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

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

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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.

漏螺旋体从接种部位，节肢动物咬合，多个组织，通常尽管宿主免疫反应仍然存在，表明与哺乳动物的相互作用并适应感染过程中的环境至关重要。莱姆病螺旋体伯氏伯氏菌（BB）产生表面蛋白p66，即几种整合素的孔蛋白和配体。 BB感染需要P66 小鼠。然而，DP66突变细菌在等于大鼠腹膜腔中的体外和保护性透析膜腔的父母菌株。这 DP66菌株比任何先天介体的父母野生型菌株（WT）更容易受到影响迄今为止测试的免疫力。通过将p66恢复到内源性基因座的互补染色体恢复小鼠的感染性。 BB不需要p66的整联蛋白结合活性小鼠感染，但需要有效的渗出和传播。缺少p66或特定整联蛋白结合结构域的氨基酸变化可降低与整合素AVB3，跨内皮的结合亲和力细菌在体外和体内的迁移，并显着降低了小鼠的传播率。表面 p66的细菌细胞和孔蛋白功能上的定位不受这些变化的影响。因此，虽然，p66的整合素结合活性的不存在确实会影响BB的某些毒力属性，但它不会解释DP66菌株的气相。我们的假设是p66的孔蛋白功能对在鼠宿主中的疏螺旋体生存。为了检验该假设，我们将采用C-MYC标记的P66突变体在体外不显示孔蛋白功能的缺陷。所有C-MYC-P66蛋白质均适当定位到细菌表面，在这个探索/发育项目中，重组B. burgdorferi菌株生成C-MYC-P66蛋白的生存和传播在体内将在体内表征老鼠。我们预测，缺乏孔蛋白的突变体在小鼠中会减弱或无毒。我们是独家具有经验，压力和感染模型的位置，以确定是否的孔蛋白功能是否是否疏浆的整体外膜蛋白p66在鼠感染中起着重要作用，并且进展到对多功能蛋白p66如何使细菌如何使细菌如何的机械理解更清晰的理解建立感染并在哺乳动物宿主中传播。这项工作将促进我们对硼螺螺旋体的致病机制，并可能指导对环境的研究伯利亚在接种现场面临的挑战以及新型治疗学的未来发展（例如，孔隙阻滞剂）专门针对伯罗利感染。