Characterization of sialic acid specific O-acetyltransferases: from neuroinvasive bacteria to human hosts

导致细菌败血症和脑膜炎的病原体经常进化出用唾液酸(SIA)装饰细胞表面的能力，唾液酸是一种糖分子，用于宿主生物体的细胞识别事件。大肠杆菌K1和B、C、W135和Y群脑膜炎双球菌被代表重要毒力因子的衣壳多糖(CPS)所覆盖。SIA特异的O-乙酰转移酶(OAT)可以进一步修饰CPS，这是一个调节囊膜抗原的特定特征的过程。在第一次授予期间，我们证明了Sia O-乙酰化的功能是在结构多样化的蛋白质折叠的基础上进化的，并成功地结晶了具有左手β-螺旋折叠的燕麦。现在，我们致力于燕麦α/β水解酶折叠的生化和结构特征的研究。为了分析SIA特异性的决定因素，将对分别由SIA和甘露糖-磷酸聚合物包裹的C群和A群脑膜炎球菌的被膜修饰燕麦进行比较分析。对大肠杆菌K1的初步研究揭示了出人意料的发现，O-乙酰化阳性而不是阴性的菌株在被膜表达上表现出动态的、生长阶段相关的变化。为了揭示其潜在的分子机制，我们建议对燕麦的细胞定位及其与胶囊生物合成和输出机制的可能相互作用进行研究。Sia O-乙酰化与宿主-病原体相互作用的相关性将在血清抵抗的背景下进行研究。需要解决的问题是：(I)O-乙酰化是否影响与Sia结合的补体调节因子H的相互作用，以及(Ii)观察到的动态被膜还原是否使细菌表面蛋白OmpA去面具，这将促进OmpA介导的补体调节因子C4BP的结合，这是一个已知的介导血清抵抗的过程。关于携带O-乙酰化Sia-胶囊的细菌的宿主与病原体的相互作用，宿主机体使用唾液酸聚糖的O-乙酰化作为调节Sia介导的识别过程的分子开关，特别是在免疫系统中。到目前为止，真核基因SIA修饰燕麦的遗传基础尚不清楚。在前期工作中，我们利用从细菌中获得的知识来鉴定人类的一种候选酶，并进行了初步的细胞实验，以证明Sia O-乙酰化参与其中。通过生成一个基于同源性的结构模型，我们确定了一个假定的催化三联体，这一特征也在脑膜炎球菌酶OatC中发现。在这项拨款申请的框架内，我们提出了人类酶的第一个生化特征，特别是在体外测定SIA特异性燕麦酶的活性。

Pathogens causing bacterial sepsis and meningitis have frequently evolved the ability to decorate their cell surfaces with sialic acid (Sia), a sugar molecule that is used for cellular recognition events in the host organism. Escherichia coli K1 and serogroup B, C, W135 and Y meningococci are covered by Sia-containing capsular polysaccharides (CPS) that represent important virulence factors. Sia-specific O-acetyltransferases (OATs) can further modify the CPS, a process that modulates the specific characteristics of the capsular antigen. During the first granting period, we demonstrated that the function of Sia O-acetylation evolved on the basis of structurally diverse protein folds and succeeded in crystallization of an OAT with left-handed beta-helix fold. Now, we aim at the biochemical and structural characterization of OATs with putative alpha/beta-hydrolase fold. To dissect the determinants of Sia-specificity, a comparative analysis will be performed based on the capsule-modifying OATs of serogroup C and A meningococci, which are encapsulated by Sia- and mannose-phosphate-polymers, respectively.Preliminary studies on Escherichia coli K1 revealed the unexpected finding that O-acetylation positive but not negative strains show dynamic, growth phase-dependent variations in capsule expression. To unravel the underlying molecular mechanism, we propose studies on the cellular localization of OATs and their putative interactions with the capsule biosynthesis and export machinery. The relevance of Sia O-acetylation on host-pathogen interactions will be studied in the context of serum resistance. Questions to be addressed are: (i) does O-acetylation impacts interactions with factor H, a Sia-binding complement-regulator, and (ii) does the observed dynamic capsule reduction demask the bacterial surface protein OmpA, which would facilitate OmpA-mediated binding of complement regulator C4BP, a process known to mediate serum resistance.Regarding host-pathogen interactions of bacteria carrying an O-acetylated Sia-capsule, it might be of particular relevance that the host organism uses O-acetylation of sialo-glycans as a molecular switch to regulate Sia-mediated recognition processes, especially in the immune system. So far, the genetic basis of eukaryotic Sia-modifying OATs is not known. In preliminary work, we used our knowledge gained on the bacterial counterparts to identify a human candidate enzyme and performed initial cellular experiments to prove an involvement in Sia O-acetylation. By generating a homology-based structural model, we identified a putative catalytic triad, a feature that was also found for the meningococcal enzyme OatC. In the frame of this grant application, we propose a first biochemical characterization of the human enzyme with particular focus on the determination of Sia-specific OAT activity in vitro.