Post-translational lipidation of proteins with mycolates in Rhodococcus equi: a novel drug target in the mycolata?

随着抗生素耐药性的增加和新的病原体的出现，迫切需要新的抗微生物药物。对致病细菌与其宿主之间相互作用尚未开发的途径进行表征，将极大地促进新型抗菌剂的开发。我们建议研究一个这样的途径，我们预计这将被证明是一个被忽视的，但在霉菌酸细菌和它们的人类或动物宿主之间相互作用的关键方面。含有霉菌酸的细菌是一类重要的细菌，它们具有基于特征脂质（霉菌酸）的蜡质细胞包膜的共同特征。许多非常重要的病原体都属于这一类细菌，包括人类的结核病、麻风病和白喉病原体，以及马驹的牛热病、牛结核病、红球菌支气管肺炎病原体和绵羊和山羊的干酪性淋巴结炎病原体。这些细菌不寻常的细胞包膜的生物合成已经被确定为有效的药物靶点（例如在结核病治疗中）。我们假设在这些细菌中存在一种途径，通过用霉菌酸修饰蛋白质，蛋白质被定位到霉菌酸细胞包膜上，这将提供一个脂质锚点，将这些蛋白质固定在蜡质细胞表层上。因此，这种类型的蛋白质修饰可能会影响含有霉菌酸的细菌的毒力，因为细胞包膜蛋白对致病菌与其宿主之间的相互作用至关重要。因此，我们建议验证这种类型的脂质修饰确实广泛存在于含有霉菌酸的细菌中，并建立其发生的机制。我们将检查含有霉菌酸的细菌的代表性范围，以证明存在霉菌酸修饰的蛋白质，使用选择性提取和化学表征方法。我们还将检查蛋白质被修饰的位点，因为这将深入了解潜在的机制，并允许基于序列的预测哪些蛋白质可能被类似地修饰。作为详细研究霉菌酸修饰途径的模型系统，我们将使用重要的马病原体马红球菌（马驹红球菌支气管肺炎的一种全球性病原体），因为我们在研究这种细菌的细胞包膜生物学方法方面有丰富的经验。我们已经确定了一种候选酶，它可能是霉菌酸修饰蛋白质的催化剂。对编码这种酶的基因进行遗传修饰将使我们能够创造出我们预测会减弱其致病能力的突变株，我们将使用新的组织培养方法来验证体外评估细菌毒力的方法。这将为该途径的重要性提供证据，该途径应与其他含有霉菌酸的病原体相关。最后，我们将从生物化学角度描述马链球菌的蛋白霉菌酸修饰机制，从而获得机制上的见解，从而使我们能够确定这一途径是否是开发新型抗菌疗法的合适靶点。作为这项工作的一部分，我们还将设计适合于高通量筛选的检测方法，可用于发现针对该途径的新型抗菌剂。总之，我们希望这个项目能够在一组重要的细菌中发现一条影响宿主-病原体相互作用的新途径，并证明这条途径代表了一个适合开发新型抗菌剂的靶标。

Novel antimicrobials are desperately needed as both antibiotic resistance increases and new pathogens emerge. The development of novel antimicrobials will be greatly facilitated by the characterisation of as yet unexploited pathways that contribute to the interactions between pathogenic bacteria and their hosts. We propose to study one such pathway, which we expect will prove to be an overlooked but crucial aspect of the interaction between mycolic acid containing bacteria and their human or animal hosts. The mycolic acid containing bacteria are an important group of bacteria which have a common feature of a waxy cell envelope based on characteristic lipids, the mycolic acids. Many very significant pathogens belong to this group of bacteria, including the causative agents of tuberculosis, leprosy and diphtheria in humans and of bovine farcy, bovine tuberculosis, rhodococcal bronchopneumonia in foals and caseous lymphadenitis in sheep and goats. The biosynthesis of the unusual cell envelopes of these bacteria is already established as valid drug target (e.g. in tuberculosis treatments). We hypothesise that there is a pathway in these bacteria by which proteins are localised to the mycolic acid cell envelope by modification of the proteins with mycolic acids, which will provide a lipid anchor holding such proteins onto the waxy cell surface layer. This type of protein modification is thus likely to influence the virulence of mycolic acid containing bacteria as cell envelope proteins are crucial to the interactions between pathogen bacteria and their hosts. We therefore propose to verify that this type of lipid modification is indeed widespread in mycolic acid containing bacteria and to establish the mechanism by which it occurs. We will examine a representative range of mycolic acid containing bacteria to demonstrate that mycolic acid modified proteins are present, using selective extraction and chemical characterisation methods. We will also examine the sites at which the proteins are modified as this should give insights into the underlying mechanism and also allow sequence based prediction of which proteins are likely to be similarly modified. As a model system in which to study the mycolic acid modification pathway in detail, we will use the important equine pathogen Rhodococcus equi (a global cause of rhodococcal bronchopneumonia in foals) as we have extensive experience of methods for studying the cell envelope biology of this bacterium. We have already identified a candidate enzyme that is likely to be the catalyst for mycolic acid modification of proteins. Genetic modifications of the gene encoding this enzyme will allow us to create mutant strains of R. equi that we predict will be attenuated in their ability to cause disease, which we will verify using novel tissue culture method for assessing bacterial virulence in vitro. This will provide evidence for the importance of this pathway that should be relevant to other mycolic acid containing pathogens. Finally, we will characterise biochemically the protein mycolic acid modification machinery in R. equi and thus gain mechanistic insights that will allow us to determine whether this pathway is a suitable target for the development of novel antimicrobial therapies. As part of this work we will also devise assays suitable for adaptation in high throughput screens that can be used for the discovery of novel antimicrobials targeting this pathway. In summary, we expect this project to identify a new pathway influencing host-pathogen interactions in an important group of bacteria and to demonstrate that this pathway represents a target suitable for the development of novel antimicrobials.