The AcasR-FadD9 regulatory axis modulates iron homeostasis and susceptibility to the anti-mycobacterial compound ACA

Mycobacterial drug resistance leads to prolonged treatment, increased mortality, and increased healthcare costs. In this study, 3-azidothiophene-2-carboxylic acid (ACA), a dual-targeting inhibitor of the cell assembly proteins CpsA1 and CpsA2, exerted its activity through an iron-dependent pathway. The screening of drug-resistant mutants revealed that a frameshift mutation in the transcriptional regulator acasR (BCG_0932/Rv0880) conferred ACA resistance. Conversely, acasR overexpression increased bacterial susceptibility. Furthermore, AcasR specifically bound to and repressed the promoter of fadD9 (Rv2590), which was its sole functionally validated target under the tested conditions. Crucially, fadD9 overexpression phenocopied ACA resistance, whereas its deletion heightened susceptibility. Integrated omics and iron assays revealed that ACA induced intracellular iron starvation, a condition exacerbated by fadD9 deletion. These findings reveal a novel resistance pathway in which AcasR modulates mycobacterial drug susceptibility by repressing fadD9, which in turn affects siderophore-mediated iron homeostasis.