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

背景呼吸道疾病综合征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，给全球畜牧业造成重大经济损失。尽管各种细菌物种与牛和绵羊呼吸道疾病相关，但溶血性曼氏菌被认为是涉及的主要细菌病原体。因此，迫切需要开发更有效的疫苗和抗生素。但由于M.对溶血性病原体了解甚少，保护性抗原也不明确。一个主要原因是，目前在体外的方法，可用于调查这种病原体与宿主呼吸道（RT）的分子相互作用是欠发达。因此，迫切需要开发可用于研究病原体与牛和绵羊RT相互作用的体外方法。一个相对较新的和非常有前途的方法涉及三维（3-D）培养的分化的原代气道上皮细胞生长在气液界面（ALI）。在这种方法中，产生了含有纤毛和非纤毛细胞的极化和完全分化的假复层上皮，这为研究短期和长期宿主-病原体相互作用提供了良好的生理相关的RT体外模拟物。在M的外膜上有大约100种蛋白质。溶血性病原体，但很少知道这些外膜蛋白（OMP）在宿主-病原体相互作用的作用。在体外和体内生长条件下，细菌蛋白质表达非常不同，并且细菌感染中的关键蛋白质是在体内特异性表达的那些。定量蛋白质组学分析的细菌细胞生长在接触的3-D上皮细胞模型在急性肺损伤代表了一个强大的和新颖的方法，将允许识别的关键目标蛋白参与与主机RT的相互作用。使用的3-D气道模型也代表了一种创新的方法，用于研究体外抗菌药物的功效。目的和目标。该项目旨在使用新的三维组织工程方法来研究M.溶血性与牛和绵羊RT。生物信息学和蛋白质组学方法将用于确定关键蛋白质，特别强调外膜蛋白，在选定的菌株与气道上皮细胞的相互作用过程中上调和下调。以这种方式鉴定的OMP在与粘附和定殖相关的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，我们将获得一个更好的理解的分子基础的主机特异性和毒力的M。溶血性和确定潜在的蛋白质目标，更有效的疾病管理。这些模型还将用于概念验证方法，以评估抗生素对M的抗菌作用。在模拟体内遇到的条件下溶血。三维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。该拟议项目是与MSD Animal Health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，这些蛋白质可以代表具有创新作用模式的新候选药物的靶点。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.