Three-dimensional analysis and modeling of the Chlamydia developmental cycle

 描述(申请人提供)：从公共卫生的角度来看，衣原体是最重要的传染病病原体之一。2011年，美国疾病控制与预防中心报告了超过140万例衣原体感染病例，使其成为美国最常见的传染病。衣原体引起的细胞内感染在病原菌中是不寻常的，因为它涉及两种特殊形式的细菌之间的转换。网状小体(RB)是一种通过二次分裂进行复制的细胞内形式，而基本体(EB)是一种可将感染传播给新细胞的感染形式。一个成功的感染周期涉及RB复制和RB到EB的转化，这两个过程都发生在称为衣原体包涵体的细胞内，但这些过程是如何调控的尚不清楚。我们开发了一种创新的方法来获得衣原体感染细胞的详细三维图像。我们首先对穿过细胞的连续切片进行电子显微镜成像，然后使用计算方法将这些扫描重建为3D图像，最后追踪每一种细菌的轮廓。使用这种方法，我们可以可视化整个衣原体包含以及所有RBS和EBS的数量和位置。在本申请的目标1中，我们建议获得并比较衣原体感染细胞在48小时发育周期中的3D重建。在目标2中，我们将使用数学和建模方法分析这些数据，以确定复制或转换的RBS比例是否随时间变化。我们还将检查RB复制和RB到EB的转化是否与外部因素相关，如包涵体的大小或包涵体膜的表面积。这一新的方法将提供关于衣原体感染细胞的前所未有的定量和空间细节。这些信息将帮助我们了解细胞内感染的基本性质，包括这种重要的病原体如何在人类细胞内复制和产生感染性后代。

 DESCRIPTION (provided by applicant): Chlamydia is one of the most important infectious agents from a public health perspective. In 2011 more than 1.4 million cases of chlamydial infections were reported to the CDC making it the most commonly reported infectious disease in the U.S. Chlamydia causes an intracellular infection that is unusual among pathogenic bacteria because it involves conversion between two specialized forms of the bacterium. The reticulate body (RB) is an intracellular form that replicates by binary fission, while the elementary body (EB) is the infectious form that can transmit the infection to a new cell. A successful infectious cycle involves both RB replication and RB-to-EB conversion within an intracellular compartment called the chlamydial inclusion, but it is not known how these processes are regulated. We have developed an innovative approach to obtain detailed three dimensional views of a Chlamydia-infected cell. We first perform electron microscopy imaging on serial sections through the cell, then use computational methods to reconstruct these scans into a 3D image and finally trace the outline of each of the bacteria. With this approach we can visualize the entire chlamydial inclusion and the numbers and locations of all the RBs and EBs. In Aim 1 of this application, we propose to obtain and compare 3D reconstructions of Chlamydia-infected cells over the course of the 48-hour developmental cycle. In Aim 2, we will analyze this data with mathematical and modeling approaches to determine if the proportion of RBs that are replicating or converting changes with time. We will also examine if RB replication and RB-to-EB conversion correlate with external factors such as the size of the inclusion or the surface area of the inclusion membrane. This novel approach will provide an unprecedented level of quantitative and spatial detail about a Chlamydia-infected cell. This information will help us to understand fundamental properties about the intracellular infection, including how this important pathogen replicates and produces infectious progeny inside a human cell.