Diagnoses of tuberculosis, 'TB,' currently rely upon non-specific techniques such as X-ray exams and acid-fast microscopy. Improved diagnostics would preferably consider specific bacterial processes to provide real-time readouts of disease burden and response to chemotherapy. This dissertation presents the cell-wall incorporation of trehalose analogues (fluorescent and radioactive) by the mycobacterial antigen 85 enzymes as a novel method to label the causative bacteria of TB, Mycobacterium tuberculosis (Mtb). The trehalose mycolyltransesterase enzymes (antigens 85A, B, and C (Ag85)) serve as essential mediators of cell envelope function and biogenesis in Mtb. We show that the Ag85 enzymes display activities so broad that they allow added non-natural carbohydrate probes to be incorporated into Mtb growing in vitro and within macrophages. Design and synthesis of a library of structurally-diverse analogs of the sugar trehalose (Tre) revealed that Ag85-enzymes catalyze esterification of a wide variety of non-natural Tre structures, even stereoisomers and those appended with charged or bulky groups (Chapter 2). A novel mass-spectrometry based Ag85 enzyme assay was developed and employed to screen the library of compounds against all three isoforms of Ag85 (Chapter 3). This screen revealed that the Ag85 enzymes exhibit preference for dissacharides over monosaccharides and a broad tolerance for most modified trehalose compounds. This activity assay also afforded full kinetic analysis and the discovery of a novel, covalent inhibitor of the Ag85 enzymes. The Ag85 activity assay informed the design of a fluorescent trehalose-based compound (FITC-Tre), which is the first, non-toxic, selective, small molecule probe for mycobacterial infection. FITC-Tre was acylated with mycolyl esters by growing mycobacteria, anchoring the probe in the cell envelope resulting in fluorescent bacteria (Chapter 4). Adding FITC-Tre to Mtb-infected macrophages allowed selective, fluorescent tagging of Mtb in vivo (Chapter 5). Colocalization studies with antibodies against a variety of phagosomal associated components have hinted at the possibility of FITC-Tre as readout of cellular trafficking of bacteria. <sup>18</sup>F-trehalose, biotin-trehalose and rhodamine-trehalose are also substrates of Ag85. <sup>18</sup>F-trehalose shows promise as Mtb selective PET probe in an infected rabbit model of tuberculosis. Future work with these probes may allow for fluorescent tracking of the Mtb during the macrophage infection process, as well as the ability to label Mtb in infected tissue. The functional differences between the three isoforms of Ag85, A, B and C, are not well understood and may have implications for the survival and persistence of mycobacteria within humans. The differences in substrate specificity and catalytic activity between the Ag85 isoforms (discussed in Chapter 3) has been further investigated (Chapter 6). Mutation of three secondary site amino acids from Ag85C into Ag85B afforded nearly a twenty-fold gain in enzyme activity. Mutation of the equivalent Ag85B residues into Ag85C triggered nearly a twenty-fold loss in activity. Dissection of the roles of these three amino acids helps to explain the previously reported large differences in catalytic activity between Ag85A, B and C. Overexpression of Ag85A, B and C under tetracycline regulation revealed that these enzymes differentially modulate incorporation of mycolates into the cell wall. The Ag85 enzymes are not functionally redundant, and instead serve unique purposes in cell wall biosynthesis. In summary, this research has demonstrated that the broad substrate tolerance of Ag85 enzymes, coupled with their extracellular location, opens the door to probes of mycobacterial infection using many imaging modalities.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:655004 |
| Date | January 2011 |
| Creators | Backus, Keriann Marie |
| Contributors | Davis, Benjamin G.; Barry, Clifton E. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:882d8560-c0d3-471b-a896-224a6b22a0f0 |
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