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机会致病菌，可引起广泛的医院感染。然而，该生物体可能最出名的是在囊性纤维化患者的肺深处形成对抗生素不敏感的粘液样菌落和生物膜，并且是导致该人群中高发病率和死亡率的原因。在过去的几十年里，细菌组装体如生物膜一直是深入研究的主题。特别是，它们已经成为许多功能基因组分析的焦点。然而，从这些方法得到的数据的解释已受到阻碍的事实，即生物膜和殖民地是复杂的结构，打破了机械干预。这意味着我们必然会丢失大量关于生物分子表达的空间分布的信息，这仅仅是由于样品收获过程的侵入性/破坏性。低通量的方法，如激光扫描共聚焦荧光显微镜分析的荧光标记的融合蛋白，揭示了生物膜很可能表现出分层的蛋白质表达。然而，直到最近，没有高通量的方法可以应用于评估全球生物分子概况如何通过和跨微生物生物组装体变化。直接研究这个问题的可能性最近随着对环境中的生物样品的实时、空间分辨分析的引入而出现（即，“湿”）条件下。所需的技术，这样做利用'DESI' MS，这是基于解吸电喷雾电离。在这里，带电溶剂液滴的细喷雾用于“挖掘”样品。然后收集所产生的解吸离子并将其传递到质谱仪中进行在线分析，并且在蛋白质的情况下，进行“自上而下”的鉴定。MSP-MS是在Graham Cooks博士的实验室中开发的，他已经将该技术应用于一系列生物样本（包括人类皮肤，干血和液体血液，植物表面，尿液样本，甚至是实体瘤的边缘）。Cooks实验室的工作也证明了DESI对蛋白质组学和代谢组学分析都是有效的。然而，在Cooks的实验室之外，DESI的应用很少，部分原因是必要的硬件最近才可用，部分原因是更广泛的生物科学界-特别是微生物学家-对该技术缺乏认识。在这个项目中，我们的目标是利用DESI来研究代谢产物和蛋白质表达谱如何在铜绿假单胞菌的菌落和生物膜之间变化。我们完全期待这种方法将提供有关在这些结构的不同部分生长的细胞的全球生物分子概况的信息。这种方法非常好地补充了我们现有的研究重点，并将为BBSRC资助的剑桥蛋白质组学中心的能力增加一个有价值的额外维度。

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