The movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA. Recent structures of MsbA and related transporters have provided insights into the molecular basis of active lipid transport; however, structural information about their pharmacological modulation remains limited. Here we report the 2.9 Å resolution structure of MsbA in complex with G907, a selective small-molecule antagonist with bactericidal activity, revealing an unprecedented mechanism of ABC transporter inhibition. G907 traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. A second allosteric mechanism of antagonism occurs through structural and functional uncoupling of the nucleotide-binding domains. This study establishes a framework for the selective modulation of ABC transporters and provides rational avenues for the design of new antibiotics and other therapeutics targeting this protein family.
The outer membrane is an essential structural component of Gram-negative bacteria that is composed of lipoproteins, lipopolysaccharides, phospholipids, and integral β-barrel membrane proteins. A dedicated machinery, called the Lol system, ensures proper trafficking of lipoproteins from the inner to the outer membrane. The LolCDE ABC transporter is the inner membrane component, which is essential for bacterial viability. Here, we report a novel pyrrolopyrimidinedione compound, G0507, which was identified in a phenotypic screen for inhibitors of growth followed by selection of compounds that induced the extracytoplasmic σ stress response. Mutations in ,, and conferred resistance to G0507, suggesting LolCDE as its molecular target. Treatment of cells with G0507 resulted in accumulation of fully processed Lpp, an outer membrane lipoprotein, in the inner membrane. Using purified protein complexes, we found that G0507 binds to LolCDE and stimulates its ATPase activity. G0507 still binds to LolCDE harboring a Q258K substitution in LolC (LolC), which confers high-level resistance to G0507 but no longer stimulates ATPase activity. Our work demonstrates that G0507 has significant promise as a chemical probe to dissect lipoprotein trafficking in Gram-negative bacteria.
There is a critical need for new antibacterial strategies to counter the growing problem of antibiotic resistance. In Gram-negative bacteria, the outer membrane (OM) provides a protective barrier against antibiotics and other environmental insults. The outer leaflet of the outer membrane is primarily composed of lipopolysaccharide (LPS). Outer membrane biogenesis presents many potentially compelling drug targets as this pathway is absent in higher eukaryotes. Most proteins involved in LPS biosynthesis and transport are essential; however, few compounds have been identified that inhibit these proteins. The inner membrane ABC transporter MsbA carries out the first essential step in the trafficking of LPS to the outer membrane. We conducted a biochemical screen for inhibitors of MsbA and identified a series of quinoline compounds that kill through inhibition of its ATPase and transport activity, with no loss of activity against clinical multidrug-resistant strains. Identification of these selective inhibitors indicates that MsbA is a viable target for new antibiotics, and the compounds we identified serve as useful tools to further probe the LPS transport pathway in Gram-negative bacteria.
In experiments on slices (from 100- to 150-g Sprague-Dawley rats) kept at 33 degreesC, we studied the effects of brief hypoxia (2-3 min) on CA1 neurons. In whole cell recordings from submerged slices, with electrodes containing only KMeSO4 and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and in the presence of kynurenate and bicuculline (to minimize transmitter actions), hypoxia produced the following changes: under current clamp, 36 cells were hyperpolarized by 2.7 +/- 0.5 (SE) mV and their input resistance (Rin) fell by 23 +/- 2.7%; in 30 cells under voltage clamp, membrane current increased by 114 +/- 22.3 pA and input conductance (Gin) by 4.9 +/- 0.9 nS. These effects are much greater than those seen previously with K gluconate whole cell electrodes, but only half those seen with "sharp" electrodes. The hypoxic hyperpolarizations (or outward currents) were not reduced by intracellular ATP (1-5 mM) or bath-applied glyburide (10 microM): therefore they are unlikely to be mediated by conventional ATP-sensitive K channels. On the other hand, their depression by internally applied ethylene glycol-bis-(beta-aminoethyl ether)-N,N, N',N'-tetraacetic acid (1.1 and 11 mM) and especially 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (11-33 mM) indicated a significant involvement of Ca-dependent K (KCa) channels. The beta-adrenergic agonist isoprenaline (10 microM) reduced hypoxic hyperpolarizations and decreases in Rin (n = 4) (and in another 11 cells corresponding changes in Gin); and comparable but more variable effects were produced by internally applied 3':5'-adenosine cyclic monophosphate (cAMP, 1 mM, n = 6) and bath-applied 8-bromo-cAMP (n = 8). Thus afterhyperpolarization-type KCa channels probably take part in the hypoxic response. A major involvement of G proteins is indicated by the near total suppression of the hypoxic response by guanosine 5'-O-(3-thiotriphosphate) (0. 1-0.3 mM, n = 23) and especially guanosine 5'-O-(2-thiodiphosphate) (0.3 mM, n = 26), both applied internally. The adenosine antagonist 8-(p-sulfophenyl)theophylline (10-50 microM) significantly reduced hypoxic hyperpolarizations and outward currents in whole cell recordings (with KMeSO4 electrodes) from submerged slices but not in intracellular recordings (with KCl electrodes) from slices kept at gas/saline interface. In further intracellular recordings, antagonists of gamma-aminobutyric acid-B or serotonin receptors also had no clear effect. In conclusion, these G-protein-dependent hyperpolarizing changes produced in CA1 neurons by hypoxia are probably initiated by Ca2+ release from internal stores stimulated by enhanced glycolysis and a variable synergistic action of adenosine.
Clinical development of antibiotics with novel mechanisms of action to kill pathogenic bacteria is challenging, in part, due to the inevitable emergence of resistance. A phenomenon of potential clinical importance that is broadly overlooked in preclinical development is heteroresistance, an often-unstable phenotype in which subpopulations of bacterial cells show decreased antibiotic susceptibility relative to the dominant population. Here, we describe a new globomycin analog, G0790, with potent activity against the Escherichia coli type II signal peptidase LspA and uncover two novel resistance mechanisms to G0790 in the clinical uropathogenic E. coli strain CFT073. Building on the previous finding that complete deletion of Lpp, the major Gram-negative outer membrane lipoprotein, leads to globomycin resistance, we also find that an unexpectedly modest decrease in Lpp levels mediated by insertion-based disruption of regulatory elements is sufficient to confer G0790 resistance and increase sensitivity to serum killing. In addition, we describe a heteroresistance phenotype mediated by genomic amplifications of lspA that result in increased LspA levels sufficient to overcome inhibition by G0790 in culture. These genomic amplifications are highly unstable and are lost after as few as two subcultures in the absence of G0790, which places amplification-containing resistant strains at high risk of being misclassified as susceptible by routine antimicrobial susceptibility testing. In summary, our study uncovers two vastly different mechanisms of resistance to LspA inhibitors in E. coli and emphasizes the importance of considering the potential impact of unstable and heterogenous phenotypes when developing antibiotics for clinical use. IMPORTANCE Despite increasing evidence suggesting that antibiotic heteroresistance can lead to treatment failure, the significance of this phenomena in the clinic is not well understood, because many clinical antibiotic susceptibility testing approaches lack the resolution needed to reliably classify heteroresistant strains. Here we present G0790, a new globomycin analog and potent inhibitor of the Escherichia coli type II signal peptidase LspA. We demonstrate that in addition to previously known mechanisms of resistance to LspA inhibitors, unstable genomic amplifications containing lspA can lead to modest yet biologically significant increases in LspA protein levels that confer a heteroresistance phenotype.
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