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）是由酵母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（HIV）感染患者相关的最常见的肌萎缩症并发症。念珠菌分离株在器械和宿主组织表面形成生物膜的能力被认为与OPC感染和义齿性口炎密切相关。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。在本申请中，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C.白色念珠菌，并确定其在真菌生物膜和宿主组织之间的相互作用的作用。我们以前建立了一个体外模型的C。白色念珠菌义齿生物膜（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）研究了念珠菌的抗真菌耐药谱。不同生长阶段的白色念珠菌生物膜（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的唑类耐药的多因素机制。白色（出版物3，附录），4）使用蛋白质组学方法鉴定在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）使用分子和生物化学方法来显示鉴定的蛋白质之一乙醇脱氢酶（alcohol dehydrogenase，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Rouabhia博士（本申请的共同研究者）最近开发的工程化人口腔粘膜（EHOM），使我们的研究更接近临床环境。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侵袭中发挥重要作用（Mandarept提交，附录）。本申请的总体假设是C.白念珠菌表达生物膜形成所必需的特异性蛋白质，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。鉴定由C.白色念珠菌目标二。确定所鉴定的蛋白质是否对C.白念珠菌通过破坏编码它们的基因在体外形成生物膜。目标3。使用体内/体外样工程化人口腔粘膜（EHOM）模型来确定所鉴定的蛋白质是否是念珠菌生物膜形成和宿主组织损伤所必需的。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提供对OPC相关C的生物学的深入了解。白色念珠菌生物膜，并可能建议潜在的治疗和诊断目标，预防，治疗和诊断口腔念珠菌感染。

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.