Characterizing Exportal by a Polymyxin B Resistance-Based Mutagenic Approach

描述（由申请人提供）：阳离子抗菌肽（CAP）是天然大分子，其广谱抗菌活性涉及与细菌膜中磷脂的相互作用;这使其成为国家过敏和传染病研究所（NIAID）支持的研究主题，以探索和开发新的更有效的治疗方法。然而，生产用于治疗用途的合成CAP的尝试大部分都是不成功的。我们认为这部分是由于缺乏对CAP与细菌膜相互作用在革兰氏阳性病原体中的具体后果以及细菌如何反过来响应CAP活性的精确理解。这一领域的研究还将使人们更好地了解宿主-病原体相互作用，这是NIAID的另一个研究目标。细菌膜含有特定磷脂的局部微区，参与组织蛋白质组分和帮助分泌元件的直接易位。在化脓性链球菌中，主要毒力因子SpeB的分泌通过ExPortal发生，ExPortal是一种富含分泌型（Sec）translocons和膜锚定丝氨酸蛋白酶HtrA的阴离子膜微结构域。ExPortal作为一个协调蛋白分泌和成熟的系统在S.化脓性链球菌和阳离子肽与带电膜相互作用的能力使ExPortal成为这些大分子杀菌作用的可能靶点。初步数据表明CAP多粘菌素B对S.化脓性链球菌导致ExPortal组织的破坏，导致链球菌因子向其不同的膜和分泌后命运的改变的运输。目前正在进行实验，以测试多粘菌素B与ExPortal相互作用的观察结果是否延伸到其他生理学相关的CAP，即人类防御素。我建议在诱变方法中利用多粘菌素B，该方法将选择参与ExPortal组织、功能和抗菌药物抗性的基因产物中的抑制突变，这将使链球菌对CAP破坏ExPortal具有抗性。这些突变基因的产物将在表达、定位、与其他蛋白质的相互作用以及在发病机制中的作用方面进一步表征。这些方法将产生关于链球菌如何对CAP作出反应的新信息，使这项研究与公共卫生相关，因为每年有大量的链球菌疾病接受治疗，并且这些病原体可能出现耐药性。

DESCRIPTION (provided by applicant): Cationic antimicrobial peptides (CAPs) are natural macromolecules whose broad-spectrum antibacterial activity involves interactions with phospholipids in bacterial membranes; this makes them a research subject of efforts supported by the National Institute of Allergy and Infectious Disease (NIAID) to explore and develop novel and more efficient therapeutics. However, attempts to produce synthetic CAPs for therapeutic use have been unsuccessful for the most part. This we feel is partially due to lack of a precise understanding of the specific consequences CAP interaction with the bacterial membrane has in gram- positive pathogens and how bacteria in turn respond to CAP activity. Research in this area will also yield a greater understanding of host-pathogen interactions, another research goal of the NIAID. Bacterial membranes contain localized microdomains of specific phospholipids involved in organizing protein components and in helping direct translocation of secreted elements. In Streptococcus pyogenes secretion of the major virulence factor SpeB occurs via the ExPortal, an anionic membrane microdomain enriched in secretory (Sec) translocons and the membrane anchored serine protease HtrA. The role of the ExPortal as a system coordinating protein secretion and maturation in S. pyogenes and the ability of cationic peptides to interact with charged membranes makes the ExPortal a likely target of the bactericidal action of these macromolecules. Preliminary data suggests that the action of the CAP Polymyxin B on S. pyogenes results in a disruption of ExPortal organization, leading to altered trafficking of streptococcal factors to their distinct membrane and post-secretion fates. Experiments are currently underway to test whether observations on polymyxin B interaction with the ExPortal extend to other physiologically relevant CAPs, the human defensins. I propose to utilize polymyxin B in a mutagenesis approach that will select for supressor mutations in gene products involved in ExPortal organization, function and antimicrobial resistance that will make streptococci resistant to disruption of the ExPortal by CAPs. The products of these mutated genes will be further characterized in terms of expression, localization, interaction with other proteins and role in pathogenesis. These approaches will yield novel information about how streptococci respond to CAPs, making this research relevant to public health given the enormous numbers treated for streptococcal disease annually and the ensuing potential for the appearance of drug resistance in these pathogens.