AUTHOR=Strong Emily , Hart Bryan , Wang Jia , Orozco Maria Gonzalez , Lee Sunhee 

TITLE=Induced Synthesis of Mycolactone Restores the Pathogenesis of Mycobacterium ulcerans In Vitro and In Vivo

JOURNAL=Frontiers in Immunology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2022.750643

DOI=10.3389/fimmu.2022.750643

ISSN=1664-3224

ABSTRACT=<p><italic>Mycobacterium ulcerans</italic> is the causative agent of Buruli ulcer (BU), the third most common mycobacterial infection. Virulent <italic>M. ulcerans</italic> secretes mycolactone, a polyketide toxin. Most observations of <italic>M. ulcerans</italic> infection are described as an extracellular milieu in the form of a necrotic ulcer. While some evidence exists of an intracellular life cycle for <italic>M. ulcerans</italic> 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 <italic>M. ulcerans</italic> pathogenesis and host-response modulation. However, this sterile system drastically simplifies the <italic>M. ulcerans</italic> infection model and assumes that mycolactone is the only relevant virulence factor expressed by <italic>M. ulcerans</italic>. Here we show that the addition of purified mycolactone to macrophages during <italic>M. ulcerans</italic> 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 <italic>M. ulcerans</italic> infection, we have developed an inducible mycolactone expression system. Utilizing the mycolactone-deficient <italic>Mul</italic>::Tn118 strain that contains a transposon insertion in the putative beta-ketoacyl transferase (<italic>mup045</italic>), we have successfully restored mycolactone production by expressing <italic>mup045</italic> in a tetracycline-inducible vector system, which overcomes <italic>in-vitro</italic> 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 <italic>M. ulcerans</italic>. This mycolactone inducible system will allow for further analysis of the roles and functions of mycolactone during <italic>M. ulcerans</italic> infection.</p>