Increased drug permeability of a stiffened mycobacterial outer membrane in cells lacking MFS transporter Rv1410 and lipoprotein LprG

Abstract

Summary The major facilitator superfamily transporter Rv1410 and the lipoprotein LprG (Rv1411) are encoded by a conserved two‐gene operon and contribute to virulence in Mycobacterium tuberculosis. Rv1410 was originally postulated to function as a drug efflux pump, but recent studies suggested that Rv1410 and LprG work in concert to insert triacylglycerides and lipoarabinomannans into the outer membrane. Here, we conducted microscopic analyses of Mycobacterium smegmatis lacking the operon and observed a cell separation defect, while surface rigidity measured by atomic force microscopy was found to be increased. Whereas Rv1410 expressed in Lactococcus lactis did not confer drug resistance, deletion of the operon in Mycobacterium abscessus and M. smegmatis resulted in increased susceptibility toward vancomycin, novobiocin and rifampicin. A homology model of Rv1410 revealed a periplasmic loop as well as a highly conserved aspartate, which were found to be essential for the operon’s function. Interestingly, influx of the fluorescent dyes BCECF‐AM and calcein‐AM in de‐energized M. smegmatis cells was faster in the deletion mutant. Our results unambiguously show that elevated drug susceptibility in the deletion mutant is caused by increased drug influx through a defective mycobacterial cell envelope and not by drug efflux mediated by Rv1410.

The ABC exporter IrtAB imports and reduces mycobacterial siderophores

“…M. smegmatis DKO and TKO cells as well as M. smegmatis TKO cells complemented with different IrtAB constructs using the pFLAG vector 24 , 50 were grown in 7H9 for 3 nights at 37°C while shaking (140 rpm). 2 ml of these pre-cultures were used to inoculate 100 ml of MM supplemented with 6.88 μM ZnSO 4 , 1.82 μM MnSO 4 , 1.64 mM MgSO 4 and 0.5% (v/v) Tween 80 (treated with aluminum-oxide) in 1-liter plastic roller bottles (Corning).…”

Repositioning rifamycins for Mycobacterium abscessus lung disease

“…M. abscessus' lipid transport machinery includes homologs of M. tuberculosis major facilitator superfamily transporter Rv1410 and lipoprotein LprG, which work together to transport triacylglycerides and lipoarabinomannans to the mycobacterial outer membrane. Deletion of the Rv1410-LprG operon results in cell wall defects, an increased influx of drugs and greater susceptibility to rifamycins [55]. M. abscessus is also thought to regulate cell wall structure and homeostasis via lipoprotein glycosylation [56].…”

A high-frequency single nucleotide polymorphism in the MtrB sensor kinase in clinical strains of Mycobacterium tuberculosis alters its biochemical and physiological properties

“…Given that the rigidity of the membrane is known to be directly proportional to its permeability [ 38 , 39 ], we first analyzed if membrane permeability is altered over 60 minutes using the EtBr uptake assay [ 40 ]. We measured the accumulation of the EtBr in all three strains and found that the mutant shows a higher mean fluorescence intensity at 60 minutes than the vector control, indicative of a higher accumulation of EtBr in the cell ( Fig 5F ).…”