Background: Fosfomycin is widely used to treat urinary tract and pediatric gastrointestinal infections ofbacteria. It is supposed that this antibiotic enters cells via two transport systems, including the bacterialGlycerol-3-phosphate Transporter (GlpT). Impaired function of GlpT is one mechanism for fosfomycinresistance.Methods: The interaction of fosfomycin with the recombinant and purified GlpT of Escherichia colireconstituted in liposomes has been studied. IC50 and the half-saturation constant of the transporter forexternal fosfomycin (Ki) were determined by transport assay of [14C]glycerol-3-phosphate catalyzed byrecombinant GlpT. Efficacy of fosfomycin on growth rates of GlpT defective bacteria strains transformed withrecombinant GlpT was measured.Results: Fosfomycin, externally added to the proteoliposomes, poorly inhibited the glycerol-3-phosphate/glycerol-3-phosphate antiport catalyzed by the reconstituted transporter with an IC50 of 6.4 mM. A kineticanalysis revealed that the inhibition was completely competitive, that is, fosfomycin interacted with thesubstrate-binding site and the Ki measured was 1.65 mM. Transport assays performed with proteoliposomescontaining internal fosfomycin indicate that it was not very well transported by GlpT. Complementationstudy, performed with GlpT defective bacteria strains, indicated that the fosfomycin resistance, besidedeficiency in antibiotic transporter, could be due to other gene defects.Conclusions: The poor transport observed in a reconstituted system together with the high value of Ki and theresults of complementation study well explain the usual high dosage of this drug for the treatment of theurinary tract infections.General significance: This is the first report regarding functional analysis of interaction between fosfomycinand GlpT.
Interaction of fosfomycin with the Glycerol 3-phosphate Transporter of Escherichia coli
CAPPELLO, Anna Rita;DOLCE, Vincenza
2011-01-01
Abstract
Background: Fosfomycin is widely used to treat urinary tract and pediatric gastrointestinal infections ofbacteria. It is supposed that this antibiotic enters cells via two transport systems, including the bacterialGlycerol-3-phosphate Transporter (GlpT). Impaired function of GlpT is one mechanism for fosfomycinresistance.Methods: The interaction of fosfomycin with the recombinant and purified GlpT of Escherichia colireconstituted in liposomes has been studied. IC50 and the half-saturation constant of the transporter forexternal fosfomycin (Ki) were determined by transport assay of [14C]glycerol-3-phosphate catalyzed byrecombinant GlpT. Efficacy of fosfomycin on growth rates of GlpT defective bacteria strains transformed withrecombinant GlpT was measured.Results: Fosfomycin, externally added to the proteoliposomes, poorly inhibited the glycerol-3-phosphate/glycerol-3-phosphate antiport catalyzed by the reconstituted transporter with an IC50 of 6.4 mM. A kineticanalysis revealed that the inhibition was completely competitive, that is, fosfomycin interacted with thesubstrate-binding site and the Ki measured was 1.65 mM. Transport assays performed with proteoliposomescontaining internal fosfomycin indicate that it was not very well transported by GlpT. Complementationstudy, performed with GlpT defective bacteria strains, indicated that the fosfomycin resistance, besidedeficiency in antibiotic transporter, could be due to other gene defects.Conclusions: The poor transport observed in a reconstituted system together with the high value of Ki and theresults of complementation study well explain the usual high dosage of this drug for the treatment of theurinary tract infections.General significance: This is the first report regarding functional analysis of interaction between fosfomycinand GlpT.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
