Molecular interactions of Mannheimia haemolytica with the bovine and ovine respiratory tracts using three-dimensional tissue engineering approaches

背景。呼吸道疾病是牛羊的一种多因素疾病，涉及不同细菌和病毒病原体之间的相互作用，给全世界的畜牧业造成重大经济损失。虽然各种细菌种类与牛和羊呼吸道疾病有关，但溶血性曼海姆病被认为是主要的细菌病原体。迫切需要开发针对溶血支原体更有效的疫苗和抗生素，但由于对溶血支原体的发病机制了解甚少，保护性抗原定义不清，改进抗菌剂的进展受到阻碍。造成这种情况的一个主要原因是，目前用于研究这种病原体与宿主呼吸道（RT）分子相互作用的体外方法尚不发达。因此，迫切需要开发可用于研究病原体与牛和羊RTs相互作用的体外方法。一种相对较新的、前景广阔的方法是在气液界面（ALI）培养分化的原代气道上皮细胞的三维（3-D）培养。在这种方法中，产生极化和完全分化的假分层上皮，其中包含纤毛细胞和非纤毛细胞，这为短期和长期宿主-病原体相互作用的研究提供了一个极好的生理相关的RT体外模拟。溶血支原体的外膜上大约有100种蛋白质，但人们对这些外膜蛋白在宿主-病原体相互作用中的作用知之甚少。在体外和体内生长条件下，细菌蛋白的表达有很大差异，细菌感染的关键蛋白是那些在体内特异性表达的蛋白。在ALI中，对与3-D上皮细胞模型接触生长的细菌细胞进行定量蛋白质组学分析是一种强大而新颖的方法，可以识别与宿主rt相互作用相关的关键靶蛋白。3-D气道模型的使用也代表了研究体外抗菌功效的一种创新方法。目的和目标。该项目旨在使用新颖的三维组织工程方法来研究溶血分枝杆菌与牛和羊RTs的分子相互作用。生物信息学和蛋白质组学方法将用于鉴定关键蛋白，特别强调在选定菌株与气道上皮细胞相互作用过程中上调和下调的omp。通过这种方式确定的omp在粘附和定植过程中的作用将通过评估气道上皮细胞模型中的敲除突变体来证实。因此，我们将大大提高对溶血支原体宿主特异性和毒力的分子基础的理解，并确定潜在的蛋白质靶点，以更有效地进行疾病管理。这些模型还将用于概念验证方法，在模拟体内遇到的条件下评估抗生素对溶血支原体的抗菌作用。3d模型将支持未来在动物临床试验之前选择新的体外化合物。应用和好处。拟议的项目是与MSD动物保健的工业合作，将导致所产生的科学数据的商业化和利用。该项目将导致细菌蛋白质的鉴定，这些蛋白质可能代表具有创新作用模式的新候选药物的靶标。此外，3d模型将允许未来选择新的体外活性化合物，这些化合物可能会在随后的动物试验中进行测试。因此，该提案可能具有重大的经济和社会效益。

Background. Respiratory disease complex is a multifactorial condition of cattle and sheep that involves interactions between different bacterial and viral pathogens and causes significant economic losses to livestock industries worldwide. Although various bacterial species are associated with bovine and ovine respiratory disease, Mannheimia haemolytica is considered to be the principal bacterial pathogen involved. There is an urgent need to develop more effective vaccines and antibiotics against M. haemolytica but progress towards improved antimicrobials is hampered because the pathogenesis of M. haemolytica is poorly understood and protective antigens are ill-defined. A major reason for this is that current in vitro methods available to investigate the molecular interactions of this pathogen with the host respiratory tract (RT) are poorly developed. Consequently, there is a compelling need to develop in vitro methods that can be used to study the interactions of pathogens with the bovine and ovine RTs. A relatively new and hugely promising approach involves the three-dimensional (3-D) culture of differentiated primary airway epithelial cells grown at an air-liquid interface (ALI). In this method, a polarized and fully differentiated pseudostratified epithelium containing both ciliated and non-ciliated cells is produced and this provides an excellent physiologically-relevant in vitro mimic of the RT for the study of both short- and long-term host-pathogen interactions. There are approximately 100 proteins in the outer membrane of M. haemolytica but very little is known about the roles of these outer membrane proteins (OMPs) in host-pathogen interactions. Bacterial protein expression is very different under in vitro and in vivo growth conditions and the key proteins in bacterial infection are those that are specifically expressed in vivo. Quantitative proteomic analysis of bacterial cells growing in contact with the 3-D epithelial cell models at an ALI represents a powerful and novel approach that will allow the identification of key target proteins involved in interactions with the host RT. The use of the 3-D airway models also represents an innovative approach for studying antimicrobial efficacy in vitro. Aims and objectives. The project aims to use novel 3-D tissue engineering approaches to investigate molecular interactions of M. haemolytica with the bovine and ovine RTs. Bioinformatics and proteomic approaches will be used to identify key proteins, with particular emphasis on OMPs, that are up- and down-regulated during interactions of selected strains with airway epithelial cells. The roles of OMPs identified in this way, in processes associated with adherence and colonization, will be confirmed by assessment of knockout mutants in the airway epithelial cell models. Consequently, we will gain a much improved understanding of the molecular basis of host-specificity and virulence of M. haemolytica and identify potential protein targets for more effective disease management. The models will also be used in a proof-of-concept approach to assess the antimicrobial effects of antibiotics on M. haemolytica under conditions that mimic those encountered in vivo. The 3-D models will support the future selection of new in vitro compounds prior to animal clinical trials.Applications and benefits. The proposed project is an industrial collaboration with MSD Animal Health and will result in the commercialisation and exploitation of the scientific data generated. The project will lead to the identification of bacterial proteins which could represent targets for new drug candidates with innovative modes-of-action. In addition, the 3-D models will allow the future selection of new in vitro-active compounds that may be tested in subsequent animal trials. Therefore, the proposal is likely to have major economic and societal benefits.