Etwa ein Drittel der Weltbevölkerung ist mit Mycobacterium tuberculosis (Mtb), der Erreger der Tuberkulose (TB), infiziert. Daher ist es unbedingt notwendig vorhandenen Behandlungsstrategien weiter zu verbessern. Diese Study beschäftigt sich mit dem Mtb Protein Ag85C, einer Mycolyltransferase, als ein neues Ziel für die medikamentöse Behandlung der TB. Ag85C ist eines von drei verwandten Proteinen, Ag85A, B und C, welche zusammen an der Biogenese der Zellwand von Mtb beteiligt sind. Eine Gruppe von chemischen Molekülen mit den Namen Ag85C-1 bis -4 wurde als Inhibitoren von Ag85C getestet. Alle Verbindungen waren in der Lage das Wachstum von Mtb in Flüssigkulturen zu inhibieren, aber nur Ag85C-3 hatte ebenfalls einen Effekt auf intrazelluläre Bakterien, welches in einem Makrophagen-Infektions-System getestet wurde. Hervorzuheben ist, dass Ag85C-3 darüber hinaus auch das in vitro Überleben eines MDR Stammes inhibierte. Dies macht dieses Molekül zu einem interessanten Kandidaten für neue anti-mycobakterielle Therapieansätze. Desweiteren wurden detaillierte, funktionelle Charakterisierungen der Effekte von Ag85C-3 auf Mtb durchgeführt. Die Verbindung modifiziert die Lipide der mykolischen Säuren in der Zellwand durch die Blockierung der Ag85 Funktionen. Dieser Effekt führt dann zu einer Veränderung in der Durchlässigkeit der Außenhülle von Mtb. Mit Hilfe der microarray Analyse wurden die Regulierungen der Signalwege durch Ag85C-3 umfassend untersucht. Es konnte gezeigt werden, dass lebensnotwendige Siderophore durch das Molekül modifiziert werden, was auf mehrere Wirkungsmechanismen schließen lässt. Diese Erkenntnisse machen Ag85C, als Ziel, und Ag85C-3, als Inhibitor, zu vielversprechende Kandidaten für zukünftige Medikamentenforschung auf dem Gebiet der TB-Therapien. Diese Studie hebt zudem die zielbasierte Identifizierung von chemischen Inhibitoren als wichtigen und wertvollen Ansatz für die Medikamentenentwicklung hervor. / Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) infects about one-third of the world’s population. Therefore there is an urgent need to improve existing intervention strategies. This study evaluated the Mtb Ag85C protein, a mycolyl transferase, as a novel target for drug mediated intervention. Ag85C belongs to a family of three cognate proteins, Ag85A, B and C. They are involved in the final steps of Mtb cell envelope biogenesis. A panel of chemical molecules, Ag85C-1-4, which bind to Ag85C were utilized as inhibitors of Ag85C. All compounds inhibited growth of Mtb in vitro in liquid medium cultures but only Ag85C-3 had an effect on intracellular bacteria in macrophage infection system. Importantly, Ag85C-3 can inhibit in vitro survival of a MDR strain of Mtb making it a relevant molecule in the search for novel classes of anti-mycobacterial compounds. Furthermore a detailed functional characterization of Ag85C-3 effect on Mtb was performed. It modified the cell wall mycolic acid containing lipid amounts by blocking Ag85 function that led to changes in permeability of Mtb envelope. A comprehensive analysis of Mtb signalling pathways regulated by Ag85C-3 was investigated through microarray analysis. It showed modification of vital siderophore biosynthesis indicating multiple mechanisms of action. Thus the target, Ag85C and the inhibitor, Ag85C-3 are promising candidates for future TB drug research aimed at combating broad spectrum resistance development. This study also reinforces target based identification of chemical inhibitors as a valid and valuable approach in drug development.

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. ¹⁸F-trehalose, biotin-trehalose and rhodamine-trehalose are also substrates of Ag85. ¹⁸F-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.

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