Functional Dissection of Toxin-Antitoxin Systems in Mycobacterium tuberculosis

描述（由申请人提供）：结核病是一种广泛和有效的人类病原体-至少有三分之一的世界人口感染结核分枝杆菌（Mtb），估计每年死亡人数为200万至300万。虽然是密集研究的主题，但这种感染非常复杂，能够引起活动性疾病或持续潜伏状态。潜伏期代表了一种适应，这种适应使结核分枝杆菌能够在人群中有效地持续存在。潜伏性感染（其中杆菌被认为在宿主巨噬细胞中持续存在）可以被重新激活，特别是在免疫受损的个体中。事实上，在艾滋病毒高发病率地区，结核病发病率上升-以及令人震惊的耐多药结核分枝杆菌菌株数量-正在记录在案。触发结核分枝杆菌潜伏期的分子开关知之甚少。大肠杆菌中的毒素-抗毒素（TA）系统/模块的研究加上其在结核分枝杆菌中丰度的生物信息学分析表明，这些模块与潜伏性结核分枝杆菌中表现出的特征性缓慢生长和休眠之间可能存在关联。TA毒素的表达赋予休眠状态，并与细菌持久性有关，这是潜伏性结核病感染固有的两种性质。TA毒素介导的休眠状态是在研究中发现的，它是大肠杆菌六种染色体编码的TA毒素之一。大肠杆菌中称为MazF（来源于mazEF TA模块）。与E.大肠杆菌和其他自由生活的生物体中，Mtb基因组具有非常高数量的TA毒素模块。而E. coli具有一个MazEF TA模块，Mtb基因组含有11个类似模块。由于Mtb细胞从活跃复制状态到休眠样状态的转化被认为与结核病的潜伏期相一致，并且因为MazF表达触发了E.大肠杆菌中，该R21应用测试了Mtb基因组特有的MazF的多个亲属有助于该生物体休眠的假设，这可能对于建立该病原体的标志性潜伏状态至关重要。第一个目的的目标是确定单个MazF-mt TA毒素的过表达或不存在是否影响Mtb生长速率和活力。第二个目标的目的是确定在已建立的Mtb非复制持久性体外模型中，一个或多个MazF对应物的缺失是否影响Mtb存活。公共卫生相关性：对Mtb感染潜伏期的分子机制了解甚少。详细了解这一过程是至关重要的，因为处于休眠状态的生物体对抗生素治疗是难治性的，混淆了对疾病的有效管理，从而使疾病能够持续传播。拟议的实验将有助于开发更有效的治疗方法，抑制触发或维持休眠或减缓生长的信号，从而提高传统结核分枝杆菌抗生素的疗效。

DESCRIPTION (provided by applicant): Tuberculosis is a widespread and potent human pathogen--at least on third of the world's population is infected with Mycobacterium tuberculosis (Mtb) yielding an annual death toll estimated at two to three million. Although the subject of intense study, this infection is extremely complex and is able to cause either an active disease or persist in a latent state. Latency represents an adaptation that has enabled the efficient persistence of Mtb in the human population. Latent infections (where the bacilli are thought to persist in the host macrophage) can be reactivated, especially in immune compromised individuals. In fact, elevated tuberculosis rates--and an alarming number of multi-drug resistant Mtb strains--are being documented in regions of high HIV incidence. The molecular switches that trigger Mtb latency are poorly understood. The study of toxin-antitoxin (TA) systems/modules in Escherichia coli coupled with bioinformatic analysis of their abundance in Mtb has suggested a possible association between these modules and the characteristic slow growth and dormancy exhibited by Mtb in latent tuberculosis. Expression of TA toxins impart a dormant state and have been linked to bacterial persistence, two properties intrinsic to latent tuberculosis infection. The TA toxin-mediated dormant state was discovered during the study the one of the six chromosomally encoded TA toxins in E. coli called MazF (derived from the mazEF TA module). In contrast to E. coli and other free-living organisms, the Mtb genome harbors a remarkably high number of TA toxin modules. While E. coli possesses a single mazEF TA module, the Mtb genome contains eleven analogous modules. Since conversion of Mtb cells from an actively replicating state to a dormant-like state is thought to coincide with the latency phase of tuberculosis, and because MazF expression triggers a type of dormancy in E. coli, this R21 application tests the hypothesis that the multiple relatives of MazF unique to the Mtb genome contribute to dormancy of this organism, which may be critical to establishing the state of latency that is the hallmark of this pathogen. The goal of the first aim is to determine if either overexpression, or the absence, of individual MazF-mt TA toxins influences Mtb growth rates and viability. The goal of the second aim is to determine if deletion of one or more MazF counterparts influences Mtb survival in established in vitro models of Mtb non-replicating persistence PUBLIC HEALTH RELEVANCE: The molecular mechanisms that underlie the latency phase of Mtb infection are poorly understood. It is crucial to understand this process in detail since organisms in a dormant state are refractory to antibiotic treatment, confounding effective management of the disease and thus enabling continued disease transmission. The proposed experiments will help in the development of more effective therapeutics that inhibit the signals which trigger or maintain dormancy or slow growth, thus increasing the efficacy of conventional Mtb antibiotics.