Abstract
Fosmidomycin is a time-dependent nanomolar inhibitor of methylerythritol phosphate (MEP) synthase, which is the enzyme that catalyzes the first committed step in the MEP pathway to isoprenoids. Importantly, fosmidomycin is one of only a few MEP pathway-specific inhibitors that exhibits antimicrobial activity. Most inhibitors identified to date only exhibit activity against isolated pathway enzymes. The MEP pathway is the sole route to isoprenoids in many bacteria, yet has no human homologs. The development of inhibitors of this pathway holds promise as novel antimicrobial agents. Similarly, analyses of the bacterial response toward MEP pathway inhibitors provides valuable information toward the understanding of how emergent resistance may ultimately develop to this class of antibiotics. We have examined the transcriptional response of Salmonella enterica serovar typhimurium LT2 to sub-inhibitory concentrations of fosmidomycin via cDNA microarray and RT-PCR. Within the regulated genes identified by microarray were a number of genes encoding enzymes associated with the mediation of reactive oxygen species (ROS). Regulation of a panel of genes implicated in the response of cells to oxidative stress (including genes for catalases, superoxide dismutases, and alkylhydrogen peroxide reductases) was investigated and mild upregulation in some members was observed as a function of fosmidomycin exposure over time. The extent of regulation of these genes was similar to that observed for comparable exposures to kanamycin, but differed significantly from tetracycline. Furthermore, S. typhimurium exposed to sub-inhibitory concentrations of fosmidomycin displayed an increased sensitivity to exogenous H2O2 relative to either untreated controls or kanamycin-treated cells. Our results suggest that endogenous oxidative stress is one consequence of exposures to fosmidomycin, likely through the temporal depletion of intracellular isoprenoids themselves, rather than other mechanisms that have been proposed to facilitate ROS accumulation in bacteria (e.g. cell death processes or the ability of the antibiotic to redox cycle).
Highlights
Fosmidomycin is an inhibitor of methylerythritol phosphate (MEP) synthase, which is the enzyme responsible for the first committed step in the biosynthesis of the isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) via the MEP pathway (Figure 1) [1,2,3,4].The MEP pathway is the sole route to isoprenoids in most bacteria, including mycobacteria, Gram-negative and Grampositive strains, in addition to some eukaryotic parasites
We do not observe tryptophan catabolism under these conditions in S. typhimurium, we have observed tryptophanases to be upregulated in similar tests of E. coli challenged with fosmidomycin
It has been clearly demonstrated that sub-inhibitory exposures to antibiotics can elicit global changes in transcription,[11,12,13,31] and these changes can play an influential role in the adaptation of the cell to the antibiotic even without mutational events or the acquisition of a de novo resistance gene
Summary
Fosmidomycin is an inhibitor of methylerythritol phosphate (MEP) synthase, which is the enzyme responsible for the first committed step in the biosynthesis of the isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) via the MEP pathway (Figure 1) [1,2,3,4]. The MEP pathway is the sole route to isoprenoids in most bacteria, including mycobacteria, Gram-negative and Grampositive strains, in addition to some eukaryotic parasites. Given that the enzymes responsible for the biosynthesis of isoprenoids are required for bacterial proliferation, and there are no human enzyme orthologs, the MEP pathway has emerged as an attractive target for the development of new broad spectrum antimicrobial agents.[5,6,7] While fosmidomycin and its interaction with MEP synthase has been thoroughly investigated,[1,2,3] much less is understood about the coordinated metabolic pathways bacterial cells evoke upon exposure to the antibiotic. This study serves as a prime example that, despite several decades of research into the compound, much information remains to be discovered
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