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

基本信息

批准号：
10198717
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
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-19 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10198717
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

项目摘要

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.

项目概要/摘要霍乱是一种腹泻病，其特征是危及生命的脱水。它是由血清型引起的革兰氏阴性细菌霍乱弧菌，产生霍乱毒素。与地方病有关的菌株之一爆发的霍乱是O1 血清型El Tor。 El Tor 霍乱弧菌形成生物膜，具有保护作用细菌免受环境压力影响的群落。第二信使双-(3'-5')- 环二鸟苷酸单磷酸 (c-di-GMP) 是运动和固着（生物膜）之间转变的关键生活方式，高水平的 c-di-GMP 与生物膜形成相关。 c-di-GMP 的合成需要二鸟苷酸环化酶。一种这样的二鸟苷酸环化酶，由VCA0956编码并命名为 CdgF 与生物膜的形成和高度感染性细菌聚集体的形成有关，在感染后期形成，并从肠道释放，为细菌重新进入肠道做准备。水环境。 cdgF 表达的调节机制尚不清楚。在这个项目中，我们将研究 cdgF 表达由不同编码的 MarR 家族控制的假设我们将其命名为 DgcR 的转录因子，DgcR 响应配体 c-di-GMP，从而产生维持 c-di-GMP 合成的正反馈回路。我们还建议 cdgF 的初始表达当 DgcR 被氧化时发生，该事件削弱了其结合 cdgF 启动子并抑制的能力转录。 DgcR 与 DNA 和配体的结合将在体外通过 DNase I 足迹法和在配体不存在和存在的情况下对 DgcR 进行生物物理分析。体内 cdgF 启动子活性的控制将可以在也表达诱导型 dgcR 的大肠杆菌中使用 cdgF 启动子报告基因构建体来解决，并且将测定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。预计完成拟议的实验描绘了一种新颖的反馈系统，其中 c-di-GMP 维持其自身的合成，并且预计定义氧化应激和 c-di-GMP 积累之间的直接联系。了解其机制产生临床相关的 CdgF 酶对于更好地了解霍乱弧菌 El Tor 非常重要致病性，它将为干扰这种调节开辟前景，从而防止形成传统抗菌剂对其无效的生物膜群落。