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.

摘要 细菌物种已经进化出多种机制来在生态、营养和化学胁迫下生存， 以及宿主细胞的防御。其中之一是进入可行但不可培养（VBNC）状态。顾名思义 表明，VBNC状态下的细菌已经失去了在常规琼脂上生长的能力，但事实上是活的，尽管在 休眠（不是孢子）状态。细菌物种进入VBNC状态以及复苏的能力 从这种状态现在被认为是一个重要的，甚至是必要的生存机制， 这些病原体是多种细菌病原体的致病机制，尤其是与慢性感染相关的病原体。 这些拟议的研究旨在从遗传和代谢方面表征VBNC状态， 从口腔病原体牙龈卟啉单胞菌的VBNC状态复苏。目标，除了定义P。 本发明的另一个目的是鉴定VBNC状态下牙龈炎基因表达和代谢的一种或多种生物标志物， 牙龈卟啉单胞菌菌株W83（PgW83）的VBNC状态。此外，敲除基因被发现是特异性的或 在VBNC期间高度表达，并将构建复苏状态。这些变种人将会 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 状态，在体内动物模型中宿主细胞侵袭和毒力。我们的假设是 具有复苏能力的VBNC亚群允许牙龈卟啉单胞菌在这些条件下存活。 它遇到的细胞内压力，从而允许牙龈卟啉单胞菌存在并持续处于慢性状态， 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 细胞和体外方法来建立PgW 83的VBNC和复苏状态。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志物。生物标志 将构建突变体并测试它们进入VBNC并在宿主细胞内复苏的能力。 最后，我们将用PgW 83野生型和突变体治疗在转化和复苏中有缺陷的小鼠。 从VBNC状态来确定牙龈卟啉单胞菌的这些状态是否是慢性牙周炎所必需的。 这些研究将为牙龈卟啉单胞菌在慢性炎症过程中发生的分子事件提供新的见解。 感染，如牙周炎，并确定新的蛋白质目标，为未来的治疗。