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Application of advanced spectroscopic techniques to monitor real-time transformations in aerosolized bovine tuberculosis bacteria

Primary Supervisor: Dr Natalie Garton, Department of Respiratory Sciences

Secondary Supervisor: Dr Andrew Hudson, Department of Chemistry

PhD project title: Application of advanced spectroscopic techniques to monitor real-time transformations in aerosolized bovine tuberculosis bacteria

University of Registration: University of Leicester

Project outline:

Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB), a devastating disease affecting global cattle populations. The disease also presents risk to other livestock, wildlife and humans and in the UK >£100m is spent annually on surveillance and control. M. bovis is transmitted via inhalation of bacterial aerosols, airborne microscopic droplets coughed up by infectious hosts. Bacteria sense and respond to changing environmental conditions by modifying their patterns of gene expression, which in turn affects their physiology. Surprisingly little is known of specific transcriptional and metabolic changes which occur in aerosolised bacteria. Investigating these in M. bovis has the potential to recognise features of the response of this pathogen to exploit for the development of new control measures to prevent the spread of bTB. In this project the student will use cutting edge techniques to study the changes that develop in aerosolized mycobacteria.

In previous studies bulk measurements have been made from sampling bacterial aerosols maintained in rotating drums. Bacterial cells in large populations can exhibit varied behaviour and approaches that interrogate individual cells are of considerable value. Using optical tweezers produced by shaped-laser beams, single cells within a population can be manipulated with exquisite precision and isolated alone or in patterned arrays. The illustration on the right shows the optical tweezing of a Mycobacterium smegmatis cell, where the rod-shaped bacterium becomes aligned along the axis of a propagating laser beam (directed into the image plane). The bacterium has been isolated from a suspension of cells, and is held by sufficiently-strong optical forces to enable the environment surrounding the cell to be varied. The response of the single cell can then be monitored by sensitive spectroscopic techniques. For example, measurements of fluorescence can be made, or a unique chemical fingerprint of cellular composition collected using Raman spectroscopy, a vibrational spectroscopy technique. We have experience of optically trapping aerosol microdroplets and in this project, aim to realise the possibility to make spectroscopic observations on a bacterial cell whilst it is suspended in an aerosol droplet.

To model the aerosol phase of transmission, single cells of M. bovis BCG will be observed in air-suspended droplets either isolated with optical tweezers or held in suspension by electrostatic forces in an electrodynamic balance. The size of the suspended aqueous droplets will match the typical size present in droplets released by coughing. Spectroscopic techniques will be used to monitor the transformation of the cells. For example, the cells can be genetically modified to express green fluorescent protein (GFP) under the control of promoters relevant to environmental changes experienced in aerosol (e.g osmotic and temperature sensitive promoters). The fluorescence of the GFP reporter is detected within the suspended droplet containing the bacterial cell by fluorescence imaging.

Initial work will involve liquid phase and aerosol model experiments using existing strains of M. smegmatis (a non-pathogenic relative of M. bovis) and development of appropriate reporter strains of M. bovis BCG. These will include transcriptional fusions reporting on osmotic stress (which is experienced by tubercle bacteria in the aerosol produced by a human TB patient coughing) and other targets informed by ongoing studies in the group, including ATP status. We have acquired Raman spectra from M. smegmatis cultured in various conditions and the student will explore the biological relevance of serial Raman spectra to examine the chemical dynamics of the aerosolised bacteria and other signals from trial experiments. Modified BCG strains will then be studied the liquid and aerosol phase in order to address specific hypotheses concerning adaptive processes taking place.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Microbiology

Techniques that will be undertaken during the project:

The cross-disciplinary project will involve a wide range of techniques, including culture of mycobacteria in defined physiological states, genetic manipulation to prepare mycobacterial strains expressing genes to report on key aspects of metabolism or the creation of deletions in key genes, biochemical characterisation, microfluidics, optical tweezing, confocal fluorescence microscopy, Raman microspectroscopy, imaging and spectroscopy.

Contact: Dr Natalie Garton, University of Leicester