rifampin and 2-nitrophenylgalactoside

The envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measure Escherichia coli envelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds and E. coli gene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinct E. coli strains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R > 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

Topics: Anti-Bacterial Agents; Bacterial Proteins; beta-Galactosidase; beta-Lactamases; Biological Transport; Cell Wall; Chromogenic Compounds; Erythromycin; Escherichia coli; Fusidic Acid; Gene Expression; High-Throughput Screening Assays; Hydrolysis; Microbial Sensitivity Tests; Mutation; Nitrophenylgalactosides; Novobiocin; Peptide Library; Peptides; Peptidomimetics; Permeability; Rifampin

The E1 gene of hepatitis C virus (HCV) has been cloned and expressed in BL21(DE3)pLys Escherichia coli strain by pET3a vector to analyze changes in membrane permeability produced by this protein. We showed that the expression of E1 (aa 192-383), as well as of two C-terminal fragments (aa 331-383 and aa 341-383) corresponding to the transmembrane (TM) region of this protein, induced a rapid lysis of cells. On the contrary, the expression of a mutant of E1 (aa 192-340), lacking the last 40 amino acids, did not cause cell lysis. The analysis of permeability changes revealed that modification of membrane permeability to several compounds were observed only in clones expressing E1 and C-terminal fragments, while the synthesis of the C-terminal-deleted mutant had little or no effect on permeability. These findings demonstrate that the TM domain of E1 protein has membrane-active properties that may be involved in some aspects of virus-cell interaction.

Topics: Bacteriolysis; Cell Membrane; Cell Membrane Permeability; Cloning, Molecular; Coloring Agents; Escherichia coli; Nitrophenylgalactosides; Nucleic Acid Synthesis Inhibitors; Peptide Fragments; Propidium; Rifampin; Viral Envelope Proteins