NhaP-type cation-proton antiporters in Vibrio cholerae and Yersinia pestis

霍乱弧菌和鼠疫耶尔森氏菌中的 NhaP 型阳离子-质子逆向转运蛋白

基本信息

批准号：
8868926
负责人：
Claudia C Hase
金额：
$ 18.25万
依托单位：
OREGON STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8868926
关键词：
Affect Alkalies Alleles Animal Model Animals Anti-Bacterial Agents Bacteria Bacterial Infections Bacterial Physiology Behavior Binding Sites Bioenergetics Ca(2+)-Transporting ATPase Calcium Cations Cell membrane Cells Chromosomes Cytoplasm Data Dependence Development Environment Escherichia coli Family Future Generations Genes Genome Goals Growth Health Homeostasis Homologous Gene Housekeeping In Vitro Infection Integral Membrane Protein Ion Pumps Ions Kinetics Knock-out Life Ligands Link Mediating Membrane Microbe Modeling Molecular Organism Outcome Pathogenesis Phase Phenotype Physiological Physiology Plasmids Play Potassium Production Property Proteins Protons Regulation Relative (related person)Reporting Research Resistance Role Site Sodium Sodium-Hydrogen Antiporter Specificity Testing Therapeutic Intervention Time Transmembrane Transport Variant Vesicle Vibrio cholerae Virulence Virulence Factors Work Yersinia pestis antimicrobial antimicrobial drug antiport antiporter base coping defined contribution drug development extracellular fitness infancy member microbial mutant novel pH Homeostasis paralogous gene pathogen pathogenic bacteria prevent trait

项目摘要

DESCRIPTION (provided by applicant): Bacteria are exposed to a variety of environmental conditions and have evolved different mechanisms to cope with changes in the extracellular milieu. The existence of bacterial K+/H+ antiporters that prevent the over- accumulation of potassium in the cytoplasm was predicted by Peter Mitchell over 50 years ago and K+/H+ antiport activity as such was demonstrated in everted membrane vesicles from Escherichia coli some time ago. However, despite the importance of K+/H+ antiport for bacterial physiology, its molecular mechanisms remain under-investigated and identification of specific "housekeeping" K+/H+ antiporters in bacteria remained elusive until recently, when we showed that the V. cholerae NhaP2 antiporter acts as a specific K+/H+ antiporter. This protein was most active at low pH and is indispensable for the growth of V. cholerae under those conditions, removing excess of internal K+ from the cytoplasm at the expense of pH. We have also begun to characterize the other two NhaP-type antiporters from V. cholerae. Here, we propose to further examine the physiological roles of the different NhaP paralogs in Vibrio cholerae. Like most pathogens, Yersinia pestis have to adapt to wide range of environments to establish an infection. One of the most peculiar features of the Y. pestis physiology and virulence is its stringent dependence on alkali/alkaline cations, primarily Ca2+. Despite the importance of calcium in the regulation of Y. pestis virulence factor production, virtually nothing is known abou the mechanism of membrane transport of calcium in this organism. In Y. pestis, several different proteins potentially mediate the transport of Ca2+ across the bacterial membrane and some of these proteins are predicted to be able to integrate Ca2+, Na+, K+ and pH homeostasis in this organism. The present project is focused on the importance of the various putative calcium transporters encoded by Y. pestis for bacterial growth under various conditions. As the function and expression of the T3SS appears to be sensitive to both Ca2+ and (indirectly) Na+, we will also examine Yop secretion in these mutants to allow the assessment of their potential as targets for the development of a novel class of antimicrobials. Our experimental approach is expected to yield the following outcomes. It is anticipated that, at the end of the requested two years of support, we will have analyzed the kinetics and cation selectivity of various mutant NhaPs, thereby testing the validity of our "ligand shading" hypothesis. In addition, we will have identified the detailed roles of the three NhaP paralogs in the overall membrane bioenergetics and physiology of V. cholerae. Lastly, we expect to have gained important information about the link between membrane calcium transport and virulence of Y. pestis. Thus, our proposed work has the potential to add significantly to our understanding of the contribution of membrane bioenergetics to bacterial physiology per se. Moreover, our work will begin to evaluate the role of NhaP-mediated cation transport in bacterial pathogenesis that could provide the basis for future testing of nhaP mutant strains in animal models. Ultimately, we anticipate the identification of transporters that might provide novel targets for drug development during later phases of the project, potentially resulting in the development of a new class of effective anti-bacterial agents with completely new mechanisms of action.

描述（由申请人提供）：细菌暴露于各种环境条件下，并进化出不同的机制来应对细胞外环境的变化。 Peter Mitchell 在 50 多年前就预测了细菌 K+/H+ 反向转运蛋白的存在，可以防止细胞质中钾的过度积累，并且 K+/H+ 反向转运活性在不久前在大肠杆菌的外翻膜囊泡中得到了证实。然而，尽管 K+/H+ 反向转运对细菌生理学很重要，但其分子机制仍未得到充分研究，并且细菌中特定“看家”K+/H+ 反向转运蛋白的鉴定仍然难以捉摸，直到最近我们证明霍乱弧菌 NhaP2 反向转运蛋白发挥作用作为特定的 K+/H+ 反向转运蛋白。这种蛋白质在低 pH 条件下最为活跃，对于霍乱弧菌在这些条件下的生长是不可或缺的，它可以以牺牲 pH 值为代价从细胞质中去除过量的内部 K+。我们还开始鉴定来自霍乱弧菌的另外两种 NhaP 型反向转运蛋白。在这里，我们建议进一步研究不同 NhaP 旁系同源物在霍乱弧菌中的生理作用。与大多数病原体一样，鼠疫耶尔森氏菌必须适应广泛的环境才能产生感染。鼠疫耶尔森氏菌生理学和毒力最独特的特征之一是其对碱金属/碱性阳离子（主要是 Ca2+）的严格依赖。尽管钙在调节鼠疫耶尔森氏菌毒力因子产生中很重要，但实际上对该生物体中钙的膜转运机制一无所知。在鼠疫耶尔森氏菌中，几种不同的蛋白质可能介导 Ca2+ 穿过细菌膜的运输，其中一些蛋白质预计能够在该生物体中整合 Ca2+、Na+、K+ 和 pH 稳态。本项目的重点是鼠疫耶尔森氏菌编码的各种假定的钙转运蛋白对于各种条件下细菌生长的重要性。由于 T3SS 的功能和表达似乎对 Ca2+ 和（间接）Na+ 敏感，我们还将检查这些突变体中的 Yop 分泌，以评估它们作为开发新型抗菌剂的靶点的潜力。我们的实验方法预计会产生以下结果。预计，在所要求的两年支持结束时，我们将分析各种突变体 NhaP 的动力学和阳离子选择性，从而测试我们的“配体遮蔽”假设的有效性。此外，我们还将确定三种 NhaP 旁系同源物在霍乱弧菌整体膜生物能学和生理学中的详细作用。最后，我们期望获得有关膜钙转运与鼠疫耶尔森氏菌毒力之间联系的重要信息。因此，我们提出的工作有可能显着增加我们对膜生物能学对细菌生理学本身的贡献的理解。此外，我们的工作将开始评估 NhaP 介导的阳离子转运在细菌发病机制中的作用，这可以为未来在动物模型中测试 nhaP 突变株提供基础。最终，我们预计转运蛋白的鉴定可能会在项目的后期阶段为药物开发提供新的靶标，从而有可能开发出具有全新作用机制的新型有效抗菌药物。