Characterizing Exportal by a Polymyxin B Resistance-Based Mutagenic Approach

通过基于多粘菌素 B 抗性的诱变方法表征输出

• 批准号: 7752900
• 负责人: Luis Alberto Vega
• 金额: $ 2.79万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7752900
• 关键词: Affect; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Antimicrobial Resistance; Appearance; Area; Bacteria; Charge; Data; Defensins; Disease; Drug resistance; Elements; Genes; Goals; Human; Mediating; Membrane; Membrane Microdomains; Mutagenesis; Mutate; Mutation; National Institute of Allergy and Infectious Disease; Pathogenesis; Peptides; Phospholipids; Polymyxin B; Polymyxin B resistance; Process; Protein Secretion; Proteins; Public Health; Research; Research Subjects; Resistance; Role; Serine Protease; Streptococcus; Streptococcus pyogenes; System; Testing; Therapeutic; Therapeutic Uses; Virulence Factors; antimicrobial; bactericide; base; insight; macromolecule; novel; pathogen; research study; secretion process; trafficking

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.

描述(申请人提供)：阳离子抗菌肽(CAPS)是一种天然大分子，其广谱抗菌活性涉及与细菌膜中的磷脂相互作用；这使它们成为美国国家过敏和传染病研究所(NIAID)支持的努力的研究对象，以探索和开发新的和更有效的治疗方法。然而，生产用于治疗用途的合成帽的尝试在大多数情况下都没有成功。我们认为，这部分是由于缺乏对CAP与细菌膜相互作用对革兰氏阳性病原体产生的具体后果以及细菌如何对CAP活性做出反应的准确了解。这一领域的研究还将产生对宿主-病原体相互作用的更好理解，这是NIAID的另一个研究目标。细菌膜含有特定磷脂的局部微域，这些微域参与组织蛋白质成分和帮助直接转移分泌的元素。在化脓性链球菌中，主要毒力因子SpeB的分泌是通过ExPortal、富含分泌型转运子(SEC)的阴离子膜微域和膜锚定的丝氨酸蛋白酶HtrA来实现的。在化脓性链球菌中，Export作为一个协调蛋白质分泌和成熟的系统，以及阳离子多肽与带电的膜相互作用的能力，使Export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对化脓性链球菌的作用导致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运输。目前正在进行实验，以测试对多粘菌素B与ExPortal相互作用的观察是否延伸到其他生理相关的帽，即人类防御素。我建议在一种突变方法中利用多粘菌素B，这种方法将在涉及ExPortal组织、功能和抗菌素耐药性的基因产物中选择抑制突变，使链球菌通过CAPS对ExPortals的破坏产生抗性。这些突变基因的产物将进一步从表达、定位、与其他蛋白质的相互作用以及在发病机制中的作用等方面进行研究。这些方法将产生关于链球菌如何对CAPS做出反应的新信息，使这项研究与公共卫生相关，因为每年有大量链球菌疾病接受治疗，以及随之而来的这些病原体出现耐药性的可能性。