Nanostructured in vitro TB latency model

描述（由申请人提供）：结核分枝杆菌潜伏在20亿人体内，代表了结核病（TB）的主要储存库，但潜伏杆菌的状态仍然是一个谜。为了根除结核病，我们需要了解潜伏期并开发药物筛选的体外模型。在这个跨学科项目中，我们将微生物结核研究与纳米技术和新材料结合起来。最近的出版物展示了一种独特的现象，即混合脂质-无机纳米结构的细胞定向组装，它驱动了一种独特的非复制持久性，保护细菌并大大延长了生存能力。我们发现这些相同的结构可以用分枝杆菌形成，并且它们表现出高度扩展的生存能力特征。我们假设潜伏结核的持续存在及其对化疗挑战的抵抗力可以通过将杆菌结合到自组装的脂质无机纳米结构中来模拟。该模型结合了体外研究和生长条件调节的能力，具有体内植入和感染的潜力。我们将建立一种新的体外模型，用于高通量筛选潜伏性结核病的靶点和药物，以补充现有的复杂体内模型，并推动这一领域向前发展。明确分枝杆菌脂质-无机纳米结构包封的最佳条件及其对分枝杆菌耐药的影响。特异性目的2确定在脂质无机纳米结构中发生的分枝杆菌代谢变化，这些变化是由非复制持久性引起的，并驱动体外存活和潜伏期。通过在小鼠中显示其感染性，证明脂质无机纳米结构包裹的分枝杆菌是有效的潜伏期模型，即使在组装后很长一段时间后也是如此。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis latently persist in 2 billion people, representing a major reservoir of tuberculosis (TB) due to reactivation, yet the state of latent bacilli remains a mystery. In order to eradicate TB, we need to understand latency and develop in vitro models for drug screening. In this interdisciplinary project we have merged microbiological tuberculosis research with nanotechnology and novel materials. Recent publications demonstrate a unique phenomenon of cell-directed assembly of hybrid lipid-inorganic nanostructures that drive a unique non-replicative persistence, and protect bacteria and drastically prolong viability. We show these same structures can be formed using mycobacteria, and that they display the highly extended viability characteristic of latency. We hypothesize that the persistence of latent TB, and its resistance to chemotherapeutic challenge, can be modeled by incorporation of bacilli into self-assembled lipid-inorganic nanostructures. This model combines the ability for in vitro study and growth condition modulation, with the potential for in vivo implantation and infection. We will generate a new in vitro model for high throughput screening for targets of, and drugs that can eliminate latent TB, to complement the existing complex in vivo models and al- low this field to move forward. Specific Aim 1 Define the conditions for optimal lipid-inorganic nanostructure-encasement of mycobacteria and its effects upon mycobacterial resistance to drugs. Specific Aim 2 Determine the changes in mycobacterial metabolism occurring upon encasement in lipid- inorganic nanostructures that result from non-replicative persistence, and drive in vitro survival and latency. Specific Aim 3 Demonstrate that lipid-inorganic nanostructure-encased mycobacteria represent valid models of latency by showing their infectivity in mice, even after extended periods of time after assembly. PUBLIC HEALTH RELEVANCE: Public Health Relevance We need to understand the way that tuberculosis is able to exist in a latent state because it is present in 2 billion people worldwide and reactivates into an active form in a significant percentage of cases. Clearing this 'reservoir' will be an essential component of an eventual eradication strategy. Although there are animal models of latency, there is no good model of in vitro latency, yet we need to have such an in vitro model so that we can understand how to attack it, and also to screen drugs with. We have developed a unique nanostructured model that mimics the extreme non-replicative persistence of latency, and want to test how effective it might be as a tool in the battle against TB.