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

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.

DESI is a newly developed mass spectrometric technique that enables biological samples to be analysed in situ. The Omni-Spray ion source required for DESI is now commercially-available, and can be attached to existing MS devices made by most major manufacturers. Crucially, operating parameters such as the output power (ES voltage) and composition (solvent) of the electrospray beam can be modulated such that DESI can be used to abrade through biological materials, allowing the user to 'mine down' and access biostrata below the surface of the sample. In addition, by rastering the ES beam across a sample, a lateral profile of biomolecule expression can be measured. DESI was introduced in October 2004 (although the commercial hardware only became available more recently), and so far, most of the studies exploiting it have been proof-of-principle technologies done in the laboratory of the inventor, Dr Graham Cooks. In the current proposal, we aim to use DESI to investigate the latent biostrata comprising bacterial biofilms and colonies. Biofilms have been the subject of many studies exploiting functional genomic analyses, yet in all cases, this has necessarily involved whole-scale mechanical perturbation of the biostructure. Because of this, we still know very little about the true nature of the biostratification in such bacterial assemblages, and it is becoming increasingly clear that this structural differentiation plays a central role in the physiology of these communities. Our existing lab priorities focus on proteomic and metabolomic analyses of Pseudomonas aeruginosa biofilms, colonies and planktonic cells, and we hope that the proposed study will complement this ongoing work. Very few other workers (on any continent) are currently exploiting DESI, so we have a unique window of opportunity in which to develop this approach further, and to exploit it in the investigation of an important microbiological and clinical problem.