Mycobacterium Fluoroquinolone Resistance Protein D (MfpD), a GTPase-Activating Protein of GTPase MfpB, Is Involved in Fluoroquinolones Potency

Tuberculosis (TB) caused by Mycobacterium tuberculosis infection remains one of the most serious global health problems. Fluoroquinolones (FQs) are an important component of drug regimens against multidrug-resistant tuberculosis, but challenged by the emergence of FQ-resistant strains. Mycobacterium fluoroquinolone resistance protein A (MfpA) is a pentapeptide protein that confers resistance to FQs. MfpA is the fifth gene in the mfp operon among most Mycobacterium, implying other mfp genes might regulate the activity of MfpA. To elucidate the function of this operon, we constructed deletion mutants and rescued strains and found that MfpD is a GTPase-activating protein (GAP) involved in FQs activity. We showed that the recombinant strains overexpressing mfpD became more sensitive to FQs, whereas an mfpD deletion mutant was more resistant to FQs. By using site-directed mutagenesis and mycobacterial protein fragment complementation, we genetically demonstrated that mfpD participated in FQs susceptibility via directly acting on mfpB. We further biochemically demonstrated that MfpD was a GAP capable of stimulating the GTPase activity of MfpB. Our studies suggest that MfpD, a GAP of MfpB, is involved in MfpA-mediated FQs resistance. The function of MfpD adds new insights into the role of the mfp operon in Mycobacterium fluoroquinolone resistance.