Novel amiloride derivatives that inhibit bacterial motility across multiple strains and stator types

Abstract

The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. The most common ion gradients that drive BFM rotation are protons (H+) and sodium ions (Na+). The sodium-powered stators, like those in the PomAPomB stator complex of Vibrio spp, can be inhibited by sodium channel inhibitors, in particular, by phenamil, a potent and widely used inhibitor. However, relatively few new sodium-motility inhibitors have been described since the discovery of phenamil. In this study, we discovered two motility inhibitors HM2-16F and BB2-50F from a small library of previously reported amiloride derivatives. Using a tethered cell assay, we showed that both HM2-16F and BB2-50F had inhibition comparable to that of phenamils on Na+ driven motors at matching concentrations, with an additional ability to inhibit rotation in H+ driven motors. The two compounds did not exhibit adverse effects on bacterial growth at the motility-inhibiting concentration of 10 uM, however toxicity was seen for BB2-50F at 100 uM. We performed higher resolution measurements to examine rotation inhibition at moderate (1 um polystyrene bead) and low loads (60 nm gold bead) and in both the presence and absence of stators. These measurements suggested that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Overall, HM2-16F and BB2-50F showed reversible inhibition of motility across a range of loads, in both Na+ and H+ stator types, and in pathogenic and non-pathogenic strains.

The rapid evolution of flagellar ion selectivity in experimental populations of E. coli

“…The potB G20V fixed variant was clearly distinguishable from Pots in this assay and maintained rotation both in the absence of sodium and in the presence of phenamil, an amiloride derivative and sodium channel blocker (Fig. 3A) ( 23 ). We confirmed the potB G20V H + -powered phenotype by introducing the same point mutation (GGG to GTG) on our plasmid vector (pPots) and testing motility in tethered cell assay when the protein was expressed in the statorless ∆ motAB RP6894 (Fig.…”

The rapid evolution of flagellar ion-selectivity in experimental populations ofE. coli

“…The potB G20V fixed variant was clearly distinguishable from Pots in this assay and maintained rotation in both the absence of sodium and in the presence of phenamil, an amiloride derivative and sodium-channel blocker (Fig. 3A) ( 22 ). We confirmed the potB G20V H + -powered phenotype by introducing the same point mutation (GGG to GTG) on our plasmid vector (pPots) and testing motility in tethered cell assay when the protein was expressed in the statorless Δ motAB RP6894 (Fig.…”

“…The tethered cell assay with anti-FliC-antibody ( 69, 70 ) was performed as previously described ( 22 ). Briefly, 1 mL of cells (OD 600 =0.5) grown in K + TB buffer (Tryptone, 85 mM KCl) was sheared by passing the cells suspension through a 26-gauge syringe needle 30 times.…”

“…Free-swimming cells were grown overnight in K + TB at 30°C to OD 600 ~0.5 then washed 3 times in 1 mL K + MB before resuspension in 500 μL of K + MB and imaging in a tunnel slide. A custom LabView software ( 17, 22, 50 ) was employed as previously reported to estimate specific rotational parameters of the tethered cells such as rotation frequency (speed), clockwise and counterclockwise bias, switching frequency and speed of swimming cells. FliC-sticky E. coli RP437 cells (Δ motAB Δ cheY Δ pilA fliC st ) ( 71 ) were used to collect data presented in Supplementary Fig.…”

Automated patch clamp screening of amiloride and 5-N,N-hexamethyleneamiloride (HMA) analogs identifies 6-iodoamiloride as a potent acid-sensing ion channel inhibitor

“…20 Amiloride 11 is an oral K + -sparing diuretic used in the management of hypertension and congestive heart failure that exerts its effects through inhibition of renal epithelial sodium channels (ENaC). 21,22 Amiloride is a promiscuous drug known to bind to a diverse range of molecular targets, including GPCRs [23][24][25] , transmembrane ion channels [26][27][28][29][30][31] and trypsin-like serine proteases 32,33 . Despite its lack of selectivity, amiloride has a good safety record with dose-related ENaC-mediated hyperkalemia the only notable risk.…”