Elevated Expression of GlpT and UhpT via FNR Activation Contributes to Increased Fosfomycin Susceptibility in Escherichia coli under Anaerobic Conditions

Abstract

Because a shortage of new antimicrobial agents is a critical issue at present, and with the spread of multidrug-resistant (MDR) pathogens, the use of fosfomycin to treat infections is being revisited as a "last-resort option." This drug offers a particular benefit in that it is more effective against bacteria growing under oxygen-limited conditions, unlike other commonly used antimicrobials, such as fluoroquinolones and aminoglycosides. In this study, we showed that Escherichia coli strains, including enterohemorrhagic E. coli (EHEC), were more susceptible to fosfomycin when grown anaerobically than when grown aerobically, and we investigated how the activity of this drug was enhanced during anaerobic growth of E. coli. Our quantitative PCR analysis and a transport assay showed that E. coli cells grown under anaerobic conditions had higher levels of expression of glpT and uhpT, encoding proteins that transport fosfomycin into cells with their native substrates, i.e., glycerol-3-phosphate and glucose-6-phosphate, and led to increased intracellular accumulation of the drug. Elevation of expression of these genes during anaerobic growth requires FNR, a global transcriptional regulator that is activated under anaerobic conditions. Purified FNR bound to DNA fragments from regions upstream of glpT and uhpT, suggesting that it is an activator of expression of glpT and uhpT during anaerobic growth. We concluded that the increased antibacterial activity of fosfomycin toward E. coli under anaerobic conditions can be attributed to elevated expression of GlpT and UhpT following activation of FNR, leading to increased uptake of the drug.

Urinary Tract Conditions Affect Fosfomycin Activity against Escherichia coli Strains Harboring Chromosomal Mutations Involved in Fosfomycin Uptake

“…Therefore, urine physiological pH values may improve the effect of FOS in these patients. Additionally, growth under anaerobic conditions has also been related to increased antibacterial activity of FOS due to elevated expression of GlpT and UhpT after activation of FNR, leading to increased FOS uptake (44). The results obtained in our study support this finding, with 2-to 32-fold MIC reductions observed under anaerobic conditions.…”

Oxygen Limitation Enhances the Antimicrobial Activity of Fosfomycin in Pseudomonas aeruginosa Following Overexpression of glpT Which Encodes Glycerol-3-Phosphate/Fosfomycin Symporter

“…This drug is rather more effective under anaerobic conditions whereas most of other drugs decrease their activities ( Inouye et al, 1989 ; Bryant et al, 1992 ; Morrissey and Smith, 1994 ; Grif et al, 2001 ; Kohanski et al, 2010 ). In our previous studies on Escherichia coli , we found that FNR (Fumarate Nitrate Reduction) and CRP (cAMP receptor protein) are positive regulators of both glpT and uhpT genes, and activities of these regulators increase under anaerobic conditions, which results in increased intracellular accumulation of fosfomycin ( Kurabayashi et al, 2015b , 2017 ).…”

Tet38 Efflux Pump Contributes to Fosfomycin Resistance in Staphylococcus aureus

“…Accumulation assay. The accumulation assay was based on the technique described by Kurabayashi et al, with some modifications (14). S. aureus cells were grown in LB medium until an optical density at 600 nm (OD 600 ) of 0.6, were harvested and resuspended in 2 ml of LB supplemented with 10-fold the MIC of fosfomycin, and then were incubated at 37°C for 1 h. The bacteria were washed thrice with phosphatebuffered saline, resuspended in 500 l of water, boiled for 3 min, and centrifuged to collect the pellets.…”

“…Accumulation of fosfomycin in S. aureus mutants and overexpressors. Using a previously described bioassay for bacterial cell accumulation of fosfomycin (14), we determined if the effects of tet38 and glpT on fosfomycin MICs could be correlated with drug accumulation. Fosfomycin accumulation was decreased 2-to 3-fold in the tet38 overexpressor strain and QT03 (glpT mutant) (Table 3), findings consistent with changes in fosfomycin MICs and supporting a role for Tet38 as an efflux transporter and GlpT as an uptake transporter of fosfomycin in S. aureus.…”

“…Fosfomycin also appears to be an efflux substrate of Tet38. Tet38 transport of fosfomycin and Tet38 substrates is affected by G3P, another substrate of GlpT, a known importer of fosfomycin (6,8,14), suggesting that G3P can act as a competitive inhibitor of Tet38 substrates, including fosfomycin. Our data indicate that fosfomycin accumulation and activity are reduced when tet38 is overexpressed and suggest that fosfomycin activity is likely to be reduced under conditions that induce the expression of tet38, such as in abscess infections (15).…”