MICA: Does phase variation contribute to virulence and immune evasion of Neisseria meningitidis?

许多细菌病原体以无害的“共生体”的形式在我们体内定居并持续存在，这些共生体不会引起疾病。即使我们的身体产生抗体来对抗表面暴露的结构，这些细菌仍然存在。在这项提议中，我们的目标是提高我们对细菌如何避免这些抗体以及这些避免机制是否影响细菌引起疾病的能力的理解。脑膜炎奈瑟菌是一种细菌，是脑膜炎和血液中毒的主要原因，但在10-30%的人群中发现。“携带者”)是上呼吸道的无害共生体。这种细菌存在于鼻腔组织和喉咙后部，尽管产生了针对细菌表面抗原的抗体，但它可以持续长达12个月。这些细菌避免抗体有害作用的一种方法是改变这些表面抗原的结构或数量。这些变化是由重复的DNA束控制的，这些DNA束以高频率突变，导致制造这些表面抗原的基因表达发生变化。我们已经证明脑膜炎奈瑟菌在上呼吸道的持久性是由这些重复DNA束的高频率突变促进的，这些突变导致一些表面抗原的表达减少。由于许多这些表面抗原帮助细菌侵入组织，并在血液中存在的细胞和分子杀死后存活下来，这一发现使我们推测，由于重复DNA的突变，表面抗原表达的减少影响了脑膜炎奈瑟菌引起血液中毒和脑膜炎的能力。我们将通过比较从患者和携带者分离的细菌中的重复DNA束来验证这一假设。我们还将测试在脑膜炎奈瑟菌持续携带期间特异性抗原表达的减少是否与针对该表面抗原的抗体存在相关。我们将使用志愿者先前研究的样本，这些志愿者在6到12个月的时间内，在三个或更多的时间点上获得细菌分离物、血清和唾液提取物。同样，我们将使用从疾病病例中收集的脑膜炎奈瑟菌分离株。多个基因的重复DNA束将通过跨越重复区域的DNA片段的大小来分析，这样我们就可以计算重复的次数，然后使用这些数字来确定一个基因是否被表达。我们将研究当细菌在携带者体内持续存在时，基因是否会发生变化，以及与携带者相比，疾病分离株中的基因是否处于不同的状态。抗体的数量将用微量测定法测定。为此，基因的蛋白产物将被纯化，与荧光微球连接，并与血清孵育。这将导致抗体与微球上的蛋白质特异性结合。然后用不同的荧光标记检测抗体，并在荧光检测机上定量。我们期待两大成果。首先，重复DNA是否产生一种特定的基因表达模式，使脑膜炎奈瑟菌引起疾病的证据。确定“毒力相关”模式将导致生产新的预防措施（即含有这些毒力决定因素的疫苗）或疾病病例管理（即确定感染源并就何时向接触者提供预防性治疗作出合理决定）。其次，证明抗体是驱动减少脑膜炎奈瑟菌表面抗原在携带者的表达。这一发现将表明含有表面抗原的疫苗可以减少致病脑膜炎奈瑟菌菌株在携带者粘膜表面的持久性，并且对于评估Bexsero（新的b型脑膜炎球菌疫苗）是否可以预防致病菌株的传播具有重要意义。

Many bacterial pathogens colonise and persist in our bodies as harmless 'commensals' that do not cause disease. These bacteria persist even though our bodies produce antibodies against exposed structures on their surface. In this proposal, we aim to improve our understanding of how bacteria avoid these antibodies and whether these avoidance mechanisms influence the ability of bacteria to cause disease.Neisseria meningitidis is a bacterium that is a major cause of meningitis and blood-poisoning but is found in 10-30% of people (i.e. 'carriers') as a harmless commensal of the upper respiratory tract. This bacterial species is present on nasal tissues and at the back of the throat where it can persist for up to twelve months despite the generation of antibodies against surface antigens of the bacteria. One way these bacteria avoid deleterious effects of antibodies is by changing the structure or amounts of these surface antigens. These changes are controlled by repetitive DNA tracts, which mutate at high frequencies resulting in alterations in the expression of genes that make these surface antigens. We have shown that persistence of N. meningitidis in the upper respiratory tract is facilitated by high frequencies of mutations in these repetitive DNA tracts causing reductions in expression of some of the surface antigens. As many of these surface antigens help the bacteria to invade tissues and to survive killing by cells and molecules present in blood, this finding has led us to speculate that reductions in expression of surface antigens, due to mutations in repetitive DNA, affects the ability of N. meningitidis to cause blood poisoning and meningitis. We will test this hypothesis by comparing the repetitive DNA tracts in bacterial isolates from patients and carriers. We will also test whether reductions in expression of specific antigens during persistent carriage of N. meningitidis is correlated with the presence of antibodies against that surface antigen.We will use samples from previous studies in volunteers from whom bacterial isolates, serum and saliva extracts were obtained at three or more time points over a six to twelve month time period. Similarly we will use pre-existing collections of N. meningitidis isolates from disease cases. The repetitive DNA tracts of multiple genes will be analysed by sizing of DNA fragments spanning the repetitive region so that we can count the numbers of repeats and then use these numbers to determine whether a gene is expressed. We will examine if a gene changes as the bacteria persist within a carrier and if the genes are in different states in the disease isolates as compared to those of carriers. The amounts of antibodies will be measured using a microassay. For this, protein products of genes will be purified, linked to fluorescent microspheres and incubated with serum. This will result in specific binding of antibodies to the proteins on the microspheres. Antibodies will then be detected with a different fluorescent tag and quantified in a fluorescence detection machine.We anticipate two major outcomes. Firstly, evidence of whether repetitive DNA produces a particular pattern of gene expression that enables N. meningitidis to cause disease. Identification of a 'virulence-associated' pattern would lead to production of novel preventive measures (i.e. vaccines containing these virulence determinants) or management of disease-cases (i.e. identification of sources of infections and rational decisions on when to provide preventive therapeutic treatment to contacts). Secondly, a demonstration that antibodies are driving reductions in expression of N. meningitidis surface antigens in carriers. This finding will indicate that vaccines containing surface antigens could reduce persistence of disease-causing N. meningitidis strains on mucosal surfaces of carriers and would be important for assessing whether Bexsero (the new MenB vaccine) can prevent spread of disease-causing strain.