Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Feb 9;4(2):185-195.
doi: 10.1021/acsinfecdis.7b00160. Epub 2017 Nov 10.

Kinetic Control of Quorum Sensing in Pseudomonas aeruginosa by Multidrug Efflux Pumps

Affiliations

Kinetic Control of Quorum Sensing in Pseudomonas aeruginosa by Multidrug Efflux Pumps

David Wolloscheck et al. ACS Infect Dis. .

Abstract

Pseudomonas aeruginosa is an important human pathogen, the physiology and virulence of which are under the control of quorum sensing signals. These signals often have dual roles, functioning as toxins to some cells and as oxidative-stress protectors for their producer cells. Hence, their internal and external concentrations should be tightly controlled. In this study, we analyzed the interplay between the multidrug efflux transporters MexEF-OprN and MexG/HI-OpmD in quorum sensing of P. aeruginosa. We found that the two transporters have overlapping substrate specificities but different efficiencies. When overproduced, both MexEF-OprN and MexG/HI-OpmD provide clinical levels of resistance to diverse fluoroquinolones and protect P. aeruginosa against toxic phenazines. However, this similarity is enabled by synergistic interactions with the outer membrane. In hyperporinated cells, MexG/HI-OpmD is saturated by much lower concentrations of fluoroquinolones but is more efficient than MexEF-OprN in efflux of phenazines. Unlike MexEF-OprN, mutational inactivation of MexG/HI-OpmD reduces the levels of pyocyanin and makes P. aeruginosa cells hypersusceptible to phenazines. Our results further show that MexG binds pyocyanin, physically associates with MexHI, and represses the activity of the transporter, revealing a negative regulatory role of this protein. We conclude that differences in kinetic properties of transporters are critical to maintain proper intra- and extracellular concentrations of phenazines and other signaling molecules and that MexG/HI-OpmD controls the steady state in the synthesis and secretion of phenazines.

Keywords: antibiotic resistance; efflux constant; hyperporination; outer membrane barrier; permeation; phenazines.

PubMed Disclaimer

Figures

Figure 1
Figure 1. The effect of hyperporination on growth of P. aeruginosa cells with different efflux capacities
A. Growth curves of the indicated P. aeruginosa strains grown in the absence of the inducer. Overnight cultures were diluted 1:100 into a fresh LB medium; the cells were grown for 18 hours and OD600 measured every 30 min. Data shown are the averages of three repeats and the error bars are SD (n=3). B. The same as in A but cultures were grown in the presence of 0.1 mM IPTG. C. After the growth curves were analyzed as described in A and B, cell aliquots were plated onto LB agar plates and CFUs counted after 24 hrs incubation at 37ºC. Averages of three repeats are shown with SD (n=3) as the error bars. D. Growth rates of indicated P. aeruginosa strains at increasing concentrations of IPTG. Averages of six repeats are shown with SD (n=3) as the error bars. E. Amounts of pyocyanin produced by indicated strains in the absence and presence of 0.1 mM IPTG. Test tubes containing overnight cultures of the indicated strains and their OD600 readings.
Figure 2
Figure 2. Fluoroquinolone susceptibility testing of cells overexpressing MexHI-OpmD, MexGHI-OpmD, and MexEF-OprN
A. Immunoblotting of membrane fractions from cells harboring the plasmids overproducing the indicated transporters. The respective outer membrane channels (OMF) are tagged with a C-terminal His-tag and visualized with anti-His monoclonal antibody. B and C. Effect of IPTG inducer concentration on MICs of ciprofloxacin in PΔ4 (B) and PΔ4-Pore (C) cells expressing indicated transporters. D and E. PΔ4 (D) or PΔ4-Pore (E) cells harboring either pMexHI-OpmD, pMexGHI-OpmD, or pMexEF-OprN are tested against a library of FQ. Fold changes in MICs are shown for cells carrying different plasmid constructs are shown. FQ for each ratio are listed from bottom to top: Ciprofloxacin, Enrofloxacin, Levofloxacin, Gatifloxacin, Moxifloxacin, Prulifloxacin, Sparfloxacin, Difloxacin, Lomefloxacin, Ofloxacin, Pazufloxacin, Norfloxacin, Pefloxacin, Sarafloxacin, and Nadifloxacin.
Figure 3
Figure 3. Kinetic uptake measurements
A. The plot of initial rates of HT accumulation in the indicated cells as a function of extracellular HT concentration. B. Graph of the initial rates of HT accumulation in pore expressing cells plotted against the extracellular HT concentration. All kinetic measurements were done in triplicate. Error bars are SD (n=3).
Figure 4
Figure 4. Expression MexG/HI-OpmD correlates with amounts of extracellular pyocyanin and is required for P. aeruginosa self-protection against its toxicity
A. Extracellular concentrations of pyocyanin in PAO1, PΔ3, PΔ4, and PΔ6 parental strains (blue) and their derivatives lacking mexGHI-opmD (ΔGHID) mutants (orange). B. Extracellular concentrations of pyocyanin in cultures of PAO1 strains overexpressing MexEF-OprN or different combinations of MexG/HI-OpmD components. All measurements were done on cultures grown to stationary phase and in triplicate. Error bars are SD (n=3). C. Spot assays with pyocyanin showing zones of inhibition in strains with and without mexGHI-opmD. D. The same as C but with PMS. E and F. Quantification of zones of inhibition of pyocyanin (E) and PMS (F) in indicated strains. Error bars are SD (n=3).
Figure 5
Figure 5. MexG binds MexHI and pyocyanin
A and B. Immunoblotting analyses of MexIHis (A) and MexHHis (B) elution fractions purified from PΔ3 cells harboring pMexGHI-FLAG and pMexGH-Flag respectively. Cells were treated with crosslinker prior to lysis as indicated. The lower panel shows the release of FLAG-tagged MexG from crosslinked samples when elution fractions are treated with a reducing agent (DTT). DSP, dithiobis(succinimidylpropionate); DTT, dithiothreitol. Top panels show the development with anti-His and the lower panels with anti-FLAG primary antibodies. C and D. Fluorescence emission at 330 nm (excitation at 290 nm) of the wild type MexG (C) and MexG-HAWA (D) incubated with increasing concentration of pyocyanin was measured and normalized as described in Methods and is plotted as a function pyocyanin concentration. Fitted line shown in black. Error bars are SD (n=3).
Figure 6
Figure 6. A proposed mechanism of MexG/HI-OpmD efflux pump
The presence of MexG negatively affects the activity of MexHI-OpmD efflux pump. MexG physically interacts with the pump and its substrates such as pyocyanin. In the ligand-bound state MexG could be in a different conformation or could dissociate from the pump, which in turn could lead to increased efflux of compounds.

Similar articles

Cited by

References

    1. Mulcahy LR, Isabella VM, Lewis K. Pseudomonas aeruginosa Biofilms in Disease. Microb Ecol. 2013;68(1):1–12. doi: 10.1007/s00248-013-0297-x. - DOI - PMC - PubMed
    1. World Health, O. Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. 2017.
    1. Zgurskaya HI, Lopez CA, Gnanakaran S. Permeability Barrier of Gram-Negative Cell Envelopes and Approaches To Bypass It. ACS Infect Dis. 2015;1(11):512–522. doi: 10.1021/acsinfecdis.5b00097. - DOI - PMC - PubMed
    1. Blair JM, Webber MA, Baylay AJ, Ogbolu DO, Piddock LJ. Molecular mechanisms of antibiotic resistance. Nat Rev Microbiol. 2015;13(1):42–51. doi: 10.1038/nrmicro3380. - DOI - PubMed
    1. Zgurskaya HI, Nikaido H. Multidrug resistance mechanisms: drug efflux across two membranes. Mol Microbiol. 2000;37(2):219–25. - PubMed

Publication types