Regulation of a gene associated with c-di-GMP production and biofilm formation in Vibrio cholerae

霍乱弧菌中与 c-di-GMP 生产和生物膜形成相关的基因的调控

• 批准号: 10038394
• 负责人: ANNE GROVE
• 金额: $ 6.94万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-19 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10038394
• 关键词: Address; Antibiotic Resistance; Antibiotics; Attenuated; Bacteria; Binding; Biological Assay; Characteristics; Cholera; Cholera Toxin; Communities; Complement; Complex; Cysteine; DNA; DNA Binding; DNase-I Footprinting; Data; Dehydration; Diarrhea; Disease Outbreaks; Electrophoretic Mobility Shift Assay; Environment; Enzymes; Epidemic; Escherichia coli; Event; Family; Feedback; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic Transcription; Gram-Negative Bacteria; Host Defense; Human; In Vitro; Infection; Intergenic DNA; Intestines; Knowledge; Lead; Life; Life Style; Ligand Binding; Ligands; Link; Mediating; Microbial Biofilms; Names; Outcome; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxides; Pathogenesis; Pathogenicity; Periodicity; Plasmids; Preparation; Process; Production; Proteins; Purines; Reactive Oxygen Species; Regulation; Reporter; Reporting; Repression; Role; Second Messenger Systems; Serotyping; Site-Directed Mutagenesis; Specificity; Stress; Surface; System; Vibrio cholerae; Virulence; antimicrobial drug; aqueous; base; biophysical analysis; cell motility; clinically relevant; diarrheal disease; diguanylate cyclase; experimental study; in vivo; insight; monomer; mortality; novel; oxidation; prevent; programs; promoter; response; transcription factor

PROJECT SUMMARY/ABSTRACT Cholera is a diarrheal disease characterized by life-threatening dehydration. It is caused by serotypes of the Gram-negative bacterium Vibrio cholerae, which produce cholera toxin. One of the strains linked to endemic cholera outbreaks is the O1 serotype El Tor. V. cholerae El Tor forms biofilms, which are protective communities in which the bacteria are shielded from environmental stress. The second messenger bis-(3'-5')- cyclic-diguanylate monophosphate (c-di-GMP) is key to the transition between motile and sessile (biofilm) lifestyles, with high levels of c-di-GMP associated with biofilm formation. Synthesis of c-di-GMP requires diguanylate cyclase enzymes. One such diguanylate cyclase, which is encoded by VCA0956 and named CdgF, has been linked to biofilm formation and to formation of the hyper-infective bacterial aggregates that form during late infection and are released from the intestinal tract in preparation for bacterial re-entry into the aqueous environment. Mechanisms by which cdgF expression is regulated are unknown. In this project, we will investigate the hypotheses that cdgF expression is controlled by the divergently encoded MarR family transcription factor that we named DgcR, and that DgcR responds to the ligand c-di-GMP, thus creating a positive feed-back loop that sustains c-di-GMP synthesis. We also propose that initial expression of cdgF occurs when DgcR is oxidized, an event that attenuates its ability to bind the cdgF promoter and repress transcription. DNA and ligand binding by DgcR will be determined in vitro by DNase I footprinting and by biophysical analyses of DgcR in absence and presence of ligand. Control of cdgF promoter activity in vivo will be addressed using cdgF promoter-reporter constructs in E. coli also expressing inducible dgcR, and the ability of c-di-GMP or oxidant to de-repress gene expression will be determined. In vitro transcription assays will be implemented to complement the in vivo assays. Completion of the proposed experiments is expected to delineate a novel feed-back system in which c-di-GMP sustains its own synthesis, and it is expected to define a direct link between oxidative stress and c-di-GMP accumulation. Knowing the mechanisms by which the clinically relevant CdgF enzyme is produced is important for a better understanding of V. cholerae El Tor pathogenicity, and it will open prospects for interfering with this regulation and hence prevent formation of the biofilm communities against which conventional antimicrobial agents are ineffective.

项目摘要/摘要 霍乱是一种以脱水为特征的慢性疾病。它是由血清型的 革兰氏阴性细菌霍乱弧菌，产生霍乱毒素。其中一种与地方病有关的菌株 霍乱爆发是O 1血清型埃尔托。霍乱弧菌El Tor形成生物膜， 在这些群落中，细菌可以免受环境压力的影响。第二信使bis-（3 '-5'）- 环二鸟苷酸单磷酸（c-di-GMP）是运动和固着（生物膜）之间转换的关键 生活方式，与高水平的c-di-GMP与生物膜形成。合成c-di-GMP需要 二鸟苷酸环化酶。其中一种二鸟苷酸环化酶由VCA 0956编码，命名为 CdgF与生物膜的形成和高感染性细菌聚集体的形成有关， 在晚期感染期间形成，并从肠道释放，为细菌重新进入肠道做准备。 水环境调节cdgF表达的机制尚不清楚。本课题 将研究cdgF表达受差异编码的马尔R家族控制的假设 转录因子，我们命名为DgcR，DgcR响应配体c-di-GMP，从而创造了一个 正反馈回路，维持c-di-GMP合成。我们还提出cdgF的初始表达 当DgcR被氧化时发生，这一事件减弱了其结合cdgF启动子和抑制cdgF表达的能力。 转录。DgcR的DNA和配体结合将通过DNA酶I足迹法和DNA印迹法在体外测定。 在不存在和存在配体的情况下的DgcR的生物物理分析。体内cdgF启动子活性的控制将 在E.大肠杆菌也表达诱导型dgcR， 将测定c-di-GMP或氧化剂去抑制基因表达的能力。体外转录测定 将实施以补充体内测定。预计将完成拟议的实验 描绘了一个新的反馈系统，其中c-di-GMP维持其自身的合成，预计 定义氧化应激和c-di-GMP积累之间的直接联系。了解了 产生临床相关的CdgF酶对于更好地理解霍乱弧菌ElTor 致病性，它将开辟干扰这种调节的前景，从而防止形成的 常规抗微生物剂对其无效的生物膜群落。