A PhoPQ-Regulated ABC Transporter System Exports Tetracycline in Pseudomonas aeruginosa

Abstract

bPseudomonas aeruginosa is an important human pathogen whose infections are difficult to treat due to its high intrinsic resistance to many antibiotics. Here, we show that the disruption of PA4456, encoding the ATP binding component of a putative ATP-binding cassette (ABC) transporter, increased the bacterium's susceptible to tetracycline and other antibiotics or toxic chemicals. Fluorescence spectroscopy and antibiotic accumulation tests showed that the interruption of the ABC transporter caused increased intracellular accumulation of tetracycline, demonstrating a role of the ABC transporter in tetracycline expulsion. Site-directed mutagenesis proved that the conserved residues of E170 in the Walker B motif and H203 in the H-loop, which are important for ATP hydrolysis, were essential for the function of PA4456. Through a genome-wide search, the PhoPQ twocomponent system was identified as a regulator of the computationally predicted PA4456-4452 operon that encodes the ABC transporter system. A >5-fold increase of the expression of this operon was observed in the phoQ mutant. The results obtained also show that the expression of the phzA1B1C1D1E1 operon and the production of pyocyanin were significantly higher in the ABC transporter mutant, signifying a connection between the ABC transporter and pyocyanin production. These results indicated that the PhoPQ-regulated ABC transporter is associated with intrinsic resistance to antibiotics and other adverse compounds in P. aeruginosa, probably by extruding them out of the cell.

PA3225 Is a Transcriptional Repressor of Antibiotic Resistance Mechanisms in Pseudomonas aeruginosa

“…aeruginosa resistance to tetracycline, ciprofloxacin, chloramphenicol, and trimethoprim (39). Unlike PA3228, which encodes both the ATP-binding and permease domains, PA4456 contributes the ATP-binding domain, while PA4455 possesses the permease domain (13, 39). Characterization of the putative ABC multidrug efflux pump encoded by PA3228 is under way in our laboratory.…”

Evolution toward maximum transport capacity of the Ttg2 ABC system inPseudomonas aeruginosa

“…The orthologous Mla system in P. aeruginosa is encoded by the PA4452-PA4456 operon (locus tags corresponding to PAO1) and the isolated gene PA2800 (MlaA ortholog, also known as VacJ). Proteins encoded by this gene cluster are highly similar to those encoded by operon ttg2 (toluene tolerance genes) in Pseudomonas putida 32, 33 . Although it is unlikely that organic solvents themselves are substrates of this transporter, this system was initially linked to toluene tolerance in that bacterium 34 .…”

“…Although it is unlikely that organic solvents themselves are substrates of this transporter, this system was initially linked to toluene tolerance in that bacterium 34 . Accordingly, components of the P. aeruginosa ABC transporter encoded by the PA4452-PA4456 have been named Ttg2A (MlaF), Ttg2B (MlaE), Ttg2C (MlaD), Ttg2D (MlaC) and Ttg2E (MlaB) 32 . Recent studies of mutant strains with disrupted ttg2 or vacJ genes support the contribution of this ABC transport system to the intrinsic resistance of P. aeruginosa to antimicrobials 24, 28, 32, 35 .…”

“…Accordingly, components of the P. aeruginosa ABC transporter encoded by the PA4452-PA4456 have been named Ttg2A (MlaF), Ttg2B (MlaE), Ttg2C (MlaD), Ttg2D (MlaC) and Ttg2E (MlaB) 32 . Recent studies of mutant strains with disrupted ttg2 or vacJ genes support the contribution of this ABC transport system to the intrinsic resistance of P. aeruginosa to antimicrobials 24, 28, 32, 35 . Yet, one of these studies has challenged the role of this system in P. aeruginosa as an ABC importer mediating phospholipid intermembrane trafficking 32 .…”

“…In addition, based on mutational studies of the ttg2 operon, we have validated the contribution of the Ttg2 system to the intrinsic basal resistance of P. aeruginosa to several antibiotic classes and other damaging compounds. Although the role of other components of this ABC transport system in multi-drug resistance has been already established for P. aeruginosa 24, 32 , this is the first study focusing on the soluble periplasmic SBP component, Ttg2D Pae . Among the components of the Ttg2 system, this SPB could be the most promising candidate for an antimicrobial intervention based on the specific blocking of this trafficking pathway.…”