Identification of early phase C. albicans biofilm proteins

描述（由申请人提供）：口咽念珠菌病（OPC，鹅口疮）是念珠菌引起的机会性口腔感染的术语，是与人类免疫缺陷病毒（HlV）感染患者相关的最常见真菌并发症。念珠菌分离物在器械和宿主组织表面形成生物膜的能力被认为与OPC感染和假牙口炎有着复杂的联系。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鉴定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体外白色念珠菌义齿生物膜模型（出版物1，附录）。使用这个模型，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抗性谱（出版物2，附录），3)研究了白色念珠菌形成的早期和成熟生物膜耐唑的多因子机制（出版物3，附录）。4)使用蛋白质组学方法鉴定早期生物膜中差异表达的潜在靶蛋白（见出版物4，附录），5)使用分子和生化方法表明鉴定的蛋白质之一（酒精脱氢酶，Adhlp）是假丝酵母生物膜的负调节因子（出版物4，附录），6)通过使用工程人口腔黏膜（EHOM）使我们的研究更接近临床环境。最近由Rouabhia博士（该应用程序的共同研究者）开发。申请人与Rouabhia博士的合作研究表明，Adhlp在念珠菌生物膜的形成和宿主粘膜组织的侵袭中都起着重要作用（手稿提交，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，并在念珠菌与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：鉴定白色念珠菌表达的早期生物膜特异性蛋白。目标2。通过破坏编码白色念珠菌的基因，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in v/ vo样工程人口腔黏膜（EHOM）模型来确定鉴定的蛋白质是否对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。本应用程序中描述的实验将深入了解opc相关白色念珠菌生物膜的生物学特性，并可能为预防、治疗和诊断口腔念珠菌感染提供潜在的治疗和诊断靶点。

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.