Community behavior of Yersinia pseudotuberculosis within microcolonies

 描述(申请人提供)：几十年来，众所周知细菌群体是异质性的，然而，开发有效消除细菌群体所有成员的疗法一直是极其困难的。这一困难很大程度上在于单个细菌在一个群体中以不同的速度复制，这导致了抗菌素敏感性的差异。目前尚不清楚是什么原因导致感染期间细菌生长速度的差异，以及宿主免疫反应是否可以减缓细菌亚群的增长。荧光成像的最新进展使得在单细胞水平上研究细菌种群内的异质性成为可能。使用荧光转录记者，我们已经表明，假结核杆菌根据单个细胞对宿主来源的压力的反应，在单个细菌复制地点形成多个亚群。单个细菌与宿主细胞的接近推动了基因表达的变化，导致复制集群(称为微克隆)外围有少量细菌对宿主来源的压力做出专门反应。寄主压力对单个细菌的影响尚不清楚；这是否会导致微菌落外围产生缓慢增长的种群？这项建议中描述的研究将利用荧光报告结构来可视化细菌在感染过程中对宿主应激的反应，并确定宿主衍生的应激对细菌生长速度的影响。 我们假设微菌落外围的单个细菌同时对多个宿主压力做出反应，这导致细菌在这个亚群中的复制速度较慢。为了验证这一假说，我们将：1)利用稳定和不稳定的荧光蛋白来确定宿主施加的应激是暂时的，还是在感染过程中保持的；2)建立假结核杆菌肠道感染的口服喂养模型，以确定肠道组织内基因表达的空间调节是否影响细菌向深层组织传播的能力；以及3)开发微流体模型来实时成像应激反应中的细菌复制，以确定单个细菌在应对单一应激或同时对多种应激做出反应时会发生什么。虽然这些研究将集中在假结核杆菌的生长上，但在这一建议中开发的模型可以很容易地应用于进一步研究其他肠道病原体和其他与医学相关的细菌病原体，以更好地了解驱动缓慢生长的细菌种群形成的机制。通过更好地了解这一过程，我们希望为开发新的治疗方法以消除所有细菌群体成员，从而清除感染提供相关信息。

 DESCRIPTION (provided by applicant): It has been known for decades that bacterial populations are heterogeneous, however, it has been extremely difficult to develop therapeutics that effectively eliminate all members of bacterial populations. Much of this difficulty lies in th fact that individual bacteria are replicating at different rates within a population, and this lead to differences in antimicrobial susceptibility. In remains unclear what drives differences in bacteria growth rates during infection, and whether the host immune response can slow the growth of subpopulations of bacteria. Recent advances in fluorescence imaging have enabled the study of heterogeneity within bacterial populations at the single cell level. Using fluorescent transcriptional reporters, we have shown that Y. pseudotuberculosis forms multiple subpopulations within a single site of bacterial replication, based on the response of individual cells to host-derived stresses. The proximity of individual bacteria to host cells drove gene expression changes, resulting in a small population of bacteria around the periphery of replicating clusters (called microcolonies) that specifically responds to host-derived stress. The impact of host stress on individual bacteria remains unclear; does this lead to the generation of a slow growing population on the periphery of microcolonies? The studies described in this proposal will utilize fluorescent reporter constructs to visualize bacterial responses to host stress over the course of infection, and determine the effects of host-derived stresses on bacterial growth rates. We hypothesize that individual bacteria around the periphery of microcolonies simultaneously respond to multiple host stresses, which results in a slower rate of bacterial replication within this subpopulation. To test this hypothesis, we will: 1) utilize stable and unstable fluorescent proteins to determine whether the stresses imparted by the host are transient, or maintained over the course of infection; 2) develop an oral feeding model of Y. pseudotuberculosis intestinal infection, to determine if spatial regulation of gene expression within intestinal tissues impacts the ability of bacteria to disseminate to deep tissues; and 3) develop a microfluidics model to live image bacterial replication during stress responses, to determine what happens to individual bacteria as they respond to single stresses or multiple stresses simultaneously. Although these studies will focus on Y. pseudotuberculosis growth, the models developed within this proposal can easily be applied to further the study of other enteric pathogens, and additional medically-relevant bacterial pathogens, to better understand the mechanisms driving the formation of slow growing bacterial populations. By gaining a better understanding of this process, we hope to provide pertinent information for the development of novel therapeutics to eliminate all members of bacterial populations, and thus clear infection.