Structural Analysis of Bordetella pertussis Biofilms by Confocal Laser Scanning Microscopy
|
Author:
Date:
2018-08-05
[Abstract] Biofilms are sessile communities of microbial cells embedded in a self-produced or host-derived exopolymeric matrix. Biofilms can both be beneficial or detrimental depending on the surface. Compared to their planktonic counterparts, biofilm cells display enhanced resistance to killing by environmental threats, chemicals, antimicrobials and host immune defenses. When in biofilms, the microbial cells interact with each other and with the surface to develop architecturally complex multi-dimensional structures. Numerous imaging techniques and tools are currently available for architectural analyses of biofilm communities. This allows examination of biofilm development through acquisition of three-dimensional images that can render structural features of the sessile community. A frequently ...
[摘要] 生物膜是嵌入自生或宿主衍生的外聚合物基质中的微生物细胞的固着群落。根据表面,生物膜可以是有益的或有害的。与浮游生物相比,生物膜细胞表现出更强的抗环境威胁,化学物质,抗菌药物和宿主免疫防御能力。当处于生物膜中时,微生物细胞彼此相互作用并与表面相互作用以形成结构复杂的多维结构。目前,许多成像技术和工具可用于生物膜群落的建筑分析。这允许通过获取可以呈现无柄群落的结构特征的三维图像来检查生物膜的发展。经常使用的工具是共聚焦激光扫描显微镜。我们提出了一个详细的协议,以生长,观察和分析呼吸道人类病原体,百日咳博德特氏菌的生物膜在空间和时间。
【背景】百日咳博德特氏菌(Bordetella pertussis)是上呼吸道的专性人类病原体,引起百日咳或百日咳(Mooi,2010; Dorji et al。,2018)。 B的生物膜。百日咳在各种人造表面上以及静态,摇动和流体流动条件下形成(Mishra et al。,2005; Sloan et al。,2007 ; Serra et al。,2011)。对这些生物膜的显微评估表明,这种细菌产生不规则形状的微集落,由流体通道分隔,嵌入由细胞外DNA(eDNA),蛋白质和多糖组成的外聚合物基质中(Parise et al。,2007; Sloan et al。,2007; Serra et al。,2008; ...
|
|
Quantification of Hydrogen Sulfide and Cysteine Excreted by Bacterial Cells
|
Author:
Date:
2018-05-20
[Abstract] Bacteria release cysteine to moderate the size of their intracellular pools. They can also evolve hydrogen sulfide, either through dissimilatory reduction of oxidized forms of sulfur or through the deliberate or inadvertent degradation of intracellular cysteine. These processes can have important consequences upon microbial communities, because excreted cysteine autoxidizes to generate hydrogen peroxide, and hydrogen sulfide is a potentially toxic species that can block aerobic respiration by inhibiting cytochrome oxidases. Lead acetate strips can be used to obtain semiquantitative data of sulfide evolution (Oguri et al., 2012). Here we describe methods that allow more-quantitative and discriminatory measures of cysteine and hydrogen sulfide release from bacterial cells. An ...
[摘要] 细菌释放半胱氨酸以调节细胞内池的大小。它们也可以通过硫的氧化形式的异化还原或通过细胞内半胱氨酸的故意或无意降解来释放硫化氢。这些过程会对微生物群落产生重要影响,因为排泄的半胱氨酸会自动氧化生成过氧化氢,而硫化氢是一种潜在的毒性物种,可通过抑制细胞色素氧化酶来阻断有氧呼吸。醋酸铅条可用于获得硫化物演化的半定量数据(Oguri et al。,2012)。在这里,我们描述的方法,允许更多的定量和歧视措施半胱氨酸和硫化氢释放细菌细胞。提供了一个说明性实例,其中当暴露于外源性胱氨酸时,大肠杆菌迅速产生半胱氨酸和硫化物(Chonoles Imlay等人,2015; Korshunov等人, ,2016)。
【背景】微生物通过几种途径产生了减少的硫物质。硫酸盐还原菌利用还原过程作为能量生成的组成部分。其他细菌释放硫化物,作为硫物质(包括半胱氨酸)的蓄意或偶然降解的副产物。我们观察到半胱氨酸本身是在细胞内水平异常高时排泄的,这种情况可能通过不受控制的氨基酸输入或半胱氨酸合成失调发生。这些硫物质具有非同寻常的反应性,因为它们以高亲和力与金属结合,也是与分子氧发生化学反应的少数生物分子之一。结果是减少的硫化合物可以对细胞产生重要影响。因此,跟踪各种情况下含硫化合物的动态变化是非常重要的。
硫醇试剂 - 特别是5,5-二硫代双(2-硝基苯甲酸)(DTNB) ...
|
|
Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System
|
Author:
Date:
2018-04-20
[Abstract] The hepatitis B virus (HBV) is an important global human pathogen and represents a major cause of hepatitis, liver cirrhosis and liver cancer. The HBV capsid is composed of multiple copies of a single viral protein, the capsid or core protein (HBc), plays multiple roles in the viral life cycle, and has emerged recently as a major target for developing antiviral therapies against HBV infection. Although several systems have been developed to study HBV capsid assembly, including heterologous overexpression systems like bacteria and insect cells, in vitro assembly using purified protein, and mammalian cell culture systems, the requirement for non-physiological concentrations of HBc and salts and the difficulty in manipulating host regulators of assembly presents major limitations ...
[摘要] 乙型肝炎病毒(HBV)是一种重要的全球人类病原体,并且是肝炎,肝硬化和肝癌的主要原因。 HBV衣壳由单个病毒蛋白的多个拷贝组成,衣壳或核心蛋白(HBc)在病毒生命周期中起着多重作用,并且最近已经成为开发抗HBV病毒疗法的主要靶标。尽管已经开发了几种用于研究HBV衣壳组装的系统,包括异源过表达系统如细菌和昆虫细胞,使用纯化蛋白质和哺乳动物细胞培养系统进行体外组装,但对非生理浓度HBc和盐以及难以操纵装配的宿主调节物在生理相关条件下的衣壳装配的详细研究方面存在主要限制。我们最近开发了基于兔网织红细胞裂解物(RRL)的哺乳动物无细胞系统,其中HBc以生理浓度表达并在近生理条件下组装成衣壳。该系统已经揭示了HBc装配要求,这是以前装配系统所不能预料的。此外,该系统中的衣壳组装受可容易操作的内源宿主因子调控。在这里,我们提供了这种无细胞衣壳装配系统的详细协议,包括如何操纵调节装配的宿主因子的说明。
【背景】乙型肝炎病毒(HBV)是一种重要的全球人类病原体,其长期感染全世界数以亿计的人并且代表病毒性肝炎,肝硬化和肝癌的主要原因(Seeger等人, 2013; Trepo et。,2014)。 HBV通过逆转录RNA中间体(所谓的前基因组RNA(pgRNA))在核衣壳内(NC)复制其基因组DNA(一种宽松的环状部分双链DNA(RC ...
|
|