Functional Dissection of Toxin-Antitoxin Systems in Mycobacterium tuberculosis

描述（由申请人提供）：结核病是一种广泛而有效的人类病原体——世界上至少有三分之一的人口感染结核分枝杆菌（Mtb），每年死亡人数估计为200万至300万。虽然是研究的重点，但这种感染非常复杂，既可以引起活动性疾病，也可以持续处于潜伏状态。潜伏期代表了一种适应，使结核分枝杆菌能够在人群中有效地持续存在。潜伏感染（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，在艾滋病毒高发地区，结核病发病率上升，耐多药结核菌株数量惊人。人们对触发结核分枝杆菌潜伏期的分子开关知之甚少。大肠杆菌毒素-抗毒素（TA）系统/模块的研究以及对其在结核分枝杆菌中丰度的生物信息学分析表明，这些模块可能与结核分枝杆菌在潜伏性结核病中表现出的缓慢生长和休眠特征有关。TA毒素的表达赋予了一种休眠状态，并与细菌的持久性有关，这是潜伏性结核感染的两个固有特性。在研究中发现了大肠杆菌中六种染色体编码的TA毒素之一的MazF（源自mazEF TA模块）的TA毒素介导的休眠状态。与大肠杆菌和其他自由生物相比，结核分枝杆菌基因组含有大量的TA毒素模块。大肠杆菌只有一个mazEF TA模块，而结核分枝杆菌基因组包含11个类似的模块。由于Mtb细胞从积极复制状态转化为休眠样状态被认为与结核病的潜伏期相吻合，并且由于MazF表达在大肠杆菌中引发了一种休眠，因此R21应用验证了一种假设，即Mtb基因组中独特的MazF的多个亲缘关系有助于该生物体的休眠，这可能对建立作为该病原体标志的潜伏期状态至关重要。第一个目的是确定过度表达或缺乏单个MazF-mt TA毒素是否会影响结核分枝杆菌的生长速度和生存能力。第二个目标是确定在已建立的非复制持久性Mtb体外模型中，一个或多个MazF对应物的缺失是否会影响Mtb的存活。详细了解这一过程至关重要，因为处于休眠状态的生物体对抗生素治疗难以耐受，从而影响疾病的有效管理，从而使疾病继续传播。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.