Induced Synthesis of Mycolactone Restores the Pathogenesis of and .

is the causative agent of Buruli ulcer (BU), the third most common mycobacterial infection. Virulent secretes mycolactone, a polyketide toxin. Most observations of infection are described as an extracellular milieu in the form of a necrotic ulcer. While some evidence exists of an intracellular life cycle for during infection, the exact role that mycolactone plays in this process is poorly understood. Many previous studies have relied upon the addition of purified mycolactone to cell-culture systems to study its role in pathogenesis and host-response modulation. However, this sterile system drastically simplifies the infection model and assumes that mycolactone is the only relevant virulence factor expressed by . Here we show that the addition of purified mycolactone to macrophages during infection overcomes the bacterial activation of the mechanistic target of rapamycin (mTOR) signaling pathway that plays a substantial role in regulating different cellular processes, including autophagy and apoptosis. To further study the role of mycolactone during infection, we have developed an inducible mycolactone expression system. Utilizing the mycolactone-deficient ::Tn118 strain that contains a transposon insertion in the putative beta-ketoacyl transferase (), we have successfully restored mycolactone production by expressing in a tetracycline-inducible vector system, which overcomes growth defects associated with constitutive complementation. The inducible mycolactone-expressing bacteria resulted in the establishment of infection in a murine footpad model of BU similar to that observed during the infection with wild-type . This mycolactone inducible system will allow for further analysis of the roles and functions of mycolactone during infection.