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

研究牙龈卟啉单胞菌的存活但不可培养 (VBNC) 状态

• 批准号: 10531137
• 负责人: Ann Progulske-Fox
• 金额: $ 36.22万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-13 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10531137
• 关键词: Agar; Animal Model; Bacteria; Biological Markers; Cells; Chemicals; Chronic; Complement; Data; Disease model; Event; Future; Gene Expression; Genes; Genetic Transcription; Goals; Growth; In Vitro; Infection; Invaded; Metabolic; Metabolism; Methods; Modeling; Molecular; Mus; Names; Nutritional; Pathogenesis; Pathway interactions; Pattern; Periodontal Diseases; Periodontitis; Population; Porphyromonas gingivalis; Proteins; Research; Resuscitation; Role; Site; Stress; Testing; Virulence; chronic infection; design; experimental study; in vivo; in vivo Model; insight; knockout gene; metabolomics; mouse model; mutant; oral pathogen; pathogenic bacteria; therapeutic protein; transcriptome sequencing

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.

抽象的 细菌种类已经发展了多种机制，可以生存生态，营养和化学应激，因为 以及宿主细胞防御。其中包括进入可行但不可培养（VBNC）状态。作为名称 表明VBNC状态的细菌失去了在常规琼脂上成长的能力，但实际上是活着的，尽管在 休眠（不是孢子）状态。细菌物种进入VBNC状态并复苏的能力 现在，从这个状态被认为是生存的重要机制，甚至是必需的机制 几种细菌病原体的发病机理，尤其是与慢性感染相关的病原体。 这些提出的研究旨在从遗传和代谢上表征VBNC状态和 口腔病原体的VBNC状态，牙龈疟原虫的复苏。除了定义P之外的目标。 VBNC状态下的牙龈牙龈基因表达和代谢也是识别一个或多个生物标志物 牙龈疟原虫菌株W83（PGW83）的VBNC状态。此外，发现基因的敲除特定于或 将在VBNC和复苏状态中高度表达。这些突变体将是 在体外和体内模型中测试以确定对这些基因的需求，因此VBNC 状态，在体内动物模型中的宿主细胞侵袭和毒力中。我们的假设是 具有复苏能力的VBNC亚群可以使牙龈疟原虫在那些条件下生存 它遇到的细胞内应力，从而使牙龈疟原虫的存在并持续存在 感染。我们已经在体外和体内实验设计了这一假设。首先，我们建议使用 细胞和体外方法来建立PGW83的VBNC和复苏状态。接下来，我们将表演 RNASEQ和代谢组学识别VBNC和复苏状态的可能生物标志物。生物标志物 突变体将被构造并测试其进入VBNC并在宿主细胞内复苏的能力。 最后，我们将用PGW83野生型和突变体在过渡到复苏方面有缺陷的小鼠 从VBNC状态确定这些牙龈疟原虫状态是否对于慢性牙周炎是必需的。 这些研究将为慢性期间发生的牙龈牙周假单胞菌的分子事件提供新的见解 感染（例如牙周炎）并确定未来疗法的新蛋白质靶标。