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

Abstract

Fosfomycin is resurfacing as a “last resort drug” to treat infections caused by multidrug resistant pathogens. This drug has a remarkable benefit in that its activity increases under oxygen-limited conditions unlike other commonly used antimicrobials such as β-lactams, fluoroquinolones and aminoglycosides. Especially, utility of fosfomycin has being evaluated with particular interest to treat chronic biofilm infections caused by Pseudomonas aeruginosa because it often encounters anaerobic situations. Here, we showed that P. aeruginosa PAO1, commonly used in many laboratories, becomes more susceptible to fosfomycin when grown anaerobically, and studied on how fosfomycin increases its activity under anaerobic conditions. Results of transport assay and gene expression study indicated that PAO1 cells grown anaerobically exhibit a higher expression of glpT encoding a glycerol-3-phosphate transporter which is responsible for fosfomycin uptake, then lead to increased intracellular accumulation of the drug. Elevated expression of glpT in anaerobic cultures depended on ANR, a transcriptional regulator that is activated under anaerobic conditions. Purified ANR protein bound to the DNA fragment from glpT region upstream, suggesting it is an activator of glpT gene expression. We found that increased susceptibility to fosfomycin was also observed in a clinical isolate which has a promoted biofilm phenotype and its glpT and anr genes are highly conserved with those of PAO1. We conclude that increased antibacterial activity of fosfomycin to P. aeruginosa under anaerobic conditions is attributed to elevated expression of GlpT following activation of ANR, then leads to increased uptake of the drug.

Harnessing Metabolic Regulation to Increase Hfq-Dependent Antibiotic Susceptibility in Pseudomonas aeruginosa

“…This might not only allow to lower the effective dose of antibiotics but also to re-sensitize P. aeruginosa strains toward certain antibiotics, e.g., for fosfomycin. In this context it is worth noting that PAO1 was recently shown to be more susceptible to fosfomycin under hypoxic conditions due to overexpression of glpT in an ANR dependent manner (Hirakawa et al, 2018 ). As CrcZ was the predominant RNA bound to Hfq in anoxic biofilms (Pusic et al, 2016 ) and Hfq impacts on glpT expression (Supplementary Figure S2 ) both might as well contribute to anaerobic regulation of glpT .…”

Biochemistry, genetics and biotechnology of glycerol utilization in Pseudomonas species

“…aeruginosa PAO1 and P . fluorescens SBW25) seems to act as a dual sn ‐glycerol‐3‐ P /fosfomycin symporter (Hirakawa et al., ). Such a mechanism has not been identified in P .…”

Adsorption of Phenazines Produced by Pseudomonas aeruginosa Using AST-120 Decreases Pyocyanin-Associated Cytotoxicity

“…To construct the in-frame deletions of phzA1 and phzB1 in the PAO1 background, we performed a gene replacement strategy based on homologous recombination using the pEX18Gm vector as previously described [ 38 , 39 ]. We amplified a flanking DNA fragment including the upstream region of phzA1 and the downstream region of phzB1 by sequence overlap extension polymerase chain reaction (PCR) with primer pairs, delta1/delta2 and delta3/delta4 ( Table 1 ).…”

“…To measure the promoter activities of lasI and rhlA in PAO1, we PCR-amplified the 365- and 425-bp regions upstream of lasI and rhlA in PAO1, respectively, using the primer pairs listed in Table 1 and ligated each product into the NsiI and KpnI-digested pBBR1MCS4lacZ reporter plasmid [ 38 ]. The PAO1 strains carrying each reporter plasmid, designated pBBRlasI (PAO1)-P or pBBRrhlA (PAO1)-P, were grown in the presence and absence of AST-120 and 10% of chloroform was added to lyse the cells.…”