animals are expected to lower the incidence of human infections and improve animal health. Whilst vaccines exist for the control of Salmonella in poultry, they confer weak and variable protection against different serovars and it is widely agreed that effective vaccines or treatments are needed for use in other livestock species. Furthermore, not all S. enterica serovars found in animals pose a threat to humans and tools are needed to predict the epidemic potential of such strains. The development of novel vaccines, treatments and diagnostics for Salmonella requires an understanding of the molecular events underlying intestinal colonisation. Our laboratories have identified some S. Typhimurium factors needed for colonisation of chickens, pigs and calves, but the function of less than one fifth of all Salmonella genes has so far been examined. S. Typhimurium infections in calves and pigs result in acute gastroenteritis whilst carriage in chickens tends to be asymptomatic. The reasons why Salmonella causes disease in one host but not another are not understood. Furthermore, it is not known why some S. enterica serovars cause systemic disease in a given host whereas others are restricted to the alimentary tract. We have invented a new method that permits the simultaneous screening of thousands of individual Salmonella mutants, each lacking a particular character, during infection of animals. The method (transposon-mediated differential hybridisation, TMDH) can rapidly describe the role of virtually all Salmonella genes during infection and relies on detecting signals from each mutant on a chip on which all the bacterial genes are arrayed. Bacterial genes are mutated at random then the mutants are assembled into large pools that can be inoculated into animals. The use of large pools dramatically reduces the number of animals that are needed. If a mutant is represented in the inoculum, but not in a pool recovered after the bacteria have passed through the animal, it can be inferred that the character disrupted in that mutant is important for colonisation. TMDH has been validated in mice, but it now needs to be used in food-producing animals. The method supersedes existing approaches, since it can inform the researcher when a gene is, or is not, required during infection without the need to isolate and examine the mutated gene. We propose to screen 10,000 S. Typhimurium mutants by this method for their ability to colonise the intestines of chickens, pigs and cattle. This will provide insights that cannot be obtained using surrogate rodent or cell-based assays and relies on the unique expertise and facilities at the applicants' laboratories. TMDH will greatly accelerate the identification of factors needed for Salmonella pathogenesis, some of which may be suitable as targets for drugs or included in a vaccine. Treatments developed for use in livestock may also be suitable for use in humans against typhoid fever.