cIn Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane. Here we describe the discovery and characterization of novel pyridineimidazole compounds that inhibit this process. Escherichia coli mutants resistant to the pyridineimidazoles show no cross-resistance to other classes of antibiotics and map to either the LolC or LolE protein of the LolCDE transporter complex. The pyridineimidazoles were shown to inhibit the LolA-dependent release of the lipoprotein Lpp from E. coli spheroplasts. These results combined with bacterial cytological profiling are consistent with LolCDE-mediated disruption of lipoprotein targeting to the outer membrane as the mode of action of these pyridineimidazoles. The pyridineimidazoles are the first reported inhibitors of the LolCDE complex, a target which has never been exploited for therapeutic intervention. These compounds open the door to further interrogation of the outer membrane lipoprotein transport pathway as a target for antimicrobial therapy.T he most distinguishing feature of Gram-negative bacteria is their cell envelope, which is comprised of both an inner and an outer membrane bilayer. The outer membrane has a unique composition of lipoproteins, ␤-barrel proteins, lipopolysaccharides, and phospholipids. Lipoproteins, membrane proteins that are covalently modified with lipids, are involved in a variety of integral cellular functions, such as the synthesis and maintenance of the cell surface and the transport of substrates (reviewed in reference 1). Lipoproteins are synthesized as precursors in the cytoplasm. Upon transit across the inner membrane by either the Sec or Tat machinery, the export signal peptide is cleaved, and attached to its amino terminus is a lipid moiety. This lipid serves as a membrane anchor for the lipoprotein. Some lipoproteins remain in the outer leaflet of the inner membrane, while others must cross the hydrophilic periplasmic space to the outer membrane. This sorting of lipoproteins to the outer membrane is achieved by the Lol system, which consists of five proteins (Fig. 1) (reviewed in reference 1). In this process, lipoproteins destined for the outer membrane are released from the inner membrane by the LolCDE complex, an inner membrane ABC transporter. LolCDE transfers the lipoproteins to LolA, a diffusible periplasmic chaperone (2, 3). LolA then transfers the lipoprotein to LolB, the outer membrane lipoprotein receptor, which incorporates these lipoproteins into the inner leaflet of the outer membrane (1, 4). This is in contrast to Grampositive bacteria, which have a single membrane bilayer; therefore, localization of lipoproteins to the cell surface requires only export through the cytoplasmic membrane and acylation (5).Infections c...

Disconnections between in vitro responses and those observed in whole cells confound many attempts to design drugs in areas of serious medical need. A method based on 1D 1H NMR spectroscopy is reported that affords the ability to monitor the hydrolytic decomposition of the carbapenem antibiotic meropenem inside Escherichia coli cells expressing New Delhi metallo-β-lactamase subclass 1 (NDM-1), an emerging antibiotic-resistance threat. Cell-based NMR studies demonstrated that two known NDM-1 inhibitors, L-captopril and ethylenediaminetetraacetic acid (EDTA), inhibit the hydrolysis of meropenem in vivo. NDM-1 activity in cells was also shown to be inhibited by spermine, a porin inhibitor, although in an in vitro assay, the influence of spermine on the activity of isolated NDM-1 protein is minimal. This new approach may have generic utility for monitoring reactions involving diffusible metabolites in other complex biological matrices and whole-cell settings, including mammalian cells.

A family of benzimidazole derivatives (BI) was shown to possess potent and selective activity against Helicobacter pylori, although the precise cellular target of the BIs is unknown. Spontaneous H. pylori mutants were isolated as resistant to a representative BI (compound A). Genomic DNA was isolated from a BI-resistant mutant, transformed into a BI-sensitive strain, and found to be sufficient to confer BI resistance. The resistance determinant was localized to a 17-kb clone after screening a lambda-based genomic library constructed from the BI-resistant strain. Upon sequencing and mapping onto the H. pylori strain J99 genome, the 17-kb clone was shown to contain the entire nuo operon (NADH:ubiquinone oxidoreductase). Further subcloning and DNA sequencing revealed that a single point mutation in nuoD was responsible for BI resistance. The mutation resulted in a G398S amino acid change at the C terminus of NuoD. Thirty-three additional spontaneous BI-resistant mutants were characterized. Sequencing of nuoD from 32 isolated mutants revealed three classes of missense mutation resulting in amino acid changes in NuoD: G398S, F404S, and V407M. One BI-resistant isolate did not have a mutation in nuoD. Instead, a T27A amino acid change was identified in NuoB. MIC testing of the wild-type H. pylori strain and four classes of nuo mutants revealed that all NuoD mutant classes were hypersensitive to rotenone, a known inhibitor of complex I (NADH:ubiquinone oxidoreductase) suggested to bind to NuoD. Further, a nuoD knockout verified that it is essential in H. pylori and may be the target of the BI compounds.

Disconnections between in vitro responses and those observed in whole cells confound many attempts to design drugs in areas of serious medical need. A method based on 1D 1H NMR spectroscopy is reported that affords the ability to monitor the hydrolytic decomposition of the carbapenem antibiotic meropenem inside Escherichia coli cells expressing New Delhi metallo‐β‐lactamase subclass 1 (NDM‐1), an emerging antibiotic‐resistance threat. Cell‐based NMR studies demonstrated that two known NDM‐1 inhibitors, L‐captopril and ethylenediaminetetraacetic acid (EDTA), inhibit the hydrolysis of meropenem in vivo. NDM‐1 activity in cells was also shown to be inhibited by spermine, a porin inhibitor, although in an in vitro assay, the influence of spermine on the activity of isolated NDM‐1 protein is minimal. This new approach may have generic utility for monitoring reactions involving diffusible metabolites in other complex biological matrices and whole‐cell settings, including mammalian cells.