Phenotype to genotype: dissecting meningococcal disease and carriage traits

细菌通过结合多种不同的行为导致疾病。脑膜炎球菌，即脑膜炎奈瑟菌，可引起脑膜炎和败血症。2016-2017年，尽管在婴儿和青少年免疫接种计划中引入了新的脑膜炎球菌疫苗，但英格兰仍有747例确诊的脑膜炎球菌病病例。为了引起疾病，脑膜炎球菌首先从一个人传播到另一个人，然后粘在喉咙后部和扁桃体周围的组织上。这种细菌可以避开人体产生的抗体等效应分子存活数天。当细菌从粘膜组织侵入血液时，细菌细胞迅速生长，抵抗补体和白细胞的杀死，疾病就发生了。从血液中，这种可怕的病原体传播并引起大脑组织的炎症。疾病过程的每一步都是由脑膜炎球菌细菌基因组中编码的分子或毒力因子的组合介导的。目前，我们知道脑膜炎球菌引起疾病需要胶囊（细菌细胞外部存在的多糖）和其他几个因素。但是，我们也有关于1000种脑膜炎球菌病和携带菌株的基因组的大量信息。这些基因组包含有关致病菌株的特性、菌株如何随时间变化以及每种菌株中存在的基因数量/类型的广泛信息。虽然这些基因组使我们能够将某些序列与疾病状态联系起来，但将基因组变异与疾病所需的表型变异联系起来的努力有限，因此我们目前的大部分信息都是推断性的，而不是基于实验的。我们打算通过一系列12项模拟各种疾病和携带行为的试验，测试多达330个分离株，来探索表型与基因型之间的联系。我们将开发高通量检测方法，使多种分离物能够快速处理，并利用这些方法研究来自患者和携带者的地方性Y血清群和高侵入性W血清群脑膜炎球菌分离物。多种表型的定量输出将为统计关联测试提供重要的力量，将表型与疾病和携带性状联系起来，并确定这些性状的特定遗传决定因素。在这些测试之后，将对特定遗传决定因素进行分子测试，以确认它们对特定行为的影响。这一步骤将涉及构建具有单个基因或多个基因改变的突变体，这样我们就可以看到表型组合如何促进疾病过程。我们还将探讨毒性和无毒变异之间的相互作用。我们已经观察到，来自一种载体的分离物具有相反的和潜在的拮抗行为，其中一种导致人类细胞单层的破坏，而另一种则加强这些细胞之间的相互作用。我们将测试这些细菌变体是否相互拮抗，以及这是否是影响多种分离株和其他疾病表型的普遍现象。我们希望获得的不仅是更好地了解脑膜炎球菌如何引起疾病，而且还改进了对脑膜炎球菌菌株疾病属性流行程度的评估。这将是重要的，因为我们监测新的MenB疫苗Bexsero对英国疾病的影响，因为避免疫苗反应可能通过改变疫苗靶向抗原或增强毒力来发生。我们的信息还将通过确定将疾病的关键决定因素纳入新疫苗，促进第二代疫苗的开发。最后，我们的方法将适用于其他细菌病原体，增强基因型到表型的翻译，用于不断扩大的致病菌基因组数据库。

Bacteria cause disease by combining multiple different behaviours. Neisseria meningitidis, the meningococcus, causes both meningitis and septicaemia. In 2016-2017, there were 747 confirmed meningococcal disease cases in England despite new meningococcal vaccines being introduced into the infant and teenage immunisation schedules. In order to cause disease, the meningococcus first transmits from one person to another and then sticks to tissues at the back of the throat and around the tonsils. The bacterium survives for multiple days avoiding effector molecules produced by our bodies such as antibodies. Disease occurs when the organism invades by moving from the mucosal tissues into the blood where the bacterial cells grow rapidly and resist being killed by complement and white blood cells. From the blood, this fearsome pathogen spreads and causes inflammation in the tissues lining the brain. Each step of the disease process is mediated by combinations of molecules or virulence factors encoded in the genomes of meningococcal bacteria. Currently, we know that the capsule (polysaccharides present on the outside of bacterial cells) and several other factors are required for meningococci to cause disease. But we also have large amounts of information about the genomes of 1,000s of meningococcal disease and carriage isolates. These genomes contain extensive information about identities of disease-causing strains, how strains change over time and on numbers/types of genes present in each strain. While these genomes have enabled us to link certain sequences to the disease state, there has only been limited efforts to link genomic variation to variation in the phenotypes required for disease so that much of our current information is inferential rather than experimentally-based. We intend to explore phenotype-to-genotype links by testing up to ~330 isolates in a series of 12 assays that mimic various disease and carriage behaviours. We will develop high throughput assays, enabling rapid processing of multiple isolates, and utilise these methodologies to study endemic serogroup Y and hyperinvasive serogroup W meningococcal isolates from patients and carriers. Quantitative outputs from multiple phenotypes will provide us with significant power for statistical association tests to link phenotypes to disease and carriage traits and to identify specific genetic determinants of these traits. These tests will be followed up by molecular testing of specific genetic determinants to confirm their contributions to a specific behaviour. This step will involve construction of mutants with alterations in a single gene or in multiple genes so that we can see how combinations of phenotypes contribute to disease processes.We will also explore interactions between virulent and avirulent variants. We have already observed that isolates from one carrier had opposing and potentially antagonistic behaviours with one causing disruption of monolayers of human cells while the other tightened interactions between these cells. We will test whether these bacterial variants antagonise each other and if this is a general phenomenon affecting multiple isolates and other disease phenotypes.What we hope to gain is not only a better understanding of how meningococci cause disease but also improved assessment of the prevalence of disease-attributes of meningococcal strains. This will be important as we monitor the impact of the new MenB vaccine, Bexsero, on disease in the UK as avoidance of vaccine responses could occur by either changes in vaccine-targeted antigens or enhanced virulence. Our information will also facilitate development of second generation vaccines by identifying key determinants of disease for inclusion in new vaccines. Finally, our approaches will be applicable to other bacterial pathogens enhancing translation of genotypes into phenotypes for the ever-expanding genomic databases of pathogenic bacteria.