Disulfide bond formation in Enterococcus faecalis

项目摘要/摘要 疾控中心认为粪肠球菌和白色念珠菌是最严重的 全球抗菌素耐药性威胁。虽然这两个物种通常作为人类的共生体而存在，但它们都是 几种口腔疾病的病原体是由于对常见治疗形式的高度耐药性所致。 然而，目前，大多数抗菌剂针对的是产生细胞壁和/或膜的必要过程。 耐药性的产生仍然是成功治疗结果的主要威胁。因此，治疗方法 针对毒力特征而不是基本过程可能是更可取的抗击方法 抗药性的发展。初步结果表明，粪肠球菌硫代还蛋白DsbA具有 以前没有意识到的作用，催化氧化蛋白质折叠，是毒力所必需的。的目标是 本研究旨在确定DsbA对粪肠球菌致病力的贡献。该项目 将检验以下假设，即DsbA是ENTV翻译后二硫键形成所必需的，a 细菌素在抑制白色念珠菌菌丝形态发生中的作用以及在其他蛋白质中的作用 是致命性所必需的。目标1将确定DsbA对ENTV成熟的贡献。基因突变 二硫键结合半胱氨酸将被插入到ENTV中，以确定对其功能的影响。此外，我们还将 测试DsbA是否是ENTV中二硫键形成的来源。其他受以下因素影响的蛋白质 毒力所需的dsbA的丢失将在目标2中进行测试。首先，转座子(TN)的插入 预测需要DsbA的蛋白质的突变将通过透射电子显微镜和 形态与dsbA缺失突变体的比较。此外，生物膜在各种介质上的形成也将 已评估。为了确定新发现的蛋白(S)对粪肠球菌毒力的贡献，Tn- 突变株将在线虫感染模型中进行测试。接下来，在框架缺失的蛋白质突变预测 需要DsbA用于翻译后二硫键的形成，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会是 插入到已识别的蛋白质中，以确定对其功能的影响。接下来，将测试DsbA以确定 如果它是已鉴定蛋白质中二硫键形成的来源。最后，为了确认新确认的 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。在其结束时，本项目预期(目标1)证明DsbA是邮局所需的- 在ENTV中形成翻译二硫键，对粪肠球菌的毒力也是必需的(目标2)。 了解DsbA的贡献，以及对其致病性要求的新观察将有助于 对白色念珠菌和粪肠球菌有效的新疗法的未来发展。

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.