Resistance of Francisella Novicida to Fosmidomycin Associated with Mutations in the Glycerol-3-Phosphate Transporte

Abstract

The methylerythritol phosphate (MEP) pathway is essential in most prokaryotes and some lower eukaryotes but absent from human cells, and is a validated target for antimicrobial drug development. The formation of MEP is catalyzed by 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR). MEP pathway genes have been identified in many category A and B biothreat agents, including Francisella tularensis, which causes the zoonosis tularemia. Fosmidomycin (Fos) inhibits purified Francisella DXR. This compound also inhibits the growth of F. tularensis NIH B38, F. novicida and F. tularensis subsp. holarctica LVS bacteria. Related compounds such as FR900098 and the lipophilic prodrug of FR900098 (compound 1) have been developed to improve the bioavailability of these DXR inhibitors. In performing disk-inhibition assays with these compounds, we observed breakthrough colonies of F. novicida in the presence of Fos, suggesting spontaneous development of Fos resistance (FosR). FosR bacteria had decreased sensitivity to both Fos and FR900098. The two most likely targets for the development of mutants would be the DXR enzyme itself or the glycerol-3-phosphate transporter (GlpT) that allows entry of Fos into the bacteria. Sensitivity of FosR F. novicida bacteria to compound 1 was not abated suggesting that spontaneous resistance is not due to mutation of DXR. We thus predicted that the glpT transporter may be mutated leading to this resistant phenotype. Supporting this, transposon insertion mutants at the glpT locus were also found to be resistant to Fos. DNA sequencing of four different spontaneous FosR colonies demonstrated a variety of deletions in the glpT coding region. The overall frequency of FosR mutations in F. novicida was determined to be 6.3 × 10−8. Thus we conclude that one mechanism of resistance of F. novicida to Fos is caused by mutations in GlpT. This is the first description of spontaneous mutations in Francisella leading to FosR.

Lipophilic Prodrugs of FR900098 Are Antimicrobial against Francisella novicida In Vivo and In Vitro and Show GlpT Independent Efficacy

“…DXR is an essential enzyme, and while mutations could theoretically occur in the active site that would alter drug activity, loss of this enzyme is unlikely. However, the same cannot be said for the GlpT transporter, as demonstrated by our use of a glpT mutant [5], [32]. GlpT is an inorganic phosphate/glycerol-3-phosphate antiporter that depends on the phosphate gradient, so any environmental conditions that affect this gradient could also affect the activity of the transporter [39].…”

Screen of FDA-approved drug library identifies maprotiline, an antibiofilm and antivirulence compound with QseC sensor-kinase dependent activity in Francisella novicida

“…The membrane potential of F. novicida was measured using the fluorescence probe [3,3 0 -Dipropylthiadicarbocyanine iodide] (DiSC3 [5]), as previously described, 39 and following manufacturer's instructions (Invitrogen). An overnight culture of Francisella was inoculated 1:50 in TSBC and grown at 37 C for 5 h. The cells were then centrifuged twice at 4000 £ g for 10 min at 4 C and washed (5 mM HEPES, 5 mM glucose).…”

“…An overnight culture of Francisella was inoculated 1:50 in TSBC and grown at 37 C for 5 h. The cells were then centrifuged twice at 4000 £ g for 10 min at 4 C and washed (5 mM HEPES, 5 mM glucose). The cells were then resuspended (in 5 mM glucose, 0.2 mM EDTA, 20 nM DiSC3 [5]), and incubated at room temperature for 15 min. 90 mL of the cell suspensions were put into wells of a black 96-well plate and 10 mL of drug (5 mM) or carrier (5% DMSO in PBS) was added.…”

“…3 Current and emerging antibiotic resistance represents a crisis on a global scale. 4 The recent detection of Francisella's antibiotic resistance, 5 and reports relating to the potential emergence of fluoroquinolone resistance [6][7][8] in Francisella species, underscores the urgent need for new antibiotics, or different antimicrobial strategies. 9 There are numerous examples of successful drug repurposing.…”

Potent, specific MEPicides for treatment of zoonotic staphylococci

“…As a result, many microorganisms, such as Mycobacterium tuberculosis and Toxoplasma gondii , are inherently resistant to FSM (due to poor cellular uptake) even though FSM potently inhibits their DXR orthologs in vitro (16,18,26). In Gram-negative organisms, FSM import is dependent on a glycerol-3-phosphate/Pi antiporter (GlpT), and FSM resistance can be achieved by reduced expression or activity of GlpT(27,28).…”