Dxr is essential in Mycobacterium tuberculosis and fosmidomycin resistance is due to a lack of uptake

Abstract

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.ResultsWe 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.ConclusionTaken 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.

Preparation of gem-difluorinated retrohydroxamic-fosmidomycin

“…The mevalonate-independent pathway of isoprenoid biosynthesis is widely found in many microorganisms [1][2][3][4] as well as in higher plants, 5,6 but it is missing in human, which uses mevalonate pathway for isoprenoid biosynthesis. The unique property of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), the second enzyme of the DOXP pathway, can therefore be considered as a remarkable and safe target for the discovery of new herbicides.…”

DXR Inhibition by Potent Mono- and Disubstituted Fosmidomycin Analogues

“…For example, while both 1 and 2 inhibit the DXRs of the protozoan Plasmodium falciparum (PfDXR) 6 and Mycobacterium tuberculosis (MtDXR), 7-10 the Plasmodium species are sensitive to these antibiotics in vitro and in vivo, 6 but the mycobacteria are not. 11 The use of fosmidomycin as a single-drug treatment for P. falciparum malaria has been hampered by low bioavailability, rapid clearance from the parasite and recrudescent infection, 12 although the compound has been used more successfully in combination with clindamycin. 13 Failure to obtain biological activity against mycobacteria appears to result from poor uptake.…”

Design, Synthesis, and X-ray Crystallographic Studies of α-Aryl Substituted Fosmidomycin Analogues as Inhibitors of Mycobacterium tuberculosis 1-Deoxy-d-xylulose 5-Phosphate Reductoisomerase

“…The absence of activity on M. tuberculosis is not due to the presence of exporters or to modification of fosmidomycin inside the cell. 10 Since the lack of uptake of this compound into the mycobacterial cell most likely results from its polar character, it would be of interest to explore analogues of fosmidomycin with modified hydrophobic/hydrophilic properties as potential antimycobacterial drugs, similar to the approach followed in the pursuit of more bioavailable antimalarial agents. These include modifications of the phosphonate group that yield phosphonate prodrugs expected to enhance oral availability.…”

“…12,13 The compound has been shown to efficiently inhibit DXRs from E. coli 14 (EcDXR) and the malaria parasite Plasmodium falciparum 11 (PfDXR), and the activities of various fosmidomycin analogues on these two enzymes seem to be well correlated. 15 Furthermore, fosmidomycin has antibacterial activity on E. coli 10,14 as well as inhibiting the growth of P. falciparum in cell culture. 11,16 The acetyl derivative of fosmidomycin, 3-(N-hydroxyacetamido)-1-propylphosphonic acid 2 (FR900098, Figure 1), has also been evaluated in many studies, and shown to be twice as active as fosmidomycin against P. falciparum in vitro and in the P. vinckei mouse model.…”