Investigation of the Porin Function of B. burgdorferi P66

伯氏疏螺旋体 P66 孔蛋白功能的研究

• 批准号: 9891001
• 负责人: Jenifer L Coburn
• 金额: $ 23.1万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-11 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9891001
• 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; tick bite

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.

疏螺旋体螺旋体从接种部位（节肢动物叮咬）传播到多个组织， 持续存在，尽管宿主免疫反应，表明与哺乳动物的相互作用和适应 在感染过程中，环境是至关重要的。莱姆病螺旋体伯氏疏螺旋体（Bb）产生 表面蛋白P66，是一种孔蛋白和几种整联蛋白的配体。Bb感染需要P66 对小鼠然而，Dp66突变细菌存活、复制并改变蛋白质产生，其水平相当于 亲本菌株在体外和大鼠腹膜腔中的保护性透析膜室中。的 Dp66菌株不比亲本野生型菌株（WT）更易受任何先天免疫调节因子的影响。 免疫力测试至今通过将p66恢复到细胞上的内源基因座来互补突变体。 染色体在小鼠中恢复了传染性。Bb不需要P66的整合素结合活性 感染小鼠，但需要有效的外渗和传播。缺乏P66或特异性 整联蛋白结合结构域中的氨基酸变化降低了与整联蛋白AVB 3的结合亲和力， 细菌在体外和体内的迁移，并显着降低小鼠中的传播率。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，在本发明中， 尽管P66整合素结合活性的缺失确实影响Bb的某些毒力属性，但它并不 解释了Dp66菌株的无毒力。我们的假设是，P66的孔蛋白功能是关键， 疏螺旋体在鼠宿主中的存活。为了验证这一假设，我们将使用c-Myc标记的p66突变体， 在体外显示和不显示孔蛋白功能缺陷。所有的c-Myc-P66蛋白都被适当地定位 在这个探索性/开发项目中，重组B。伯氏菌株 将在体内对c-Myc-P66蛋白的产生进行存活和传播方面的表征 小鼠我们预测孔蛋白缺陷突变体将在小鼠中减毒或无毒。我们在独家报道 有经验的立场，菌株，和感染模型，以确定是否孔蛋白的功能， 疏螺旋体的完整外膜蛋白P66在鼠感染期间起重要作用， 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和传播。这项工作将促进我们对 致病机制的疏螺旋体螺旋体，并可能指导调查的环境 疏螺旋体在接种部位面临的挑战以及未来可能开发的新疗法 (e.g.孔阻断剂）特异性对抗疏螺旋体感染。