Investigating the viable but not culturable (VBNC) state in P. gingivalis

摘要 细菌物种进化出多种机制来生存生态、营养和化学压力，如 以及宿主细胞的防御。其中之一是进入可生存但不可培养(VBNC)状态。作为我的名字 表明，VBNC状态的细菌已经失去了在常规琼脂上生长的能力，但实际上是活的，尽管在 休眠(不是孢子)状态。细菌进入VBNC状态和复苏的能力 这种状态现在被认为是一种重要的甚至是必要的生存和 病原体几种细菌病原体的致病机制，尤指与慢性感染有关的病原体 这些拟议的研究旨在从遗传学和代谢学的角度描述VBNC状态和 从口腔病原体牙龈假单胞菌的VBNC状态复苏。目标是，除了定义P. VBNC状态下牙周炎基因的表达和代谢也是为了确定一个或多个 牙龈假单胞菌W83株(PgW83)的VBNC状态。此外，对被发现为或特异的基因的敲除 在VBNC期间高度表达，复苏状态将被构建。这些突变体将会是 在体外和体内模型中进行测试，以确定对这些基因的需求，从而确定VBNC 在体内动物模型中，宿主细胞的侵袭和毒力状态。我们的假设是， 具有复苏能力的VBNC亚群使牙龈假单胞菌能够在 它遇到的细胞内压力，从而允许牙龈假单胞菌存在并持续在一种慢性状态 感染。我们设计了体外和体内实验来验证这一假设。首先，我们建议使用 建立PgW83的VBNC和复苏状态的细胞和体外方法。接下来，我们将表演 RNAseq和代谢组学，以确定VBNC和复苏状态的可能生物标记物。生物标志物 将构建并测试突变体进入VBNC并在宿主细胞内复苏的能力。 最后，我们将处理PgW83野生型和突变体在过渡和复苏过程中存在缺陷的小鼠 从VBNC状态来确定这些状态的牙龈假单胞菌是否对慢性牙周炎是必需的。 这些研究将为了解慢性牙周炎患者牙龈假单胞菌的分子事件提供新的见解。 例如牙周炎等感染，并为未来的治疗确定新的蛋白质靶点。

ABSTRACT Bacterial species have evolved multiple mechanisms to survive ecological, nutritional and chemical stresses as well as host cell defenses. Among these is to enter a viable but non-culturable (VBNC) state. As the name indicates, bacteria in the VBNC state have lost the ability to grow on routine agar, but are in fact, alive, albeit in a dormant (not spore) state. The ability of a bacterial species to enter the VBNC state and also resuscitation from this state is now recognized to be an important or even required mechanism for the survival and pathogenesis of several bacterial pathogens, especially those that are associated with chronic infections. These proposed studies are designed to genetically and metabolically characterize the VBNC state and resuscitation from the VBNC state of the oral pathogen, P. gingivalis. The goal, in addition to defining the P. gingivalis gene expression and metabolism in the VBNC state is to also identify one or more biomarkers for the VBNC state of P. gingivalis strain W83 (PgW83). In addition, knockouts of genes found to be specific for or highly expressed during the VBNC and resuscitation states will be constructed. These mutants will then be tested in both in vitro and in vivo models to determine the requirement for these genes, and thus the VBNC state, in host cell invasion and virulence in an in vivo animal model. Our hypothesis is that the existence of VBNC subpopulations with the ability to resuscitate allows P. gingivalis to survive those conditions of intracellular stress that it encounters thus allowing P. gingivalis to exist and persist in a chronic state of infection. We have designed in vitro and in vivo experiments to test this hypothesis. First, we propose to use cellular and in vitro methods to establish the VBNC and resuscitation states of PgW83. Next, we will perform RNAseq and metabolomics to identify possible biomarkers for VBNC and resuscitation states. Biomarker mutants will be constructed and tested for their abilities to enter the VBNC and resuscitate within host cells. Finally, we will treat mice with PgW83 wildtype and mutants defective in transitioning into and resuscitation from the VBNC state to determine whether these states of P. gingivalis are necessary for chronic periodontitis. These studies will provide new insights into the molecular events of P. gingivalis occurring during chronic infections such as periodontitis and identify new protein targets for future therapies.