The Glycerol-3-Phosphate Permease GlpT Is the Only Fosfomycin Transporter in Pseudomonas aeruginosa

Fosfomycin is transported into Escherichia coli via both glycerol-3-phosphate (GlpT) and a hexose phosphate transporter (UhpT). Consequently, the inactivation of either glpT or uhpT confers increased fosfomycin resistance in this species. The inactivation of other genes, including ptsI and cyaA, also confers significant fosfomycin resistance. It has been assumed that identical mechanisms are responsible for fosfomycin transport into Pseudomonas aeruginosa cells. The study of an ordered library of insertion mutants in P. aeruginosa PA14 demonstrated that only insertions in glpT confer significant resistance. To explore the uniqueness of this resistance target in P. aeruginosa, the linkage between fosfomycin resistance and the use of glycerol-3-phosphate was tested. Fosfomycin-resistant (Fos-R) mutants were obtained in LB and minimal medium containing glycerol as the sole carbon source at a frequency of 10⁻⁶. However, no Fos-R mutants grew on plates containing fosfomycin and glycerol-3-phosphate instead of glycerol (mutant frequency, ≤5 × 10⁻¹¹). In addition, 10 out of 10 independent spontaneous Fos-R mutants, obtained on LB-fosfomycin, harbored mutations in glpT, and in all cases the sensitivity to fosfomycin was recovered upon complementation with the wild-type glpT gene. The analysis of these mutants provides additional insights into the structure-function relationship of glycerol-3-phosphate the transporter in P. aeruginosa. Studies with glucose-6-phosphate and different mutant derivatives strongly suggest that P. aeruginosa lacks a specific transport system for this sugar. Thus, glpT seems to be the only fosfomycin resistance mutational target in P. aeruginosa. The high frequency of Fos-R mutations and their apparent lack of fitness cost suggest that Fos-R variants will be obtained easily in vivo upon the fosfomycin treatment of P. aeruginosa infections.Pseudomonas aeruginosa is an opportunistic, life-threatening bacterial pathogen that especially affects critically ill patients in intensive care units or those suffering from chronic respiratory diseases such as cystic fibrosis (19, 40). Its 6.3-Mb genome supports its enormous metabolic versatility and, consequently, its adaptability to almost any challenging environment. One of the consequences of this versatility is the rapid adaptation to stressful environmental conditions, including starvation, desiccation, and antibiotic treatments (14, 40). Mutants resistant to one or several antibiotics will evolve during sufficiently prolonged treatments, this being a process facilitated by the presence of hypermutable alleles (31, 32). After years of treating cystic fibrosis patients with antibiotics, P. aeruginosa became unavoidably resistant to many or all of them (5). Multidrug-resistant strains of P. aeruginosa are an important problem for the treatment of nosocomial outbreaks and cystic fibrosis patients (27, 37). Currently, the treatment of multidrug-resistant P. aeruginosa requires the combination of various antimicrobial agents. Fosfomycin (Fos) has been reported to be effective in combination with other antipseudomonal agents (6, 29, 42, 44). The proportion of Fos-resistant (Fos-R) strains in clinical isolates of P. aeruginosa currently is not well known, and even the mechanisms that support Fos resistance in P. aeruginosa are not clear. Thus, the knowledge of the molecular bases involved in the development of spontaneous Fos resistance in P. aeruginosa is of particular interest.Fos is a unique broad-spectrum bactericidal antibiotic that is chemically unrelated to any other known antimicrobial agent used to treat urinary tract and gastrointestinal infections in humans (9, 35). It binds UDP-GlcNAc enol-pyruvyltransferase (MurA), acting as a phosphoenolpyruvate analogue and avoiding the formation of UDP-N-acetylglucosamine-3-O-enolpyruvate from UDP-N-acetylglucosamine and phosphoenolpyruvate (12, 33). Fos is taken up actively into bacterial cells via transport systems. In Escherichia coli, Fos is imported through two nutrient transport systems, the glycerol-3-phosphate (glycerol-3-P) transporter (GlpT) and glucose-6-phosphate (glucose-6-P) transporter (UhpT), to achieve its target and inhibits the initial step in cell wall synthesis (12, 17). The expression of these transport systems is induced by their substrates (glycerol-3-P and glucose-6P) and requires the presence of the cyclic AMP receptor protein (cAMP-CRP) complex (23, 30). Additionally, the high-level expression of UhpT requires the regulatory genes uhpA, uhpB, and uhpC (12, 30). Therefore, Fos-R strains isolated in E. coli contain mutations that prevent Fos transport using GlpT or UhpT (23, 30). Plasmid-encoded resistance also has been described previously (4, 41).In this paper, we describe the screening and analysis of Fos-R clones in a P. aeruginosa PA14 ordered insertional library (18). In addition, we studied the mutations responsible for the spontaneous resistance to Fos in P. aeruginosa PA14, the effect of these mutations on the in vitro growth rate, and the uniqueness of the mutational target.