Humoral immunity against the M. tuberculosis kasB persistent mutant

摘要 结核分枝杆菌(Mtb)非常善于在受感染的宿主体内建立临床沉默 潜伏状态可随后重新激活，对结核病(TB)控制构成巨大阻碍。它是 普遍认为，据估计，世界上三分之一的人口感染了结核分枝杆菌 窝藏潜伏杆菌。后者的人口为结核分枝杆菌的永久化提供了一个巨大的蓄水池。潜伏者 持续性的结核分枝杆菌通常对药物耐药，因此难以治疗。考虑到Mtb的进入倾向 休眠，很可能是敏感宿主吸入的感染性接种物中含有潜伏的杆菌 除了那些正在积极复制的物种。充分的证据支持这样一种观点，即结核分枝杆菌在接触到 有利于建立潜伏期的条件，表现出与之不同的基因表达谱 积极复制细菌的能力。因此，潜伏的持续体的抗原谱很可能不同于 它的快速增长的同行。事实上，已经证明耐酸牢度的损失存在于Mtb中 慢性持续状态，被认为至少部分是细胞包膜结构异常的结果 在休眠杆菌中。基于以上原因，一种有效的结核病疫苗理所当然地应该引起一种免疫。 既可以针对主动复制的，也可以针对潜在的持久杆菌的反应。事实上，卡介苗是唯一一种抗结核药物 目前可获得的疫苗和在与有助于促进持久性的条件无关的条件下产生的疫苗 (因此可能不表达潜伏结核杆菌特有的抗原(AGs))，可能不能 诱导一种免疫反应，以最佳方式保护免受持久性有机体的侵害。 雅各布斯小组最近鉴定了一组mtb kasB的突变体，它编码一种-酮酰基-酰基 载体蛋白合成酶参与分枝杆菌酸(分枝杆菌的主要家族)的生物合成途径 细胞包膜脂质)。研究表明，KasB的活性是通过两个位点的磷酸化来调节的 苏氨酸残基，并且特定的KasB缺乏突变体表现出与 持久性，包括耐酸牢度的丧失和在接种到小鼠体内时无法复制。重要的是 包括抗酸阴性kasB持久突变体的免疫方案--哪些磷酸化位点 在上述两个苏氨酸残基上，每个都被一个天冬氨酸(KasB-DD)取代--保护 与单独使用卡介苗的方案相比，抗结核分枝杆菌效果更好。耐酸阴性kasB-DD菌株表现出 涉及真菌酸类以外的物种的异常脂谱。细胞信封的异常包装是由于 异常的表面脂类，如霉菌酸，可能会暴露其他大分子，包括蛋白质和 碳水化合物，否则会被遮盖住。总的来说，这些观察结果支持这样一种观点，即坚持不懈 结核分枝杆菌可以表达不同的AGS和靶向这些部分，除了那些典型的活跃存在的 复制细菌，可能会提高疫苗的效力。因此，我们假设以AGS为目标 由Mtb持久器差异表达和/或不同于Mtb持久器代表了一种开发反 结核病战略，包括免疫疗法和疫苗。为了开始检验这一假设，我们建议 鉴定抗酸阴性kasB-DD持续体诱导的体液免疫反应。选择 这一方法是基于新出现的证据，表明抗体在保护中发挥重要作用 抗结核分枝杆菌和调节感染结果。此外，对抗体应答的严格表征 可以预期KasB-Dd(以及在该过程中，控制WT Mtb的KasB-Dd)将生成广泛的Mtb集合 Ag特异性单抗(MAbs)构成了一套最有价值的工具，用于推动我们的 对MTB研究的三个重要方面的理解：(一)WT和PERSISTER的体液反应 结核杆菌，(Ii)难以追踪的持久性结核分枝杆菌的生物学，以及(Iii)调节机制 结核潜伏期。因此，这些研究产生的信息有可能导致 开发新的战略以更好地控制结核分枝杆菌，包括有效的疫苗。

Abstract Mycobacterium tuberculosis (Mtb) is remarkably adept to establishing in an infected host a clinically silent latent state that can subsequently reactivates, posing a formidable hindrance to tuberculosis (TB) control. It is generally thought that the majority of the one-third of the world's population estimated to be infected with Mtb harbor latent bacilli. This latter population affords a large reservoir for the perpetuation of Mtb. The latent persistent form of Mtb is often drug-tolerant and thus difficult to treat. Given the propensity of Mtb to enter dormancy, it is likely that the infectious inoculum inhaled by a susceptible host contains latent form of bacilli in addition to those that are actively replicating. Ample evidence support the notion that Mtb, when exposed to conditions conducive to the establishment of a latent state, displays a gene expression profile distinct from that of actively replicating bacilli. Thus, it is likely that the antigenic profile of a latent persister is different than that of its rapidly growing counterpart. Indeed, loss of acid fastness has been demonstrated in Mtb existing in a chronic persistent state, which is thought to be, at least in part, the result of an aberrant cell envelope structure in dormant bacilli. Based on the above, it stands to reason that an effective TB vaccine should elicit an immune response that can target both actively replicating and latent persistent bacilli. In fact, BCG, the only anti-TB vaccine currently available and generated in conditions unrelated to those conducive to promote persistence (and therefore likely not expressing antigens (Ags) unique to latent tubercle bacilli), may not be capable of inducing an immune response that optimally protects against persistent organisms. The Jacobs group has recently characterized a set of mutants of Mtb kasB, which encodes a -ketoacyl-acyl carrier protein synthase involved in the biosynthetic pathway of mycolic acids (a major family of mycobacterial cell envelope lipids). The study has revealed that KasB activity is regulated via phosphorylation at two threonine residues, and that specific KasB-deficient mutants display phenotypes consistent with features of persisters, including a loss of acid fastness and inability to replicate when inoculated into mice. Importantly, immunization protocol that includes an acid-fast negative kasB persistent mutant -- which phosphorylation sites at the two aforementioned threonine residues have each been replaced by an asaparte (kasB-DD) -- protects against Mtb better than regimens that use BCG alone. The acid-fast negative kasB-DD strain displays an aberrant lipid profile involving species beyond mycolic acids. Aberrant packing of the cell envelope due to abnormal surface lipids such as mycolic acids may expose other macromolecules, including proteins and carbohydraes, that are otherwise masked. Collectively, these observations support the notion that persistent Mtb can express distinct Ags and that targeting such moieties, in addition to those typically present in actively replicating bacilli, might lead to enhanced vaccine efficacy. We therefore hypothesize that targeting Ags differentially expressed by and/or distinct to Mtb persisters represents an effective approach to developing anti- TB strategies including immunotherapeutics and vaccines. To begin testing this hypothesis, we propose to characterize the humoral immune response elicited by the acid-fast negative kasB-DD persister. The choice of this approach is based on emerging evidence suggesting antibodies (Abs) play a significant role in protection against Mtb and in modulating infection outcome. In addition, rigorous characterization of the Ab response to kasB-DD (and in the process, that of the control WT Mtb) can be expected to generate an extensive set of Mtb Ag-specific monoclonal Abs (mAbs) that constitutes a most valuable set of tools for advancing our understanding of three important aspects of Mtb research: (i) the humoral response of WT and persister tubercle bacilli, (ii) the biology of the difficult-to-track persistent Mtb, and (iii) the mechanisms that regulate tuberculous latency. Thus, the information yielded by these studies have the potential to lead to the development of novel strategies for better control of Mtb, including efficacious vaccines.