Role of the Multidrug Resistance Efflux Pump MexCD-OprJ in the Pseudomonas aeruginosa Quorum Sensing Response

Abstract

Multidrug efflux pumps constitute a category of antibiotic resistance determinants that are a part of the core bacterial genomes. Given their conservation, it is conceivable that they present functions beyond the extrusion of antibiotics currently used for therapy. Pseudomonas aeruginosa stands as a relevant respiratory pathogen, with a high prevalence at hospitals and in cystic fibrosis patients. Part of its success relies on its low susceptibility to antibiotics and on the production of virulence factors, whose expression is regulated in several cases by quorum sensing (QS). We found that overexpression of the MexCD-OprJ multidrug efflux pump shuts down the P. aeruginosa QS response. Our results support that MexCD-OprJ extrudes kynurenine, a precursor of the alkyl-quinolone signal (AQS) molecules. Anthranilate and octanoate, also AQS precursors, do not seem to be extruded by MexCD-OprJ. Kynurenine extrusion is not sufficient to reduce the QS response in a mutant overexpressing this efflux pump. Impaired QS response is mainly due to the extrusion of 4-hydroxy-2-heptylquinoline (HHQ), the precursor of the Pseudomonas Quinolone Signal (PQS), leading to low PQS intracellular levels and reduced production of QS signal molecules. As the consequence, the expression of QS-regulated genes is impaired and the production of QS-regulated virulence factors strongly decreases in a MexCD-OprN P. aeruginosa overexpressing mutant. Previous work showed that MexEF-OprJ, another P. aeruginosa efflux pump, is also able of extruding kynurenine and HHQ. However, opposite to our findings, the QS defect in a MexEF-OprN overproducer is due to kynurenine extrusion. These results indicate that, although efflux pumps can share some substrates, the affinity for each of them can be different. Although the QS response is triggered by population density, information on additional elements able of modulating such response is still scarce. This is particularly important in the case of P. aeruginosa lung chronic infections, a situation in which QS-defective mutants are accumulated. If MexCD-OprJ overexpression alleviates the cost associated to triggering the QS response when un-needed, it could be possible that MexCD-OprJ antibiotic resistant overproducer strains might be selected even in the absence of antibiotic selective pressure, acting as antibiotic resistant cheaters in heterogeneous P. aeruginosa populations.

Keywords: MexCD-OprJ; Pseudomonas aeruginosa; antibiotic resistance; multidrug efflux pump; quorum sensing.

FIGURE 1

Overexpression of the MexCD-OprJ efflux pump results in a decrease in the production of different virulence factors regulated by the QS system. The elastase (A), protease IV (B), pyocyanin (C), rhamnolipids, (D) and swarming (E) assays were conducted as described in Methods using cultures of the PAO1, nfxB^∗ and nfxB^∗ΔmexD strains. Statistical significances were evaluated by using a Student’s two-tailed test and considered significant if P < 0.05, with a confidence interval of 95% (^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001). nfxB^∗ presented a lower production of all tested virulence factors that the parental wild-type PAO1. The deletion of mexD in strain nfxB^∗mexD restores the phenotypes to the levels of the wild-type strain, indicating that the defects in the expression of virulence factors were solely due to the activity of the mexCD-oprJ efflux pump.

FIGURE 2

Overexpression of the MexCD-OprJ efflux system affects the expression levels of QS-regulated genes. Transcriptional analysis by real-time RT-PCR of (A) genes regulated by quorum sensing (QS) response (lasA, lasB, rhlA, rhlB, lecA, phzB1, phzB2, phzS, and mexG) and (B,C) genes responsible for QSSMs production (lasI, rhlI, pqsA, pqsD, pqsE, phnB, and pqsH) from samples obtained in (B) exponential (OD₆₀₀ = 0.6) and (A,C) early stationary phase of growth (OD₆₀₀ = 2.5) in PAO1, nfxB^∗ and nfxB^∗ΔmexD strains grown in LB medium. Statistical significances were evaluated by using a Student’s two-tailed test and considered significant if P < 0.05, with a confidence interval of 95% (^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001). The results showed that the genes implicated in the synthesis of PQS pqsA and pqsE were expressed at lower level in the nfxB^∗ strain than in the wild-type PAO1 strain at exponential phase of growth (B). In early stationary growth phase (A,C), the expression levels of the PQS-biosynthesis genes (pqsD, pqsE, phnB, and pqsH), as well as all of the analyzed QS-regulated genes, were significantly lower in the nfxB^∗ strain than in the PAO1 wild-type. The deletion of mexD in strain nfxB^∗mexD restores or even increase the levels of expression to those of the wild-type strain, indicating that these defects were solely due to the activity of the MexCD-OprJ efflux pump.

FIGURE 3

PQS and HHQ production is impaired in the strain that overproduces the MexCD-OprJ efflux pump. (A) To determine the accumulation levels of the autoinducers synthesized by Pseudomonas aeruginosa, a technique based on TLC coupled with a PqsR-based biosensor was used. The samples were extracted from cultures in early stationary phase (OD₆₀₀ = 2.5). (B) The TLC-spots corresponding to HHQ were quantified by densitometry and the ratio between the HHQ present in the supernatant respect to cell extract was calculated and represented. Statistical significances were evaluated by using a Student’s two-tailed test and considered significant if P < 0.05, with a confidence interval of 95% (^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001). As shown, overexpression of the MexCD-OprJ efflux system in nfxB^∗ strongly reduces the production of PQS and HHQ as compared with PAO1 and nfxB^∗ΔmexD strains. Furthermore, the analysis by densitometry of the HHQ ratio shows that this defect in AQs production is likely caused by an excessive extrusion of HHQ through MexCD-OprJ.

FIGURE 4

The nfxB^∗ mutant displays minor alterations in the kinetic of accumulation of 3-oxo-C12-HSL autoinducer. TLCs (A,D) and time course accumulation assays (B,C,E) were used to determine the accumulation of either 3-oxo-C12-HSL or C4-HSL autoinducer compounds. The samples for the TLC assays were extracted from cultures in late exponential phase (OD₆₀₀ = 1.7) and the samples for the time course assay were taken at different time along the cell cycle (4, 5, 6, and 7 h post-inoculation). The area under the curve of each time course assay was calculated (F) and statistical significances were evaluated by using a Student’s two-tailed test with a confidence interval of 95% (^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001). As shown, the overexpression of MexCD-OprJ has a slightly but significant effect on 3-oxo-C12-HSL accumulation. The nfxB^∗ strain presented higher levels of 3-oxo-C12-HSL than PAO1 and nfxB^∗ΔmexD both outside (A,B,F) and inside the cells (A,C,F). In contrast, the supernatant accumulation of C4-HSL in late exponential phase was lower in the MexCD-OprJ overexpressing mutant as compared with PAO1 and nfxB^∗ΔmexD strains (D,E). However, the quantification of total area under the curves (F) only showed a significant increase in nfxB^∗ΔmexD strain respect to both PAO1 and nfxB^∗.

FIGURE 5

Impaired intracellular accumulation of anthranilate produced by an excessive kynurenine extrusion through MexCD-OprJ is not the cause for lower AQs production of the nfxB^∗ mutant. Statistical significances were evaluated by using a Student’s two-tailed test and considered significant if P < 0.05, with a confidence interval of 95% (^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001). (A) The duplication time of PAO1, nfxB^∗, and nfxB^∗ΔmexD strains growing in minimal medium with succinate (control), tryptophan or kynurenine (both anthranilate precursors) as sole carbon sources was determined. As shown, the nfxB^∗ mutant presents an impaired growth in both tryptophan and kynurenine, and deletion of mexD gene restored the growth rate in nfxB^∗ mutant, suggesting these compounds might be substrates of MexCD-OprJ. (B) Anthranilate and kynurenine accumulation in cell-free supernatants was quantified by HPLC-MS from PAO1 and nfxB^∗ cultures grown along 24 h in M63 minimal medium with succinate (10 mM) and tryptophan (10 mM) as sole carbon sources. Left panels anthranilate, right panels kynurenine. As shown, the supernatants from nfxB^∗ cultures contained more kynurenine and less anthranilate than those from the wild-type PAO1 strain, indicating that kynurenine is a substrate of MexCD-OprJ and anthranilate is not extruded by this efflux pump. (C) The production of AQs in PAO1 and nfxB^∗ strains growing in LB medium supplemented with anthranilate 1 mM was analyzed in early stationary phase (OD₆₀₀ = 2.5) by TLC. (D) The extracellular vs. intracellular HHQ ratios were calculated measuring each one of the HHQ spots obtained in the TLC-assays by densitometry. (E) Real-time pqsABCDE expression was analyzed in both PAO1 and nfxB^∗ strains growing in LB medium and LB supplemented with anthranilate 4 mM respectively, using a chromosomal insertion of the reporter construction PpqsA::luxCDABE. The results show that anthranilate supplementation of LB medium does not restore the AQs production in the nfxB^∗ strain (C,E), reinforcing our hypothesis that HHQ extrusion (D) rather than kynurenine extrusion through MexCD-OprJ is the main cause for the QS-defective response of the nfxB^∗ strain.

FIGURE 6

LB supplementation with octanoate increased the AQs production in PAO1, nfxB^∗, and nfxB^∗ΔmexD strains but, in the case of nfxB^∗, but the accumulation levels remain being lower in nfxB^∗ than observed in the other strains. To determine the time course production of AQs in PAO1, nfxB^∗, and nfxB^∗ΔmexD strains, we extracted these compounds from both the cells (A) and the cell-free supernatants (B) at different times along the cell cycle (4, 5, 6, and 7 h post-inoculation). Additionally, the last points of time course extractions were analyzed by TLC (C) in order to know the proportion of PQS and HHQ present on each AQs-extracts. The total area under each time course accumulation curve was quantified (D) and statistical significances were evaluated by using a Student’s two-tailed test and considered significant if P < 0.05, with a confidence interval of 95% (^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001). The results show that supplementation of LB with 5 mM octanoate, even allowing nfxB^∗ strain to accumulate levels of AQs out of the cells near to those in PAO1 and nfxB^∗ΔmexD (B–D), was insufficient to restore the intracellular accumulation of PQS and HHQ (A,C,D). Furthermore, the fact that in TLC assay (C), the spot corresponding with HHQ present in nfxB^∗ supernatant is slightly higher than that in PAO1 and nfxB^∗ΔmexD, together with the evident low intracellular accumulation of HHQ in the nfxB^∗ strain, confirm our hypothesis that MexCD-OprJ is able to extrude HHQ.

FIGURE 7

LB supplementation with octanoate increased the pyocyanin production in all analyzed strains, but nfxB^∗ remained producing lower levels than those observed in PAO1 when grew in LB medium. For pyocyanin assay, the strains were grown in LB medium with or without octanoate (5 mM) along a time lapse of 20 h and the pyocyanin was extracted with chloroform-based protocol as is described in Methods. Statistical significances were evaluated by using a Student’s two-tailed test and considered significant if P < 0.05, with a confidence interval of 95% (^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001). The results show that supplementation of LB with 5 mM octanoate, even allowing nfxB^∗ strain increase the pyocyanin production, was insufficient to reach the levels produced by PAO1 in LB without octanoate. This indicate that a hypothetical low intracellular accumulation of octanoate is not the main cause for defective pyocyanin production observed in nfxB^∗ strain.