Genetic Requirements for the survival of Tubercle Bacilli in Nonhuman Primates

描述(申请人提供)：结核病(TB)，由结核分枝杆菌(Mtb)引起，是一种全球性紧急传染病。据估计，每年有800万人罹患结核病，约200万人死于结核病。与艾滋病的协同作用、抗药性的出现以及缺乏有效的抗结核病药物和疫苗使这种情况更加恶化。迫切需要新的药物和疫苗来有效控制结核病。这需要更好地了解结核分枝杆菌如何适应在感染的不同阶段不可避免地面临的各种环境条件。可以说，非人类灵长类动物(HHP)是结核病关键方面的最佳模型。因此，分析结核分枝杆菌成功感染NHP肺部并持续存在的机制将是非常有用的。我们研究了结核分枝杆菌转座子突变体库中暴露于高剂量气溶胶的NHP肺中结核分枝杆菌生长/存活所必需的基因。在这个急性结核病模型中，与只有6%的突变株在体内生长的小鼠模型相比，所有被测试的突变体中有33.13%的突变株在体内生长减弱。Mtb突变体在灵长类动物体内的存活能力减弱，参与了脂类毒力因子的运输；细胞壁阿拉伯糖和多肽、脂肪酸和多酮的生物合成；DNA修复；甾醇代谢和哺乳动物细胞进入(MCE)。我们的研究强调了结核分枝杆菌用于感染和克服NHP肺部感染期间所遇到的恶劣环境的各种毒力机制。我们希望利用我们的能力来模拟人类结核病的不同临床阶段-急性、肺结核病、慢性进展性结核病和NHP中的潜伏性、无症状结核病-以研究结核分枝杆菌突变株的生长/生存表型。此外，我们希望利用NHP模型更好地了解两个Mtb通路在毒力和致病机制中的作用。这些操纵子包括mce1/mce4操纵子，其成员是在NHP肺中因生长而减弱的突变体；以及DOS调节子的成员，尽管它们在潜伏期、持久性和耐低氧防御中的作用明确，但令人惊讶的是，它们在NHP肺中没有减弱。 公共卫生相关性：结核病(TB)是一种全球紧急情况，每年造成约200万人死亡。猴子由于在基因和生理上与人类相似，是建立结核病模型的一个非常好的动物系统。在结核杆菌染色体中存在的数百个基因中，我们已经确定了哪些基因是在猴子的肺部成功感染所必需的。这些结果将与从患有结核病/艾滋病的动物身上获得的结果进行比较。来自MCE(哺乳动物细胞进入)家族的基因是在NHP肺中衰减的基因之一。我们将使用结核分枝杆菌(Mtb)进行功能研究，并使用我们的猴子模型定义mce基因的突变，以更好地了解它们在疾病过程中的作用。

DESCRIPTION (provided by applicant): Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a global infectious disease emergency. Each year an estimated 8 million people develop, and about two million people die of TB. Synergy with AIDS, the emergence of drug-resistance and the lack of effective anti-TB drugs and vaccines has worsened this situation. New drugs and vaccines are urgently needed to effectively control TB. This requires a better understanding of how Mtb adapts to a wide-variety of environmental conditions, inevitably faced by it during the various stages of infection. Nonhuman Primates (NHPs), arguably, best model critical aspects of TB. Analysis of the mechanisms employed by Mtb to successfully infect and persist in NHP lungs would therefore be very useful. We studied genes essential for growth/survival of Mtb in the NHP lungs experimentally exposed to high doses of aerosols of an Mtb transposon mutant library. In this acute model of TB, 33.13% of all tested mutants were attenuated for in-vivo growth compared to the mouse model where only ~6% of all mutants are attenuated. The Mtb mutants attenuated for in-vivo survival in primates were involved in the transport of lipid virulence factors; biosynthesis of cell-wall arabinan and peptidoglycan, fatty-acids and polyketides; DNA repair; sterol metabolism and mammalian cell-entry (mce). Our study highlights the various virulence-mechanisms employed by Mtb for infection and to overcome the hostile environment encountered during infection of NHP lungs. We would like to leverage our ability to model the various clinical phases of human TB - acute, pulmonary TB, chronic-progressive TB and latent, asymptomatic TB in NHPs - to study the growth/survival phenotype profiles of Mtb mutants. Further, we would like to better understand the role of two Mtb pathways crucial for virulence and pathogenesis, using the NHP model. These include the mce1/mce4 operons, whose members were among mutants that were attenuated for growth in NHP lungs; and members of the dos regulon, which were surprisingly not attenuated in NHP lungs, in-spite of their well-defined roles in latency, persistence and defense against hypoxia. PUBLIC HEALTH RELEVANCE: Tuberculosis (TB) is a global emergency, responsible for the death of an estimated 2 million people annually. Monkeys, due to their genetic and physiological similarities to humans, are a very good animal system to model TB. Out of the several hundreds of genes present in the chromosome of the tubercle bacillus, we have determined which ones are needed to establish a successful infection in the lungs of monkeys. These results will be compared to those obtained from animals with TB/AIDS co-infection. Genes from the mce (mammalian cell entry) family were among those which were attenuated in the NHP lungs. We will perform functional studies using Mycobacterium tuberculosis (Mtb) and defined mutants in mce genes, using our monkey model, to better understand their role in disease process.