Investigation of the Porin Function of B. burgdorferi P66

伯氏疏螺旋体 P66 孔蛋白功能的研究

基本信息

批准号：
9762522
负责人：
Jenifer L Coburn
金额：
$ 19.25万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-03-11 至 2021-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9762522
关键词：
Adopted Affect Affinity Amino Acids Ampicillin Antibiotic Resistance Antibiotics Arthropods Attenuated Bacteria Binding Biology Bite Blood Borrelia Borrelia Infections Borrelia burgdorferi Caliber Cells Chromosomes Complement DBL Oncoprotein Data Development Dialysis procedure Drug Controls Endothelium Environment Extravasation Future Gene Expression Geographic Distribution Gram-Negative Bacteria Greater sac of peritoneum Growth Human Immune response In Vitro Infection Integrin Binding Integrins Investigation Kinetics Knowledge Laboratories Laboratory culture Lead Ligands Lipid Bilayers Lyme Disease Maintenance Mammalian Cell Mediator of activation protein Membrane Membrane Proteins Modeling Molecular Mouse Strains Mus Natural Immunity Nutrient Order Spirochaetales Pathogenicity Penetration Phenotype Play Positioning Attribute Predisposition Production Proteins Rattus Recombinants Relapsing Fever Role Serum Site Skin Stress Surface Testing Ticks Tissues VDAC1 gene Vancomycin Vancomycin Resistance Virulence Work antimicrobial peptide c-myc Genes chronic infection experience in vivo insight intravital microscopy migration mouse model mutant normal microbiota novel strategies novel therapeutics preference prevent receptor restoration

项目摘要

Borrelia spirochetes disseminate from the site of inoculation, an arthropod bite, to multiple tissues, and often persist despite the host immune response, indicating that interactions with and adaptation to the mammalian environment are critical during infection. The Lyme disease spirochete Borrelia burgdorferi (Bb) produces a surface protein, P66, that is a porin and a ligand for several integrins. P66 is required for Bb infection of mice. Dp66 mutant bacteria, however, survive, replicate, and alter protein production at levels equivalent to the parental strain both in vitro and in the protective dialysis membrane chambers in rat peritoneal cavities. The Dp66 strains are not more susceptible than the parental wild-type strain (WT) to any mediator of innate immunity tested to date. Complementation of the mutants by restoration of p66 to the endogenous locus on the chromosome restores infectivity in mice. The integrin-binding activity, specifically, of P66 is not required for Bb infection of mice, but is required for efficient extravasation and dissemination. The absence of P66 or specific amino acid changes in the integrin-binding domain reduces binding affinity to integrin avb3, transendothelial migration of the bacteria in vitro and in vivo, and significantly reduced rates of dissemination in mice. Surface localization on the bacterial cell and the porin function of P66 are not affected by these changes. Thus, although, the absence of integrin binding activity of P66 does affect some virulence attributes of Bb, it does not account for the avirulence of the Dp66 strains. Our hypothesis is that the porin function of P66 is critical to Borrelia survival in the murine host. To test this hypothesis, we will employ c-Myc-tagged p66 mutants that do and do not display deficits in porin function in vitro. All of the c-Myc-P66 proteins are appropriately localized to the bacterial surface, and in this exploratory/developmental project, the recombinant B. burgdorferi strains producing the c-Myc-P66 proteins will be characterized in vivo with regard to survival and dissemination in mice. We predict that porin-deficient mutants will be attenuated or avirulent in mice. We are in the exclusive position of having the experience, strains, and infection models to determine whether the porin function of the integral outer membrane protein P66 of Borrelia plays a significant role during murine infection, and to progress to a clearer mechanistic understanding of how the multifunctional protein P66 enables the bacteria to establish infection and disseminate in mammalian hosts. This work will advance our understanding of the pathogenic mechanisms of Borrelia spirochetes, and may guide investigations into the environmental challenges faced by Borrelia at the site of inoculation and perhaps future development of novel therapeutics (e.g. pore blockers) specifically against Borrelia infections.

疏螺旋体螺旋体从节肢动物咬伤的接种部位传播到多个组织，并且经常尽管存在宿主免疫反应，但仍持续存在，表明与哺乳动物的相互作用和适应感染期间的环境至关重要。莱姆病螺旋体伯氏疏螺旋体 (Bb) 产生表面蛋白 P66，它是一个孔蛋白和几个整合素的配体。 Bb 感染需要 P66 的老鼠。然而，Dp66 突变细菌能够存活、复制并改变蛋白质产量，其水平相当于亲代菌株在体外和大鼠腹膜腔的保护性透析膜室中。这 Dp66 菌株并不比亲本野生型菌株 (WT) 对先天性介导物更敏感。迄今为止进行了免疫力测试。通过将 p66 恢复到内源基因座来补充突变体染色体恢复小鼠的感染性。 Bb 不需要整合素结合活性，特别是 P66 小鼠感染，但需要有效的外渗和传播。缺乏 P66 或特定的整合素结合域中的氨基酸变化降低了与整合素 avb3 的结合亲和力，跨内皮细胞细菌在体外和体内的迁移，并显着降低了小鼠体内的传播率。表面 P66 在细菌细胞上的定位和孔蛋白功能不受这些变化的影响。因此，尽管 P66 缺乏整合素结合活性确实会影响 Bb 的某些毒力属性，但它不会影响 Bb 的某些毒力属性。解释了 Dp66 菌株的无毒力。我们的假设是 P66 的孔蛋白功能对于伯氏疏螺旋体在小鼠宿主中存活。为了检验这个假设，我们将使用带有 c-Myc 标签的 p66 突变体在体外表现出和不表现出孔蛋白功能缺陷。所有 c-Myc-P66 蛋白均已适当定位到细菌表面，在这个探索/开发项目中，重组伯氏疏螺旋体菌株产生的 c-Myc-P66 蛋白将在体内对其存活和传播进行表征老鼠。我们预测孔蛋白缺陷突变体在小鼠中将减毒或无毒。我们在独家具有经验、菌株和感染模型的立场，以确定孔蛋白功能是否疏螺旋体的整合外膜蛋白 P66 在小鼠感染过程中发挥着重要作用，并且对多功能蛋白 P66 如何使细菌能够在哺乳动物宿主中建立感染并传播。这项工作将加深我们对疏螺旋体螺旋体的致病机制，并可指导环境研究伯氏疏螺旋体在接种部位面临的挑战以及新疗法的未来发展（例如毛孔阻滞剂）专门针对疏螺旋体感染。