Identification of early phase C. albicans biofilm proteins

描述(申请人提供)：口咽念珠菌病(OPC，鹅口疮)是指由酵母菌引起的机会性口腔感染，是与人类免疫缺陷病毒(HLV)感染患者相关的最常见的真菌并发症。念珠菌在设备和宿主组织表面形成生物膜的能力被认为与OPC感染和义齿口炎有复杂的联系。因此，了解生物膜在发病机制和宿主-Cand/CFA相互作用中的作用是至关重要的。在这项应用中，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与宿主组织之间相互作用中的作用。我们之前已经建立了白念珠菌义齿生物膜的体外模型(发表1，附录)。利用这个模型，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)调查了不同生长阶段白色念珠菌生物膜的抗菌谱(出版物2，附录)，3)研究了由白色念珠菌形成的早期和成熟生物膜对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鉴定了在早期生物膜中差异表达的潜在靶蛋白(见出版物4，附录)，5)使用分子和生物化学方法证明了已鉴定的蛋白质之一(酒精脱氢酶，Adhlp)是念珠菌生物被膜的负调控因子(出版物4，附录)。6)通过使用Rouabhia博士(该应用的联合研究员)最近开发的工程化人类口腔黏膜(EHOM)，使我们的研究更接近临床环境。申请人和Rouabhia博士的这项合作研究表明，Adhlp在念珠菌生物膜的形成和宿主粘膜组织的侵袭中都发挥着重要作用(提交的手稿，附录)。这一应用的总体假设是，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的相互作用中发挥关键作用。我们将通过以下具体目标来验证我们的假设：目的1.鉴定白色念珠菌表达的早期生物被膜特异蛋白。目的2.通过干扰编码白念珠菌的基因，确定已鉴定的蛋白质是否对白念珠菌在体外形成生物膜的能力起关键作用。目的3.利用In v/Vo样工程化人类口腔黏膜(EHOM)模型，确定已鉴定的蛋白质是否对念珠菌生物膜的形成和宿主组织损伤是必需的。目的4.用小鼠口腔假丝酵母菌生物被膜模型验证体外和体内EHOM结果。本申请中描述的实验将为OPC相关白色念珠菌生物膜的生物学提供洞察力，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.