Dxr is essential in Mycobacterium tuberculosis and fosmidomycin resistance is due to a lack of uptake.
Fosmidomycin is a phosphonic antibiotic which inhibits 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr), the first committed step of the non-mevalonate pathway of isoprenoid biosynthesis. In Mycobacterium tuberculosis Dxr is encoded by Rv2870c, and although the antibiotic has been shown to inhibit the recombinant enzyme 1, mycobacteria are intrinsically resistant to fosmidomycin at the whole cell level. Fosmidomycin is a hydrophilic molecule and in many bacteria its uptake is an active process involving a cAMP dependent glycerol-3-phosphate transporter (GlpT). The fact that there is no glpT homologue in the M. tuberculosis genome and the highly impervious nature of the hydrophobic mycobacterial cell wall suggests that resistance may be due to a lack of cellular penetration.


RESULTS
We demonstrated that dxr (Rv2780c) is an essential gene in M. tuberculosis, since we could not delete the chromosomal copy unless a second functional copy was provided on an integrating vector. This confirmed that the intracellular target of fosmidomycin was essential as well as sensitive. We looked at the uptake of fosmidomycin in two mycobacterial species, the slow-growing pathogenic M. tuberculosis and the fast-growing, saprophytic Mycobacterium smegmatis; both species were resistant to fosmidomycin to a high level. Fosmidomycin was not accumulated intra-cellularly in M. tuberculosis or M. smegmatis but remained in the extra-cellular medium. In contrast, fosmidomycin uptake was confirmed in the sensitive organism, Escherichia coli. We established that the lack of intra-cellular accumulation was not due to efflux, since efflux pump inhibitors had no effect on fosmidomycin resistance. Finally, we demonstrated that fosmidomycin was not modified by mycobacterial cells or by extracts but remained in a fully functional state.


CONCLUSIONS
Taken together, these data demonstrate that fosmidomycin resistance in M. tuberculosis and M. smegmatis results from a lack of penetration of the antibiotic to the site of the sensitive target.
Brown
Amanda C
AC
Institute for Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK. a.c.brown@qmul.ac.uk <a.c.brown@qmul.ac.uk>
Parish
Tanya
T
D019747:Aldose-Ketose Isomerases / Q000235:genetics*; D000904:Antibiotics, Antitubercular / Q000493:pharmacokinetics / Q000494:pharmacology; D003434:Crossing Over, Genetic; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D005578:Fosfomycin / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D017353:Gene Deletion; D005798:Genes, Bacterial; D020043:Genes, Essential; D008658:Inactivation, Metabolic; D026901:Membrane Transport Proteins; D009097:Multienzyme Complexes / Q000235:genetics*; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D009876:Operon; D010088:Oxidoreductases / Q000235:genetics*
D000904:Antibiotics, Antitubercular; D026901:Membrane Transport Proteins; D009097:Multienzyme Complexes; D005578:Fosfomycin; C024640:fosmidomycin; D010088:Oxidoreductases; C114231:1-deoxy-D-xylulose 5-phosphate reductoisomerase; D019747:Aldose-Ketose Isomerases