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 霍乱是一种以威胁生命的脱水为特征的腹泻疾病。它是由血清型引起的 革兰氏阴性细菌弧菌霍乱，产生霍乱毒素。与流行有关的菌株之一 霍乱爆发是O1血清型El Tor。 V. Cholerae el tor形成生物膜，具有保护性 细菌免受环境压力的社区。第二个Messenger bis-（3'-5'） - 环状二甘氨酸单磷酸盐（C-DI-GMP）是摩托车和无柄（生物膜）之间过渡的关键 生活方式，具有与生物膜形成相关的高水平C-DI-GMP。 C-DI-GMP的合成需要 二烷基酸环化酶。一个这样的二烷基酸环化酶，由VCA0956编码并命名 CDGF已与生物膜形成以及与超感染细菌聚集体的形成有关 在晚期感染期间形成形式，并从肠道中释放出来，准备细菌重新进入 水环境。 CDGF表达的机制尚不清楚。在这个项目中，我们 将研究CDGF表达的假设由发散编码的MARR家族控制 我们命名为DGCR的转录因子，该DGCR对配体C-DI-GMP响应，从而创建了一个 维持C-DI-GMP合成的正馈回循环。我们还建议CDGF的初始表达 当DGCR被氧化时发生，这种事件减弱了其结合CDGF启动子并抑制的能力 转录。 DGR通过DNase I足迹在体外确定DNA和配体结合，并通过 在不存在和存在配体的情况下，DGCR的生物物理分析。在体内控制CDGF启动子活性将 在大肠杆菌中使用CDGF启动子培养剂构建体，也表达诱导型DGCR，并以此 C-DI-GMP或氧化剂具有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。预计提出的实验的完成 描绘出一个新颖的饲料式系统，其中C-DI-GMP维持其自身的合成，并有望 定义氧化应激与C-DI-GMP积累之间的直接联系。知道哪种机制 生产临床上相关的CDGF酶对于更好地理解V.霍乱El tor很重要 致病性，它将为干扰这种调节而打开前景，从而阻止形成 常规抗菌剂无效的生物膜群落。