Mass spectrometry-based 'omic mining through the biostrata of Pseudomonas aeruginosa colonies and biofilms

通过铜绿假单胞菌菌落和生物膜的生物地层进行基于质谱的“组学挖掘”

• 批准号: BB/F01581X/1
• 负责人: Martin Welch
• 金额: $ 11.64万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FF01581X%2F1
• 关键词: Mass; spectrometry; based; omic; mining

Pseudomonas aeruginosa is a Gram-negative opportuinistic human pathogen, which is responsible for causing a wide range of nosocomial infections. However, the organism is perhaps best known for forming antibiotic-insensitive mucoid colonies and biofilms deep within the lungs of patients with cystic fibrosis, and is responsible for causing high rates of morbidity and mortality among this population. Bacterial assemblies such as biofilms have been the subject of intensive investigation over the last few decades. In particular, they have been the focus of numerous functional genomic analyses. However, interpretation of the data derived from these approaches has been hampered by the fact that biofilms and colonies are complex structures which break down upon mechanical intervention. This means that we necessarily lose a lot of information about the spatial distribution of biomolecule expression simply due to the invasive/destructive nature of the sample harvesting procedure. Low through-put approaches like laser scanning confocal fluorescence microscopic analysis of fluorescently-labelled fusion proteins has revealed that biofilms are highly likely to exhibit stratified protein expression. However, until very recently, no high through-put approach could be applied to assess how global biomolecule profiles vary through and across microbial bioassemblies. The potential to investigate this issue directly came recently with the introduction of real-time, spatially-resolved analysis of biological samples in ambient (i.e., 'wet') conditions. The technology required to do this exploits 'DESI' MS, which is based on desorption electrospray ionization. Here, a fine spray of charged solvent droplets is used to 'mine in' to the sample. The resulting desorbed ions are then collected and passed into a mass spectrometer for on-line analysis, and, in the case of proteins, 'top-down' identification. DESI-MS was developed in the lab of Dr Graham Cooks, who has applied the technique to a range of biological samples (including human skin, dried- and liquid blood, plant surfaces, urine samples and even the margins of solid tumours). Work from Cooks' lab has also demonstrated that DESI is effective for both proteomic and metabolomic profiling. However, outside Cooks' lab, DESI has seen very little uptake, partly because the necessary hardware has only recently become available, and partly due to lack of awarness of the technology amongst the wider biological sciences community - especially microbiologists. In this project, we aim to exploit DESI to study how the metabolite and protein expression profiles vary across and through colonies and biofilms of P. aeruginosa. We fully anticipate that this approach will provide information about the global biomolecule profiles of cells growing in different parts of these structures. This approach complements our existing research priorities extremely well, and will add a valauble extra dimension to the capabilities of the BBSRC-funded Cambridge Centre for Proteomics.

铜绿假单胞菌是一种革兰氏阴性的人类病原体，负责引起广泛的医院感染。但是，该生物可能以形成抗生素不敏感的粘液菌落和生物膜而闻名，并在患有囊性纤维化的患者的肺部深处，并负责在该人群中引起高发病率和死亡率。在过去的几十年中，细菌组件（例如生物膜）一直是进行深入研究的主题。特别是，它们一直是许多功能基因组分析的重点。但是，生物膜和菌落是在机械干预时崩溃的复杂结构，从而阻碍了从这些方法得出的数据的解释。这意味着，仅由于样本收获程序的侵入性/破坏性性质，我们必然会丢失有关生物分子表达的空间分布的大量信息。诸如荧光标记融合蛋白的激光扫描共聚焦荧光显微镜分析之类的低渗透方法表明，生物膜很可能表现出分层蛋白的表达。但是，直到最近，还不能应用高渗透方法来评估全球生物分子曲线如何通过微生物生物组合而变化。最近直接调查该问题的潜力是由于对环境（即“湿”）条件中生物样品的实时，空间分析的分析引入。进行此功能的“ DESI” MS所需的技术，该技术基于解吸电离电离。在这里，使用了精细的充电溶剂液滴将其用于样品中的“矿山”。然后收集所得的解吸离子并将其传递到质谱仪中进行在线分析，在蛋白质的情况下，“自上而下”的识别。 DESI-MS是在Graham Cooks博士的实验室中开发的，Graham Cooks博士已将该技术应用于一系列的生物样品（包括人皮肤，干燥和液体血液，植物表面，尿液样本，甚至实体瘤的边缘）。库克斯实验室的工作还表明，DESI对蛋白质组学和代谢组分析有效。但是，在库克斯的实验室之外，Desi几乎没有吸收，部分原因是由于最近才能使用，部分原因是由于在更广泛的生物科学社区（尤其是微生物学家）中缺乏该技术的可取性。在这个项目中，我们旨在利用DESI研究代谢产物和蛋白质表达的图谱如何在铜绿假单胞菌的菌落和生物膜之间变化。我们完全预料到，这种方法将提供有关这些结构不同部分生长的细胞全球生物分子谱的信息。这种方法对我们现有的研究优先级的补充非常好，并将为BBSRC资助的剑桥蛋白质组学中心的功能增加效果。

项目成果

Ligand binding kinetics of the quorum sensing regulator PqsR.

群体感应调节剂 PqsR 的配体结合动力学。

• DOI: 10.1021/bi400315s
• 发表时间: 2013
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Welch M

Microbial biofilm as a smart material.

• DOI: 10.3390/s150204229
• 发表时间: 2015-02-12
• 期刊: Sensors (Basel, Switzerland)
• 作者: Garde C;Welch M;Ferkinghoff-Borg J;Sams T
• 通讯作者: Sams T

Kinetic model for signal binding to the Quorum sensing regulator LasR.

• DOI: 10.3390/ijms140713360
• 发表时间: 2013-06-27
• 期刊: International journal of molecular sciences
• 影响因子: 5.6
• 作者: Claussen A;Jakobsen TH;Bjarnsholt T;Givskov M;Welch M;Ferkinghoff-Borg J;Sams T
• 通讯作者: Sams T

Comparative microarray analysis reveals that the core biofilm-associated transcriptome of Pseudomonas aeruginosa comprises relatively few genes.

• DOI: 10.1111/j.1758-2229.2010.00158.x
• 发表时间: 2010-06
• 期刊: Environmental microbiology reports
• 影响因子: 3.3
• 作者: Sanaya Patell;M. Gu;P. Davenport;M. Givskov;Richard D. Waite;M. Welch