Phenotype to genotype: dissecting meningococcal disease and carriage traits

细菌通过结合多种不同的行为而致病。脑膜炎奈瑟菌，脑膜炎双球菌，可引起脑膜炎和败血症。2016-2017年间，尽管新的脑膜炎双球菌疫苗被引入婴儿和青少年免疫计划，但英国仍有747例确诊脑膜炎双球菌疾病病例。为了致病，脑膜炎球菌首先从一个人传播到另一个人，然后粘在喉咙后面和扁桃体周围的组织上。这种细菌可以存活数天，躲避我们身体产生的抗体等效应器分子。当微生物从粘膜组织进入血液，细菌细胞在血液中快速生长并抵抗补体和白细胞的杀伤时，就会发生疾病。这种可怕的病原体从血液中传播，并在脑组织中引起炎症。疾病过程的每一步都是由脑膜炎双球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致病需要被膜(存在于细菌细胞外的多糖)和其他几个因素。但我们也有关于1000多种脑膜炎双球菌和携带者分离株基因组的大量信息。这些基因组包含关于致病菌株的身份、菌株如何随时间变化以及每个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表型的变异联系起来的努力有限，因此我们目前的许多信息都是推论的，而不是基于实验的。我们打算通过在一系列12种模拟各种疾病和运输行为的检测中测试多达330个分离物来探索表型与基因型之间的联系。我们将开发高通量分析方法，使多种分离株能够快速处理，并利用这些方法研究来自患者和携带者的地方性Y群和高侵袭性W群脑膜炎球菌分离株。来自多种表型的定量结果将为我们提供强大的统计关联检验能力，以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，将对特定的基因决定因素进行分子测试，以确认它们对特定行为的贡献。这一步将涉及构建单基因或多基因改变的突变体，以便我们能够看到表型组合如何对疾病过程做出贡献。我们还将探索毒力和无毒变种之间的相互作用。我们已经观察到，来自一种载体的分离株具有相反的和潜在的拮抗行为，其中一种引起人类细胞单层的破坏，而另一种则加强了这些细胞之间的相互作用。我们将测试这些细菌变异是否相互拮抗，以及这是否是影响多个菌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导致疾病，而且还改进了对脑膜炎球菌菌株疾病属性的流行率的评估。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，因为可以通过改变疫苗靶向抗原或增强毒力来避免疫苗反应。我们的信息还将通过确定新疫苗中包含的疾病关键决定因素来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，为不断扩大的病原菌基因组数据库加强基因类型到表型的翻译。

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.