Biogenesis and modification of the Bordetella cell envelope

博德特氏菌细胞包膜的生物发生和修饰

• 批准号: RGPIN-2022-05073
• 负责人: Fernandez, Rachel
• 金额: $ 2.33万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=761027
• 关键词: Biogenesis; modification; Bordetella; cell; envelope

The envelope of Gram negative bacteria is complex. It comprises the inner membrane and the cell wall. The cell wall consists of a thin peptidoglycan structure surrounded by an asymmetric outer membrane bilayer consisting of phospholipids and lipopolysaccharides (LPS) within which are attached or embedded various proteins. The space between the inner and outer membranes is called the periplasm. In addition to functioning as a selective barrier to protect bacteria from harsh conditions, the envelope plays a critical role in determining the outcome of interactions between the bacterium and its environment. Indeed, several components of the cell envelope are regulated in response to environmental conditions. This proposal addresses components comprising the envelope of Bordetella species. Many Bordetella species are human or animal pathogens and there is much to learn about how the Bordetella envelope is made. We will focus on two aspects of the Bordetella envelope - a system that modifies the lipid A portion of lipopolysaccharide and the biogenesis of an unusual member of the autotransporter class of outer membrane proteins. We have discovered a 5 gene locus, called lgm, that modifies lipid A with glucosamine. These genes are arranged in what appears to be a non-contiguous operon, a recently described genetic architecture that has been shown to coordinate bacterial gene expression. Here, lgmA-lgmD are found in the sense strand, with lgmE found in the antisense strand partially overlapping lgmB. All genes are expressed which suggests that the lgm locus undergoes posttranscriptional control. Surprisingly, the anti-sense lgmE appears to encode a membrane protein whose expression may be involved in posttranslational control of this locus. We will examine how the lgm locus is regulated temporally and in response to various stimuli. We also propose that the Lgm proteins function together as a complex and in a coordinated manner. We have developed an expression system that is able to make 10-20mg/L quantities of components of this system. This will allow us to characterize these proteins structurally and develop hypotheses of how this locus is regulated transcriptionally, post-transcriptionally, translationally, and post-translationally. Autotransporter protein biogenesis requires that the protein be suitably targeted to the outer membrane while being maintained in a translocation-competent state. TcfA is an unusual autotransporter protein that is only found in Bordetella. It is mis-targeted when expressed in E. coli, suggesting that Bordetella possess a unique system to localize TcfA to the surface. We will determine the requirements of TcfA localization. Uncovering fundamental principles of how the envelope is built in both model and non-model organisms can lead to improved methods for cell surface manipulation that can be used in bio and nanotechnology applications.

革兰氏阴性菌的包膜是复杂的。它包括内膜和细胞壁。细胞壁由薄的肽聚糖结构组成，该结构被由磷脂和脂多糖（LPS）组成的不对称外膜双层包围，磷脂和脂多糖内附着或嵌入各种蛋白质。内外膜之间的空间称为周质。除了作为选择性屏障保护细菌免受恶劣条件的影响外，包膜在决定细菌与其环境之间相互作用的结果方面起着关键作用。事实上，细胞被膜的几个组成部分是受环境条件调节的。该提案涉及构成博德特氏菌属种包膜的组分。许多博德特氏菌属物种是人类或动物的病原体，关于博德特氏菌属的包膜是如何形成的，还有很多东西需要了解。我们将集中在两个方面的博德特氏菌包膜-一个系统，修改脂多糖的脂质A部分和一个不寻常的成员的自转运类外膜蛋白的生物发生。我们已经发现了一个5基因位点，称为lgm，它用葡萄糖胺修饰脂质A。这些基因排列在一个非连续的操纵子中，这是一种最近描述的遗传结构，已被证明可以协调细菌的基因表达。在此，IgmA-IgmD存在于有义链中，IgmE存在于与IgmB部分重叠的反义链中。所有基因的表达表明，lgm基因座进行转录后控制。令人惊讶的是，反义lgmE似乎编码膜蛋白，其表达可能参与该基因座的翻译后控制。我们将研究lgm基因座是如何在时间上和对各种刺激的反应中进行调节的。我们还提出，LGM蛋白作为一个复杂的功能，并在一个协调的方式。我们已经开发了一个表达系统，能够产生10- 20 mg/L的量的该系统的组件。这将使我们能够从结构上表征这些蛋白质，并提出关于该基因座如何在转录、转录后、转录后和转录后调节的假设。自转运蛋白的生物合成需要蛋白质被适当地靶向外膜，同时保持在易位能力状态。TcfA是一种不寻常的自转运蛋白，仅在博德特氏菌中发现。当在E.表明博德特氏菌具有独特的TcfA定位系统。我们将确定TcfA本地化的要求。揭示包膜如何在模型和非模型生物体中构建的基本原理可以导致用于生物和纳米技术应用的细胞表面操作的改进方法。