Disulfide bond formation in Enterococcus faecalis

粪肠球菌中二硫键的形成

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要疾病预防控制中心认为粪肠球菌和白色念珠菌是最严重的细菌之一全球抗菌药物耐药性威胁。虽然这两个物种通常作为人类共生体存在，但两者都是由于对常见治疗形式的高度耐药性，导致多种口腔疾病的病原体。目前，大多数抗菌剂都针对生成细胞壁和/或细胞膜的基本过程，然而耐药性的产生仍然是治疗成功的主要威胁。因此，治疗方法针对毒力特征而不是关键过程可能是对抗病毒的更可取的方法抵抗力的发展。初步结果表明粪肠球菌硫氧还蛋白 DsbA 具有以前未认识到催化氧化蛋白折叠的作用，并且是毒力所必需的。的目标该研究计划旨在确定 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 细菌素参与抑制白色念珠菌菌丝形态发生以及其他蛋白质需要毒力。目标 1 将确定 DsbA 对 EntV 成熟的贡献。突变在二硫键半胱氨酸将被插入 EntV 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。受到负面影响的其他蛋白质目标 2 中将测试 dsbA 的丢失以及毒力所需的损失。首先，转座子 (Tn) 插入预计需要 DsbA 的蛋白质突变体将通过透射电子显微镜观察到与 dsbA 缺失突变体相比的形态。此外，各种介质上生物膜的形成也将受到影响。评价。为了确定新鉴定的蛋白质对粪肠球菌毒力的贡献，Tn- 突变体将在秀丽隐杆线虫感染模型中进行测试。接下来，预测蛋白质的框内缺失突变体将生成翻译后二硫键形成所需的 DsbA，并以类似的方式进行测试作为 Tn 突变体来证实我们最初的观察。二硫键半胱氨酸的突变将被插入已识别的蛋白质中以确定对其功能的影响。接下来，将测试 DsbA 以确定如果它是已识别蛋白质中二硫键形成的来源。最后，确认新识别的 DsbA 底物有助于粪肠球菌毒力，每个缺失突变体将在小鼠模型中进行测试腹膜炎。最后，该项目预计将（目标 1）证明 DsbA 是后勤保障所必需的。 EntV 中翻译二硫键的形成，也是粪肠球菌毒力所必需的（目标 2）。了解 DsbA 的贡献以及对其致病性要求的新观察将提供信息未来开发有效对抗白色念珠菌和粪肠球菌的新疗法。