Mechanism of methylthioadenosine nucleosidase-mediated inhibition of biofilm form

甲硫腺苷酶介导的生物膜形成抑制机制

• 批准号: 8098992
• 负责人: Jorge Antonio Benitez
• 金额: $ 29.02万
• 依托单位: SOUTHERN RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8098992
• 关键词: Anabolism; Antibiotic Therapy; Antibiotics; Bacteria; Bacterial Physiology; Biochemical Reaction; Biocide; Biology; Cell Density; Chemical Structure; Chemicals; Cholera; Chronic; Clinical; Communicable Diseases; Communities; Confocal Microscopy; Disease; Disinfectants; Environment; Enzymes; Feeds; Food; Frameshift Mutation; Genes; Gifts; Homologous Gene; Horizontal Gene Transfer; Host Defense; Infection; Measurement; Mediating; Medicine; Microbial Biofilms; Microbial Genetics; Nucleoside Hydrolases; Organism; Pathway interactions; Pharmacologic Substance; Prevention; Reaction; Relapse; Role; Second Messenger Systems; Sensory; Signal Transduction; Signal Transduction Pathway; System; Transcription Coactivator; VAI-2; Vibrio; Vibrio cholerae; Vibrio cholerae O1; Vibrio vulnificus; Virulence; Virulence Factors; analog; bacterial resistance; chemical genetics; college; design; improved; in vitro Assay; infectious disease treatment; inhibitor/antagonist; mutant; novel; pathogenic bacteria; prevent; public health relevance; quorum sensing; resistance mechanism; response; second messenger; small molecule; tool; waterborne; waterborne infection

DESCRIPTION (provided by applicant): The formation of biofilm communities poses a major challenge to the prevention and treatment of infectious diseases. It is well established that bacteria within biofilm communities are highly recalcitrant to elimination with biocides, classical antibiotics and disinfectants. Biofilm formation has been suggested to enhance the survival and persistence of numerous pathogenic bacteria in their natural reservoirs. Furthermore, treatment of biofilm-related infections require long-term antibiotic therapies that favor the acquisition of resistance mechanisms while the high cell density in the biofilm environment provides an ideal scenario for horizontal gene transfer. Small molecules capable of inhibiting biofilm formation can provide new means to prevent and treat infectious disease. Vibrio cholerae of serogroups O1 and O139 causes the diarrheal disease cholera, while the closely related species Vibrio vulnificus can cause fatal systemic food and waterborne infections. Both species have been shown to produce quorum sensing regulated biofilms and possess analogous signal transduction pathway responding to autoinducer 2. Recently, transition state analogs of the enzyme methylthioadenosine nucleosidase (MTAN or pfs nucleosidase) that block the biosynthesis of autoinducer 2 were found to inhibit biofilm formation in V. cholerae. Transition state analogs are compounds with a chemical structure that resembles the "high energy" intermediate to which a substrate is converted in a catalytic reaction but to not undergo further conversion to products and act as potent inhibitors. In this application we will use the transition state inhibitors of MTAN MT- immucillin-A and MT-DADMe-immucillin-A as a tool to improve our understanding of the role of MTAN and autoinducer 2 in quorum sensing and biofilm formation in V. cholerae and V. vulnificus. In Aim 1 we will determine the mechanism by which inhibition of MTAN diminishes biofilm formation in V. cholerae. To this end, we will combine chemical inhibition studies, quorum sensing mutants and the manipulation/measurement of the intracellular concentration of second messengers and key metabolites to explain the effect of these inhibitors on biofilm formation. Quorum sensing co-regulates biofilm formation and the expression of virulence factors in pathogenic Vibrios. Therefore, in Aim 2 we will determine if transition state inhibitors of MTAN act through the same pathway in V. cholerae and V. vulnificus and study their effect on the expression of virulence factors in the two pathogenic Vibrios. Again, we will combine chemical inhibition studies, microbial genetics and in vitro assays of virulence to establish the role of MTAN in the expression of virulence factors. PUBLIC HEALTH RELEVANCE: The formation of bacterial biofilms poses a major challenge to the prevention and treatment of infectious diseases since bacteria within biofilm communities are highly recalcitrant to elimination with biocides, classical antibiotics and disinfectants. A better understanding of the environmental signals and response pathways leading to biofilm formation could facilitate the rational design of biofilm inhibitors to prevent and treat infectious disease. In this application we propose to use a combined chemical biology and microbial genetic approach to increase our understanding of the mechanism that triggers biofilm formation in two pathogenic Vibrios (Vibrio cholerae and Vibrio vulnificus) responsible for major food and waterborne illness.

描述（由申请人提供）：生物膜群落的形成对预防和治疗传染病构成了重大挑战。众所周知，生物膜群落中的细菌是用杀菌剂，经典抗生素和消毒剂消除的高度顽固性。已经提出生物膜形成以增强其自然储层中众多致病细菌的生存和持久性。此外，与生物膜相关感染的治疗需要长期的抗生素疗法，这有利于获得耐药机制，而生物膜环境中的高细胞密度为水平基因转移提供了理想的情况。能够抑制生物膜形成的小分子可以提供预防和治疗传染病的新方法。血清群O1和O139的弧菌霍乱会引起腹泻病霍乱，而密切相关的物种弧菌可引起致命的全身性食物和水传播感染。两种物种均已证明可以产生构体调节的生物膜，并具有对自动诱导剂2响应的类似信号转导途径。最近，甲基甲基腺苷核苷核苷酶（MTAN或PFS核苷酶）的过渡状态类似物，可阻止自动化生物合成的生物合成2的生物合成。过渡态类似物是具有化学结构的化合物，类似于“高能量”中间体，底物在催化反应中转化为，而不是进一步转化为产品并充当有效的抑制剂。在此应用中，我们将使用MTAN MT-Immucillin-A和MT-Dadme-Immucillin-A的过渡状态抑制剂作为一种工具，以提高我们对MTAN和AutoDucter 2在Quorum Sensing和V. vulnificus中MTAN和自动诱导剂2中的作用的理解。在AIM 1中，我们将确定抑制MTAN在V.霍乱中减少生物膜形成的机制。为此，我们将结合化学抑制研究，法定感应突变体以及对第二信使的细胞内浓度的操纵/测量，以解释这些抑制剂对生物膜形成的影响。法定人感应共同调节生物膜的形成和致病性颤音中毒力因子的表达。因此，在AIM 2中，我们将确定MTAN的过渡状态抑制剂是否通过V.霍乱和V. vulnificus中的相同途径作用，并研究它们对两种致病颤音中毒力因子表达的影响。同样，我们将结合化学抑制研究，微生物遗传学和毒力的体外测定法，以确立MTAN在毒力因子表达中的作用。 公共卫生相关性：细菌生物膜的形成对预防和治疗传染病构成了重大挑战，因为生物膜群落中的细菌对消除杀害剂，经典抗生素和消毒剂的消除是高度顽固的。更好地了解导致生物膜形成的环境信号和反应途径可以促进生物膜抑制剂的理性设计，以预防和治疗传染病。在此应用中，我们建议使用一种合并的化学生物学和微生物遗传方法，以增加对触发两种致病性颤音（Vibrio Cholerae和Vibrio vibrious）中生物膜形成的机制的理解，负责主要食品和水生疾病。

项目成果

Role of methylthioadenosine/S-adenosylhomocysteine nucleosidase in Vibrio cholerae cellular communication and biofilm development.

• DOI: 10.1016/j.bbrc.2015.03.170
• 发表时间: 2015-05-22
• 期刊: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
• 影响因子: 3.1
• 作者: Silva, Anisia J.;Parker, William B.;Allan, Paula W.;Ayala, Julio C.;Benitez, Jorge A.
• 通讯作者: Benitez, Jorge A.