Identification of early phase C. albicans biofilm proteins

早期白色念珠菌生物膜蛋白的鉴定

• 批准号: 7809624
• 负责人: Mahmoud A Ghannoum
• 金额: $ 36.17万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-15 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7809624
• 关键词: Adherence; Alcohol dehydrogenase; Animal Experimentation; Animal Model; Architecture; Azole resistance; Biochemical; Biological Assay; Biology; Bromodeoxyuridine; Canada; Candida; Candida albicans; Candidiasis; Cell Death; Cell Wall; Cell membrane; Cells; Characteristics; Chromatography; Clinical; Collaborations; Collagen Type IV; Complication; Data; Denture Stomatitis; Dentures; Development; Devices; Diagnosis; Diagnostic; Early identification; Electron Microscopy; Ensure; Exhibits; Fluorescence Microscopy; Fungal Drug Resistance; Gene Proteins; Genes; Growth; HIV; Histopathology; Human Engineering; Hyphae; Illinois; In Vitro; Infection; Invaded; Laboratories; Laser Scanning Microscopy; Link; Manuscripts; Mass Spectrum Analysis; Measurement; Measures; Metabolic; Metabolic Pathway; Methods; Microbial Biofilms; Microscopy; Modeling; Molecular; Monitor; Mouth Diseases; Mucous Membrane; Mus; Opportunistic Infections; Oral; Oral candidiasis; Oral cavity; Oral mucous membrane structure; Parents; Pathogenesis; Pathway Analysis; Pathway interactions; Patients; Phase; Phenotype; Play; Prevention; Principal Investigator; Production; Proteins; Proteomics; Publications; Research; Research Personnel; Resistance profile; Role; Scanning; Shipping; Ships; Stimulus; Surface; Techniques; Testing; Therapeutic; Thick; Tissues; Tongue; Universities; Weight; Yeasts; base; candida biofilm; gel electrophoresis; genetic manipulation; in vitro Model; in vivo; in vivo Model; insight; laminin-5; liquid chromatography mass spectrometry; microorganism; mutant; oral infection; oropharyngeal thrush; programs; protein expression; research clinical testing; research study; tool; two-dimensional

DESCRIPTION (provided by applicant): Oropharyngeal candidiasis (OPC, thrush) is the term given to opportunistic oral infection caused by the yeast Candida, and is the most common mycotic complication associated with human immunodeficiency virus (HlV)-infected patients. The ability of Candida isolates to form biofilm on devices and host tissue surfaces is believed to be intricately linked with OPC infections and denture stomatitis. Therefore, understanding the role of biofilms in pathogenesis and host-Cand/cfa interactions is critical. In this application, to gain insight into these interactions, we will use a proteomics-based approach to identify specific proteins that are central to the biofilm forming ability of C. albicans and determine their role in interactions between fungal biofilm and host tissues. We have previously established an in vitro model of C. albicans denture biofilm (Publication 1, Appendix). Using this model, we: 1) identified the developmental phases of candidal biofilm formation (Publication 1, Appendix), 2) investigated the antifungal resistance profile of C. albicans biofilms at different growth phases (Publication 2, Appendix), 3) investigated the multifactorial mechanisms of azole resistance of early and mature biofilms formed by C. albicans (Publication 3, Appendix), 4) used a proteomic approach to identify potential target proteins that are differentially expressed in early phase biofilms (see Publication 4, Appendix), 5) used molecular and biochemical methods to show that one of the identified proteins (alcohol dehydrogenase, Adhlp) is a negative regulator of Candida biofilm (Publication 4, Appendix), and 6) moved our studies closer to the clinical setting by using an engineered human oral mucosa (EHOM), developed recently by Dr. Rouabhia (Co-investigator on this application). This collaborative research between the applicant and Dr. Rouabhia showed that Adhlp plays an important role in both Candida biofilm formation and invasion of host mucosal tissues (Manuscript submitted, Appendix). The overall hypothesis of this application is that C. albicans express specific proteins that are essential for biofilm formation and play critical roles in Candida-host tissue interactions. We will test our hypothesis using the following Specific Aims: Aim 1. Identify early phase biofilm-specific proteins expressed by C. albicans. Aim 2. Determine whether the identified proteins are critical to the ability of C. albicans to form biofilms in vitro by disrupting genes encoding them. Aim 3. Use an in v/Vo-like Engineered Human Oral Mucosa (EHOM) Model to determine whether the identified proteins are essential for Candida biofilm formation and host tissue damage. Aim 4. Validate the in vitro and EHOM results in vivo using a murine oral model of Candida biofilms. Experiments described in this application will provide insight into the biology of OPC-associated C. albicans biofilms and may suggest potential therapeutic and diagnostic targets for the prevention, treatment, and diagnosis of oral Candida infections.

描述（由申请人提供）：口咽念珠菌病（OPC，鹅口疮）是指由念珠菌引起的机会性口腔感染，是与人类免疫缺陷病毒（HIV）感染患者相关的最常见的真菌并发症。念珠菌分离株在设备和宿主组织表面形成生物膜的能力被认为与 OPC 感染和假牙口腔炎密切相关。因此，了解生物膜在发病机制和宿主-Cand/CFA 相互作用中的作用至关重要。在本应用中，为了深入了解这些相互作用，我们将使用基于蛋白质组学的方法来识别对白色念珠菌生物膜形成能力至关重要的特定蛋白质，并确定它们在真菌生物膜和宿主组织之间相互作用中的作用。我们之前已经建立了白色念珠菌假牙生物膜的体外模型（出版物 1，附录）。使用该模型，我们：1) 确定了念珠菌生物膜形成的发育阶段（出版物 1，附录），2) 研究了不同生长阶段白色念珠菌生物膜的抗真菌耐药性概况（出版物 2，附录），3) 研究了白色念珠菌生物膜的抗真菌耐药性。白色念珠菌形成的早期和成熟生物膜的唑类耐药性的多因素机制（出版物 3，附录），4）使用蛋白质组学方法为了识别早期生物膜中差异表达的潜在靶蛋白（参见出版物 4，附录），5），使用分子和生化方法表明，已识别的蛋白质之一（乙醇脱氢酶，Adhlp）是念珠菌生物膜的负调节因子（出版物 4，附录）和 6）通过使用由 Rouabhia 博士（该研究的联合研究员）最近开发的工程人类口腔粘膜 (EHOM)，使我们的研究更接近临床环境。 应用）。申请人和 Rouabhia 博士之间的这项合作研究表明，Adhlp 在念珠菌生物膜形成和宿主粘膜组织的侵袭中发挥着重要作用（提交的手稿，附录）。该应用的总体假设是白色念珠菌表达对生物膜形成至关重要的特定蛋白质，并在念珠菌-宿主组织相互作用中发挥关键作用。我们将使用以下具体目标来检验我们的假设： 目标 1. 识别白色念珠菌表达的早期生物膜特异性蛋白。目标 2. 通过破坏编码蛋白质的基因，确定所鉴定的蛋白质是否对白色念珠菌在体外形成生物膜的能力至关重要。目标 3. 使用类似体内工程的人类口腔粘膜 (EHOM) 模型来确定所鉴定的蛋白质是否对于念珠菌生物膜形成和宿主组织损伤至关重要。目标 4. 使用念珠菌生物膜的小鼠口腔模型验证体外和体内 EHOM 结果。本申请中描述的实验将深入了解 OPC 相关的白色念珠菌生物膜的生物学，并可能为口腔念珠菌感染的预防、治疗和诊断提出潜在的治疗和诊断靶标。