UTILIZATION OF STREPTOMYCES TO STUDY QUORUM SENSING IN MYCOBACTERIUM SMEGMATIS

利用链霉菌研究耻垢分枝杆菌的群体感应

• 批准号: 8167501
• 负责人: ANGELA MCKINNEY-WILLIAMS
• 金额: $ 3.93万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8167501
• 关键词: A-factor (Streptomyces); Actinomycetales; Animal Model; Antibiotics; Bacillus subtilis; Bacteria; Biological Assay; Bioluminescence; Candida albicans; Candidiasis; Cell physiology; Cells; Chemicals; Communication; Computer Retrieval of Information on Scientific Projects Database; DNA; Development; Disease; Enzymes; Epithelial; Exhibits; Farnesol; Fatty Acids; Fruit; Funding; Gene Expression; Gene Expression Regulation; Gene Transfer; Genes; Genome; Genus Mycobacterium; Gram-Negative Bacteria; Gram-Positive Bacteria; Grant; Growth; Guanine + Cytosine Composition; High Pressure Liquid Chromatography; Host Defense; Human; Hyphae; Infection; Institution; Invaded; Laboratories; Libraries; Life; Lung; Mass Spectrum Analysis; Mentors; Methods; Microbial Biofilms; Modeling; Molecular Weight; Mutagenesis; Mycelium; Mycobacterium leprae; Mycobacterium marinum; Mycobacterium smegmatis; Mycobacterium tuberculosis; Myxococcus; Nature; Nebraska; Nosocomial Infections; Oligopeptides; Pathogenesis; Pathogenicity; Phase; Pheromone; Pigmentation physiologic function; Pigments; Play; Population; Process; Production; Pseudomonas; Pseudomonas aeruginosa; Research; Research Design; Research Personnel; Resources; Role; Signaling Molecule; Soil; Source; Streptomyces; Streptomyces coelicolor; Streptomyces griseus; Streptomycin; Structure; System; Testing; Texas; Tissues; Toxin; Tuberculosis; United States National Institutes of Health; Universities; Vibrio; Virulence; Virulence Factors; Virulent; Work; Yeasts; analog; butyrolactone; cell motility; clinically significant; cystic fibrosis patients; density; ethyl acetate; exoenzyme; homoserine lactone; insight; mutant; mycobacterial; overexpression; pathogen; prevent; quorum sensing; research study; response; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mentor: Dr. Jeffrey Cirillo (Texas A&M) Specific Aims: Quorum sensing (QS) is a bacterial communication system that utilizes secreted chemical molecules in a density dependent manner. This type of communication system regulates a variety of cellular functions and has been shown to play a role in bacterial pathogenesis (Xavier and Bassler, 2005; Zhang and Dong, 2004). The specific aim in this study is to determine if QS occurs in mycobacteria and the possible role QS plays in mycobacterial pathogenesis. The bacterial species Streptomyces has a well-defined QS system and its QS molecule has been identified. Both Streptomyces and Mycobacterium are gram-positive Actinomycetales, have a G-C rich genome and reside in the soil, therefore, it is likely that similar QS systems exists in Mycobacterium. The hypothesis for this study is that a QS system exists in Mycobacterium. Streptomyces will be used as a model organism to study QS in Mycobacterium. This research design is aimed at isolating a biologically active QS molecule, determining the structure of the isolated QS molecule and identifying gene(s) involved in QS in Mycobacterium. Background and Significance: Quorum sensing (QS) is a type of communication between cells in a density-dependent manner. QS plays a role in a variety of cellular functions such as gene expression, gene transfer, sporulation, virulence, and antibiotic production. Many quorum sensing molecules have been found in different types of bacteria. The chemicals involved in quorum sensing are also referred to as autoinducers and allow for intraspecies communication (Xavier and Bassler, 2005). The process of QS allows bacteria to determine the numbers of identical bacteria in a population and thereby alter gene expression synchronously in the population. It has been found that QS-controlled processes are important for successful bacterial-host relationships, including symbiotic and pathogenic relationships (Xavier and Bassler, 2005). The nature and function of these QS molecules are diverse. Gram-negative bacteria such as Pseudomonas aeruginosa and Vibrio fisheri use low molecular weight substances such as N-acyl homoserine lactones (AHLs) as pheromones. Some of the functions of AHLs include conjugation, virulence enzyme production, biofilm production, bioluminescence, and motility. Gram-positive bacteria such as Bacillus subtilis, Myxococcus sp., and Streptomyces sp. use a variety of different signaling molecules that include oligopeptides, fatty acids, and butyrolactones, respectively. Processes that are regulated by QS molecules in gram-positive bacteria include sporulation, fruiting body formation, virulence, and antibiotic production (Volosin and Kaprelyants, 2004). QS has been found to play a significant role in pathogenesis in a variety of different bacterial species. Candida albicans and Pseudomonas aeruginosa are two examples. In C. albicans, the QS molecule called farnesol has been shown to suppress mycelium formation. The transition from the yeast form to the mycelium form of C. albicans is essential for its pathogenicity and if this transition can be prevented then this human pathogen could be better controlled. Development of farnesol analogs could prove to have clinical significance and could hold great potential for sufferers of Candidiasis, which occurs when C. albicans is able to adhere, colonize and invade epithelial tissues (Shchepin, Hornby, Bruger, Niessen, Dussault, and Nickerson, 2003). Pseudomonas aeruginosa, a gram-negative opportunistic human pathogen, also utilizes QS for pathogenesis and as stated earlier utilizes N-acyl homoserine lactone (AHL) as the signaling molecule. This pathogen is a common cause of nosocomial infections and a major cause of lung infections in cystic fibrosis patients. Key to the expression of many of the virulence factors found in Pseudomonas is density-dependent gene regulation. Some examples of genes expressed due to QS in Psuedomonas exoenzymes, toxins and genes needed for the development of biofilms. To emphasize the density-dependent method used by Psuedomonas it has been shown that quorum sensing molecules are produced in higher quantities late in the exponential growth phase and that once the population of bacteria reach a critical density, the bacteria are capable of overwhelming the host and sabotaging host defenses (Arevalo-Ferro, Hentzer, Reil, Gorg, Kjelleberg, Givskov, Riedel and Eberl, 2003). Due to the role of QS in pathogenesis, it is hypothesized that Mycobacterium also possesses a QS system. The specific aim in this study is to determine if QS occurs in Mycobacterium and the possible role QS plays in mycobacterial pathogenesis. In this study the non-pathogenic mycobacterial species, Mycobacterium smegmatis, will be used to study this phenomenon. The information obtained from M. smegmatis may be applicable to Mycobacterium tuberculosis and help to identify if QS plays a role in pathogenesis. The information obtained may be useful in determining how M. tuberculosis is able to overcome host defenses and help to identify a means to treat and/or prevent tuberculosis. Mycobacterium smegmatis, was chosen in this experiment for specific reasons. First, unlike many other species of bacteria from this genus, M. smegmatis is non-pathogenic. This makes it suitable and safe for human handling, essential for laboratory work at an undergraduate institution such as Nebraska Wesleyan University. Second, because it is in the same genus as pathogens such as Mycobacterium tuberculosis and Mycobacterium leprae, its quorum-sensing mechanism is most likely very similar to that of these virulent strains (El-Etr, Subbian, S. Cirillo, and J. Cirillo, 2004). If a deeper understanding of M. smegmatis can be developed, it can serve as a model for M. tuberculosis and M. leprae. In turn, providing insight into the disease-causing mechanism of these pathogenic strains, thereby holding value for humankind. One of the most thoroughly studied quorum sensing systems is that of Streptomyces. Streptomyces is known to produce many secondary metabolites, many involved in morphological differentiation such as development of aerial mycelium and sporulation (Choi, Lee, Hwang, Kinosita, and Hihira, 2003). In the early 90's A-factor (2S-isocapryloyl-3S-hydroxymethyl-g-butyrolactone) was discovered as an autoregulator in Streptomyces griseus, and its release caused the production of streptomycin, induction of aerial hyphae and pigmentation (Horinouchi and Beppu, 1992; Takano, Nihira, Hara, Jones, Gershater, Yamada, and Bibb, 2000). Mutants of Streptomyces unable to produce A-factor also lack the ability to undergo sporulation, make pigments, or antibiotics. Both Streptomyces and Mycobacterium are gram-positive bacteria with a high G-C content in their genome. They both belong to the phylum, Actinomycetales, and both live in the soil, suggesting that they come in contact with one another and have the ability to respond to one another. Because quorum sensing assays are well established for Streptomyces and the signaling molecules identified, Streptomyces will serve as a useful tool in the study of QS in Mycobacterium and be utilized to identify QS molecules in Mycobacterium. It is hypothesized that Mycobacterium smegmatis does possess a QS system and preliminary results in my laboratory indicate that a biologically active molecule isolated from Mycobacterium smegmatis does elicit a response (aerial mycelia formation and pigmentation) in Streptomyces. Research Design and Methods: The proposed project has two main objectives. The first objective is to isolate a biologically active QS molecule from Mycobacterium smegmatis using ethyl acetate extractions. The extracted molecule will be tested on lawns of Streptomyces and a biologically active extract will show increased pigmentation and/or aerial hyphae on lawns of Streptomyces. Once a biologically active molecule has been obtained, the molecule will be purified using HPLC, and the structure of the QS molecule determined using Mass Spectrometry. The second objective is to identify gene(s) involved in QS in Mycobacterium smegmatis. To accomplish this, two Mycobacterium smegmatis libraries will be used. The first is an overexpression library containing Mycobacterium marinum genes inserted into Mycobacterium smegmatis and the second library will be a library of Mycobacterium smegmatis mutants obtained by transposon mutagenesis. Clones from both libraries will be screened for increased or diminished arial hyphae and/or pigmentation when tested against lawns of Streptomyces. Positive clones will be analyzed further and DNA from these clones will be isolated and sequenced to determine which gene has been disrupted (transposon mutagenesis library) or is present in duplicate (overexpression library). Preliminary Work Performed and Results: 1. Development of QS Assay to test if Mycobacterium smegmatis secretes a biogically active QS molecule that Streptomyces can respond to. a. To test if Mycobacterium smegmatis secretes a molecule that Streptomyces coelicolor can respond to, the indicator strain (Streptomyces coelicolor M145) was streaked on one half of a Petri plate and the test strain (Mycobacterium smegmatis) was streaked on the other half of the plate. If Mycobacterium smegmatis secretes a molecule that Streptomyces coelicolor responds to, Streptomyces coelicolor will exhibit accelerated pigmentation and/or aerial hyphae formation. b. Results obtained  Streptomyces coelicolor responds to a molecule secreted by Mycobacterium smegmatis as seen in Figure 1. Aerial hyphae production and pigmentation of Streptomyces coelicolor in response to Mycobacterium smegmatis was observed.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 导师：Jeffrey Cirillo 博士（德克萨斯农工大学） 具体目标： 群体感应（QS）是一种细菌通讯系统，以密度依赖的方式利用分泌的化学分子。 这种类型的通讯系统调节多种细胞功能，并已被证明在细菌发病机制中发挥作用（Xavier 和 Bassler，2005；Zhang 和 Dong，2004）。 本研究的具体目的是确定分枝杆菌中是否发生 QS 以及 QS 在分枝杆菌发病机制中可能发挥的作用。链霉菌属细菌具有明确的 QS 系统，并且其 QS 分子已被鉴定。 链霉菌和分枝杆菌都是革兰氏阳性放线菌目，具有丰富的 G-C 基因组并存在于土壤中，因此，分枝杆菌中可能存在类似的 QS 系统。 本研究的假设是分枝杆菌中存在 QS 系统。 链霉菌将被用作研究分枝杆菌 QS 的模式生物。本研究设计旨在分离具有生物活性的 QS 分子，确定分离的 QS 分子的结构并鉴定分枝杆菌中参与 QS 的基因。 背景及意义： 群体感应（QS）是一种细胞之间以密度依赖方式进行的通信。 QS 在多种细胞功能中发挥作用，例如基因表达、基因转移、孢子形成、毒力和抗生素生产。 在不同类型的细菌中发现了许多群体感应分子。参与群体感应的化学物质也称为自诱导剂，并允许种内通讯（Xavier 和 Bassler，2005）。 QS 过程允许细菌确定群体中相同细菌的数量，从而同步改变群体中的基因表达。 研究发现，QS 控制的过程对于成功的细菌-宿主关系非常重要，包括共生关系和致病关系（Xavier 和 Bassler，2005）。 这些 QS 分子的性质和功能是多种多样的。 铜绿假单胞菌和渔弧菌等革兰氏阴性细菌使用 N-酰基高丝氨酸内酯 (AHL) 等低分子量物质作为信息素。 AHL 的一些功能包括缀合、毒力酶产生​​、生物膜产生、生物发光和运动。 革兰氏阳性细菌，例如枯草芽孢杆菌、粘球菌属和链霉菌属。使用各种不同的信号分子，分别包括寡肽、脂肪酸和丁内酯。 革兰氏阳性菌中受 QS 分子调节的过程包括孢子形成、子实体形成、毒力和抗生素生产（Volosin 和 Kaprelyants，2004）。 已发现 QS 在多种不同细菌物种的发病机制中发挥着重要作用。 白色念珠菌和铜绿假单胞菌是两个例子。 在白色念珠菌中，称为金合欢醇的 QS 分子已被证明可以抑制菌丝体的形成。 白色念珠菌从酵母形式向菌丝体形式的转变对其致病性至关重要，如果可以阻止这种转变，那么可以更好地控制这种人类病原体。 法呢醇类似物的开发可能被证明具有临床意义，并且对念珠菌病患者具有巨大的潜力，当白色念珠菌能够粘附、定殖和侵入上皮组织时就会发生念珠菌病（Shchepin、Hornby、Bruger、Niessen、Dussault 和 Nickerson， 2003）。 铜绿假单胞菌是一种革兰氏阴性机会性人类病原体，也利用 QS 进行发病机制，并且如前所述，利用 N-酰基高丝氨酸内酯 (AHL) 作为信号分子。 这种病原体是医院感染的常见原因，也是囊性纤维化患者肺部感染的主要原因。假单胞菌中发现的许多毒力因子表达的关键是密度依赖性基因调控。 假单胞菌胞外酶、毒素和生物膜发育所需基因中 QS 表达的基因的一些例子。 为了强调假单胞菌使用的密度依赖性方法，已经证明群体感应分子在指数生长期后期会大量产生，并且一旦细菌群体达到临界密度，细菌就能够压倒宿主并破坏宿主防御（Arevalo-Ferro、Hentzer、Reil、Gorg、Kjelleberg、Givskov、Riedel 和 Eberl，2003）。 由于 QS 在发病机制中的作用，假设分枝杆菌也拥有 QS 系统。 本研究的具体目的是确定分枝杆菌中是否发生 QS 以及 QS 在分枝杆菌发病机制中可能发挥的作用。 在这项研究中，非致病性分枝杆菌物种耻垢分枝杆菌将用于研究这种现象。 从耻垢分枝杆菌获得的信息可能适用于结核分枝杆菌，并有助于确定 QS 是否在发病机制中发挥作用。 获得的信息可能有助于确定结核分枝杆菌如何克服宿主防御，并有助于确定治疗和/或预防结核病的方法。 出于特定原因，本实验选择耻垢分枝杆菌。 首先，与该属的许多其他细菌不同，耻垢分枝杆菌是非致病性的。 这使得它适合且安全地进行人工操作，这对于内布拉斯加卫斯理大学等本科机构的实验室工作至关重要。 其次，由于它与结核分枝杆菌和麻风分枝杆菌等病原体属于同一属，因此其群体感应机制很可能与这些强毒菌株（El-Etr、Subbian、S. Cirillo 和 J. Cirillo）非常相似。 ，2004）。 如果能够对耻垢分枝杆菌有更深入的了解，它可以作为结核分枝杆菌和麻风分枝杆菌的模型。 反过来，可以深入了解这些致病菌株的致病机制，从而对人类具有价值。 研究最深入的群体感应系统之一是链霉菌的群体感应系统。 已知链霉菌产生许多次生代谢物，其中许多涉及形态分化，例如气生菌丝体的发育和孢子形成（Choi、Lee、Hwang、Kinosita 和 Hihira，2003）。 20 世纪 90 年代初，A 因子（2S-异辛酰基-3S-羟甲基-g-丁内酯）被发现是灰色链霉菌中的自动调节剂，其释放导致链霉素的产生、气生菌丝的诱导和色素沉着（Horinouchi 和 Beppu，1992）高野、尼希拉、原、琼斯、格沙特、山田和比伯， 2000）。 无法产生 A 因子的链霉菌突变体也缺乏形成孢子、产生色素或抗生素的能力。 链霉菌和分枝杆菌都是革兰氏阳性细菌，其基因组中 G-C 含量较高。 它们都属于放线菌门，并且都生活在土壤中，这表明它们彼此接触并具有相互反应的能力。 由于群体感应测定法已针对链霉菌和所鉴定的信号分子得到很好的建立，因此链霉菌将成为分枝杆菌中 QS 研究的有用工具，并可用于鉴定分枝杆菌中的 QS 分子。 据推测，耻垢分枝杆菌确实拥有 QS 系统，并且我实验室的初步结果表明，从耻垢分枝杆菌中分离出的生物活性分子确实引起了链霉菌的反应（气生菌丝体形成和色素沉着）。 研究设计和方法： 拟议项目有两个主要目标。 第一个目标是使用乙酸乙酯提取物从耻垢分枝杆菌中分离出具有生物活性的 QS 分子。 提取的分子将在链霉菌菌苔上进行测试，生物活性提取物将在链霉菌菌苔上显示出增加的色素沉着和/或气生菌丝。 一旦获得生物活性分子，将使用 HPLC 纯化该分子，并使用质谱法确定 QS 分子的结构。 第二个目标是鉴定耻垢分枝杆菌中参与 QS 的基因。 为了实现这一目标，将使用两个耻垢分枝杆菌文库。 第一个是包含插入耻垢分枝杆菌中的海分枝杆菌基因的过表达文库，第二个文库是通过转座子诱变获得的耻垢分枝杆菌突变体的文库。 当针对链霉菌菌苔进行测试时，将筛选来自两个文库的克隆的菌丝和/或色素沉着的增加或减少。 将进一步分析阳性克隆，并对这些克隆的 DNA 进行分离和测序，以确定哪个基因已被破坏（转座子诱变文库）或存在重复（过表达文库）。 前期工作及成果： 1. 开发 QS 测定，以测试耻垢分枝杆菌是否分泌链霉菌可以响应的生物活性 QS 分子。 一个。 为了测试耻垢分枝杆菌是否分泌天蓝色链霉菌可以响应的分子，将指示菌株（天蓝色链霉菌 M145）在培养皿的一半上划线，并将测试菌株（耻垢分枝杆菌）在培养皿的另一半上划线。 如果耻垢分枝杆菌分泌天蓝色链霉菌响应的分子，天蓝色链霉菌将表现出加速的色素沉着和/或气生菌丝形成。 b. 获得的结果 天蓝色链霉菌对耻垢分枝杆菌分泌的分子作出反应，如图 1 所示。观察到天蓝色链霉菌响应耻垢分枝杆菌而产生气生菌丝和色素沉着。