Disulfide bond formation in Enterococcus faecalis

粪肠球菌中二硫键的形成

• 批准号: 9569269
• 负责人: Armand Brown
• 金额: $ 7.09万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9569269
• 关键词: Aftercare; Anatomy; Antimicrobial Resistance; Attenuated; Bacteria; Biochemical; Bioinformatics; Biology; Caenorhabditis elegans; Candida albicans; Cell Wall; Cellular Morphology; Centers for Disease Control and Prevention (U.S.); Complement; Cysteine; Data; Development; Disease; Disulfides; Enterococcus faecalis; Firmicutes; Future; Gastrointestinal tract structure; Generations; Genetic; Gram-Negative Bacteria; Gram-Positive Bacteria; Healthcare; Human; Infection; Mediating; Membrane; Microbial Biofilms; Minor; Modeling; Morphogenesis; Morphology; Mouth Diseases; Mus; Mutation; Oral candidiasis; Oral cavity; Organism; Outcome; Oxidoreductase; Pathogenicity; Peptide Hydrolases; Peptides; Periapical Periodontitis; Peritonitis; Play; Post-Translational Protein Processing; Process; Property; Proteins; Publishing; Pulp Canals; Research Proposals; Resistance; Resistance development; Risk; Role; Source; TXN gene; Testing; Therapeutic; Therapeutic Intervention; Thiol Disulfide Oxidoreductase; Transmission Electron Microscopy; Treatment Efficacy; Treatment Failure; Treatment outcome; Virulence; Work; antimicrobial; bacteriocin; base; combat; disulfide bond; mouse model; mutant; negative affect; new therapeutic target; novel; novel therapeutics; oral infection; oropharyngeal thrush; oxidation; pathogen; pathogenic fungus; periplasm; protein folding; protein function; targeted treatment; therapeutic development; trait

PROJECT SUMMARY/ABSTRACT Enterococcus faecalis and Candida albicans are considered by the CDC to be among the most serious global antimicrobial resistant threats. While both species normally exist as human commensals, both are the causative agents of several oral diseases due to high-levels of resistance to common forms of treatment. Currently, most antimicrobials target essential processes in generating cell wall and/or membrane, however generation of resistance remains a major threat to a successful outcome of treatment. Therefore, therapies that target a virulence trait rather than an essential process may be a more desirable approach towards combating the development of resistance. Preliminary results indicate that the E. faecalis thiodredoxin DsbA has a previously unrealized role in catalyzing oxidative protein folding, and is required for virulence. The objective of this research proposal is to determine the contribution of DsbA towards E. faecalis pathogenicity. The project will test the hypothesis that DsbA is required for the post-translational disulfide bond formation of EntV, a bacteriocin implicated in the suppression of C. albicans hyphal morphogenesis, and in additional proteins required for virulence. Aim 1 will determine the contribution of DsbA towards EntV maturation. Mutations in disulfide bonding cysteines will be inserted into EntV to determine the effect on its function. In addition, we will test if DsbA is the source of disulfide bond formation in EntV. Additional proteins that are negatively affected by the loss of dsbA, and that are required for virulence will be tested in Aim 2. First, transposon (Tn) insertion mutants of proteins predicted to require DsbA will be visualized by transmission electron microscopy and morphology compared to the dsbA deletion mutant. In addition, biofilm formation on various media will also be evaluated. To determine the contribution of the newly identified protein(s) toward E. faecalis virulence, Tn- mutants will be tested within a C. elegans model of infection. Next, in frame deletion mutants of proteins predicted to require DsbA for post-translational disulfide bond formation will be generated, and tested in a similar fashion as the Tn-mutants to confirm our original observations. Mutations in disulfide bonding cysteines will then be inserted into identified proteins to determine the effect on their function. Next, DsbA will be tested to determine if it is the source of disulfide bond formation in identified proteins. Finally, to confirm that the newly identified DsbA substrates contribute to E. faecalis virulence, each deletion mutant will be tested within a mouse model of peritonitis. At its conclusion, this project is expected to (Aim 1) demonstrate that DsbA is required for post- translational disulfide bond formation in EntV, and is also required for E. faecalis virulence (Aim 2). Understanding the contribution DsbA, and the novel observation of its requirement for pathogenicity will inform the future development of new therapeutics effective against C. albicans and E. faecalis.

项目摘要/摘要 CDC认为粪便肠球菌和白色念珠菌是最严重的 全球抗菌抗性威胁。虽然这两种物种通常都作为人类的共生存在，但两者都是 由于对共同治疗形式的高水平抗性，几种口腔疾病的病因。 目前，大多数抗菌剂针对产生细胞壁和/或膜的基本过程，但是 抗药性的产生仍然是对治疗成功结果的主要威胁。因此，对此进行治疗 针对毒力性状而不是基本过程可能是对抗的一种更理想的方法 抵抗的发展。初步结果表明，粪肠球菌硫代氧蛋白DSBA具有 以前未实现的作用在催化氧化蛋白折叠中，是毒力所必需的。目的 该研究建议是确定DSBA对粪肠球菌致病性的贡献。项目 将检验以下假设：ENTV的翻译后二硫键形成需要DSBA，A 细菌素与白色念珠菌菌丝形态发生有关，并在其他蛋白 毒力需要。 AIM 1将确定DSBA对ENTV成熟的贡献。突变 二硫键键合的半胱氨酸将插入ENTV中，以确定对其功能的影响。此外，我们将 测试DSBA是否是ENTV中二硫键形成的来源。受负面影响的其他蛋白质 DSBA的丢失和毒力所需的损失将在AIM 2中进行测试。首先，Transposon（TN）插入 预测需要DSBA的蛋白质突变体将通过透射电子显微镜和 与DSBA缺失突变体相比，形态学。此外，各种媒体上的生物膜形成也将是 评估。为了确定新鉴定的蛋白质对粪肠球菌毒力的贡献 突变体将在秀丽隐杆线虫感染模型中进行测试。接下来，在蛋白质的框架缺失突变体中预测 需要DSBA进行翻译后的二硫键形成，并以类似的方式进行测试 作为TN突变者确认我们的原始观察结果。然后将是二硫键粘结性半胱氨酸的突变 插入确定的蛋白质以确定对其功能的影响。接下来，DSBA将进行测试以确定 如果它是鉴定蛋白质中二硫键形成的来源。最后，确认新近确定的 DSBA底物有助于大肠杆菌毒力，每个缺失突变体将在小鼠模型中进行测试 腹膜炎。总而言之，该项目有望（AIM 1）证明DSBA是必需的。 ENTV中的转化二硫键形成，也是粪肠球菌毒力所必需的（AIM 2）。 了解DSBA的贡献以及对致病性要求的新颖观察将为您提供信息 针对白色念珠菌和粪肠球菌有效的新疗法的未来发展。