Functional Dissection of Toxin-Antitoxin Systems in Mycobacterium tuberculosis

描述(申请人提供)：结核病是一种广泛和强有力的人类病原体--世界上至少三分之一的人口感染结核分枝杆菌(Mtb)，每年造成约200万至300万人死亡。虽然是密集研究的主题，但这种感染极其复杂，既能导致活动性疾病，也能持续潜伏状态。潜伏期代表了一种适应，它使得结核分枝杆菌在人类群体中能够有效地持续存在。潜伏感染(细菌被认为持续存在于宿主巨噬细胞中)可以重新激活，特别是在免疫受损的人身上。事实上，在艾滋病毒高发地区，结核病发病率上升--以及数量惊人的耐多药结核分枝杆菌菌株--正在被记录在案。触发MTB潜伏期的分子开关还知之甚少。对大肠杆菌中毒素-抗毒素(TA)系统/模块的研究以及对它们在结核分枝杆菌中丰度的生物信息学分析表明，这些模块与结核分枝杆菌在潜伏期结核病中表现出的缓慢生长和休眠特征之间可能存在关联。TA毒素的表达使人处于休眠状态，并与细菌持久性有关，这是潜伏性结核病感染固有的两个特性。在研究中发现了TA毒素介导的休眠状态，这是大肠杆菌中六种染色体编码的TA毒素之一，称为MazF(来源于mazEF TA模块)。与大肠杆菌和其他自由生物不同，结核分枝杆菌基因组含有相当多的TA毒素模块。而大肠杆菌只有一个mazEF TA模块，而Mtb基因组包含11个类似的模块。由于结核分枝杆菌细胞从活跃复制状态到类休眠状态的转换被认为与结核病的潜伏期相一致，并且由于MazF表达在大肠杆菌中触发了一种类型的休眠，因此R21的应用验证了这样的假设，即MazF基因组特有的MazF的多个亲属有助于该生物的休眠，这可能对于建立这种病原体的特征潜伏期状态至关重要。第一个目标是确定单个MazF-Mt TA毒素的过度表达或缺失是否会影响Mtb的生长速度和活性。第二个目标是确定在已建立的结核分枝杆菌非复制性持久性公共卫生相关性体外模型中，一个或多个MazF对应物的缺失是否会影响结核分枝杆菌的存活：结核分枝杆菌感染潜伏期的分子机制尚不清楚。至关重要的是，必须详细了解这一过程，因为处于休眠状态的生物体对抗生素治疗难以奏效，从而混淆了对疾病的有效管理，从而使疾病能够继续传播。拟议的实验将有助于开发更有效的疗法，抑制触发或维持休眠或缓慢生长的信号，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.