14 research outputs found
Altered Antibiotic Transport in OmpC Mutants Isolated from a Series of Clinical Strains of Multi-Drug Resistant E. coli
Antibiotic-resistant bacteria, particularly Gram negative species, present significant health care challenges. The permeation of antibiotics through the outer membrane is largely effected by the porin superfamily, changes in which contribute to antibiotic resistance. A series of antibiotic resistant E. coli isolates were obtained from a patient during serial treatment with various antibiotics. The sequence of OmpC changed at three positions during treatment giving rise to a total of four OmpC variants (denoted OmpC20, OmpC26, OmpC28 and OmpC33, in which OmpC20 was derived from the first clinical isolate). We demonstrate that expression of the OmpC K12 porin in the clinical isolates lowers the MIC, consistent with modified porin function contributing to drug resistance. By a range of assays we have established that the three mutations that occur between OmpC20 and OmpC33 modify transport of both small molecules and antibiotics across the outer membrane. This results in the modulation of resistance to antibiotics, particularly cefotaxime. Small ion unitary conductance measurements of the isolated porins do not show significant differences between isolates. Thus, resistance does not appear to arise from major changes in pore size. Crystal structures of all four OmpC clinical mutants and molecular dynamics simulations also show that the pore size is essentially unchanged. Molecular dynamics simulations suggest that perturbation of the transverse electrostatic field at the constriction zone reduces cefotaxime passage through the pore, consistent with laboratory and clinical data. This subtle modification of the transverse electric field is a very different source of resistance than occlusion of the pore or wholesale destruction of the transverse field and points to a new mechanism by which porins may modulate antibiotic passage through the outer membrane
Molecular Basis of Enrofloxacin Affinity to a Membrane Channel of E. Coli - When Binding Does Not Imply Translocation
Molecular Basis of Enrofloxacin Translocation through OmpF, an Outer Membrane Channel of Escherichia coli - When Binding Does Not Imply Translocation
The molecular pathway of enrofloxacin, a fluoroquinolone antibiotic, through the outer membrane channel
OmpF of Escherichia coli is investigated. High-resolution ion current fluctuation analysis reveals a strong
affinity for enrofloxacin to OmpF, the highest value ever recorded for an antibiotic-channel interaction. A
single point mutation in the constriction zone of OmpF, replacing aspartic acid at the 113 position with
asparagine (D113N), lowers the affinity to a level comparable to other antibiotics. All-atom molecular dynamics
simulations allow rationalizing the translocation pathways: wild-type OmpF has two symmetric binding sites
for enrofloxacin located at each channel entry separated by a large energy barrier in the center, which inhibits
antibiotic translocation. In this particular case, our simulations suggest that the ion current blockages are
caused by molecules occupying either one of these peripheral binding sites. Removal of the negative charge
on position 113 removes the central barrier and shifts the two peripheral binding sites to a unique central site,
which facilitates translocation. Fluorescence steady-state measurements agree with the different location of
binding sites for wild-type OmpF and the mutant. Our results demonstrate how a single-point mutation of the
porin, and the resulting intrachannel shift of the affinity site, may substantially modify translocation
The Biophysics Of Antibiotics Translocation Through OmpF Revealed By Computer Simulations
Altered antibiotic transport in OmpC mutants isolated from a series of clinical strains of multi-drug resistant E. coli
Antibiotic-resistant bacteria, particularly Gram negative species, present significant health care challenges. The permeation of antibiotics through the outer membrane is largely effected by the porin superfamily, changes in which contribute to antibiotic resistance. A series of antibiotic resistant E. coli isolates were obtained from a patient during serial treatment with various antibiotics. The sequence of OmpC changed at three positions during treatment giving rise to a total of four OmpC variants (denoted OmpC20, OmpC26, OmpC28 and OmpC33, in which OmpC20 was derived from the first clinical isolate). We demonstrate that expression of the OmpC K12 porin in the clinical isolates lowers the MIC, consistent with modified porin function contributing to drug resistance. By a range of assays we have established that the three mutations that occur between OmpC20 and OmpC33 modify transport of both small molecules and antibiotics across the outer membrane. This results in the modulation of resistance to antibiotics, particularly cefotaxime. Small ion unitary conductance measurements of the isolated porins do not show significant differences between isolates. Thus, resistance does not appear to arise from major changes in pore size. Crystal structures of all four OmpC clinical mutants and molecular dynamics simulations also show that the pore size is essentially unchanged. Molecular dynamics simulations suggest that perturbation of the transverse electrostatic field at the constriction zone reduces cefotaxime passage through the pore, consistent with laboratory and clinical data. This subtle modification of the transverse electric field is a very different source of resistance than occlusion of the pore or wholesale destruction of the transverse field and points to a new mechanism by which porins may modulate antibiotic passage through the outer membrane.</p