Limits...
Interaction of antibacterial compounds with RND efflux pumps in Pseudomonas aeruginosa.

Dreier J, Ruggerone P - Front Microbiol (2015)

Bottom Line: Subtle differences in physicochemical features of compound-pump and compound-solvent interactions largely determine how compounds are affected by efflux activity.The combination of different high-resolution techniques helps to gain insight into the functioning of these molecular machineries.This review discusses substrate recognition patterns based on experimental evidence and computer simulations with a focus on MexB, the pump subunit of the main RND transporter in P. aeruginosa.

View Article: PubMed Central - PubMed

Affiliation: Basilea Pharmaceutica International Ltd., Basel, Switzerland.

ABSTRACT
Pseudomonas aeruginosa infections are becoming increasingly difficult to treat due to intrinsic antibiotic resistance and the propensity of this pathogen to accumulate diverse resistance mechanisms. Hyperexpression of efflux pumps of the Resistance-Nodulation-Cell Division (RND)-type multidrug efflux pumps (e.g., MexAB-OprM), chromosomally encoded by mexAB-oprM, mexCD-oprJ, mexEF-oprN, and mexXY (-oprA) is often detected in clinical isolates and contributes to worrying multi-drug resistance phenotypes. Not all antibiotics are affected to the same extent by the aforementioned RND efflux pumps. The impact of efflux on antibiotic activity varies not only between different classes of antibiotics but also between members of the same family of antibiotics. Subtle differences in physicochemical features of compound-pump and compound-solvent interactions largely determine how compounds are affected by efflux activity. The combination of different high-resolution techniques helps to gain insight into the functioning of these molecular machineries. This review discusses substrate recognition patterns based on experimental evidence and computer simulations with a focus on MexB, the pump subunit of the main RND transporter in P. aeruginosa.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of efflux assay formats. (A) Increase of fluorescence intensity upon internalization of a probe. A probe with low fluorescence intensity in the medium surrounding the cells (open circles) is taken up by cells (double arrow). The fluorescence intensity of the probe increases when the probe is internalized (black circles) and interacts with cellular structures such as DNA, proteins, or membranes. Efflux of the probe (arrow through the box to the outside) causes a decrease of the total fluorescence and efflux inhibition causes an increase of the total fluorescence. (B) Quenching of fluorescence intensity upon internalization of a probe. A fluorescent probe (black circles) is added to the medium surrounding the cells. The fluorescence intensity of the probe decreases when the probe is taken up by the cells (open circles) and interacts with cellular structures (e.g., DNA and RNA). Efflux of the probe causes an increase of the total fluorescence and efflux inhibition causes a decrease of the total fluorescence. (C) Intracellular conversion of a probe into a fluorescent product. A non-fluorescent probe (rectangles with a hatched box) is added to the medium surrounding the cells. The probe can penetrate into the cells where it is enzymatically converted (e.g., cleaved or reduced) into a fluorescent product (black squares). The conversion rate of the intact probe into the fluorescent product depends on the intracellular concentration of the intact probe. Efflux of the intact probe slows down the production of the fluorescent product and efflux inhibition increases the rate of fluorescent product generation.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4495556&req=5

Figure 2: Schematic representation of efflux assay formats. (A) Increase of fluorescence intensity upon internalization of a probe. A probe with low fluorescence intensity in the medium surrounding the cells (open circles) is taken up by cells (double arrow). The fluorescence intensity of the probe increases when the probe is internalized (black circles) and interacts with cellular structures such as DNA, proteins, or membranes. Efflux of the probe (arrow through the box to the outside) causes a decrease of the total fluorescence and efflux inhibition causes an increase of the total fluorescence. (B) Quenching of fluorescence intensity upon internalization of a probe. A fluorescent probe (black circles) is added to the medium surrounding the cells. The fluorescence intensity of the probe decreases when the probe is taken up by the cells (open circles) and interacts with cellular structures (e.g., DNA and RNA). Efflux of the probe causes an increase of the total fluorescence and efflux inhibition causes a decrease of the total fluorescence. (C) Intracellular conversion of a probe into a fluorescent product. A non-fluorescent probe (rectangles with a hatched box) is added to the medium surrounding the cells. The probe can penetrate into the cells where it is enzymatically converted (e.g., cleaved or reduced) into a fluorescent product (black squares). The conversion rate of the intact probe into the fluorescent product depends on the intracellular concentration of the intact probe. Efflux of the intact probe slows down the production of the fluorescent product and efflux inhibition increases the rate of fluorescent product generation.

Mentions: Many protocols use molecules whose fluorescent properties are sensitive to their environment and change upon entry into a cell (Figures 2A,B). A prominent example is ethidium bromide (EtBr) since the quantum yield of the fluorescence increases when ethidium intercalates into DNA (LePecq and Paoletti, 1967; Mine et al., 1999; Morita et al., 2001; Li et al., 2003). Active efflux causes reduced intracellular levels of EtBr and as a consequence a decrease of fluorescence whereas inhibition of efflux pumps is recorded as a signal increase (Figure 2A). The same assay principle (Figure 2A) applies to 1-anilinonaphtalene-8-sulphonate (ANS) accumulation because the fluorescence quantum yield increases when ANS binds to hydrophobic structures (e.g., proteins, membranes) in the cell. ANS is a substrate of MexD and has been used to study this transporter in P. aeruginosa (Walmsley et al., 1994; Mao et al., 2002; Kamal et al., 2013). Another example is 1,2′-dinaphthylamine which has been used to measure AcrB efflux in E. coli because it is a substrate of AcrB and becomes strongly fluorescent when it partitions into a phospholipid bilayer (Bohnert et al., 2011a).


Interaction of antibacterial compounds with RND efflux pumps in Pseudomonas aeruginosa.

Dreier J, Ruggerone P - Front Microbiol (2015)

Schematic representation of efflux assay formats. (A) Increase of fluorescence intensity upon internalization of a probe. A probe with low fluorescence intensity in the medium surrounding the cells (open circles) is taken up by cells (double arrow). The fluorescence intensity of the probe increases when the probe is internalized (black circles) and interacts with cellular structures such as DNA, proteins, or membranes. Efflux of the probe (arrow through the box to the outside) causes a decrease of the total fluorescence and efflux inhibition causes an increase of the total fluorescence. (B) Quenching of fluorescence intensity upon internalization of a probe. A fluorescent probe (black circles) is added to the medium surrounding the cells. The fluorescence intensity of the probe decreases when the probe is taken up by the cells (open circles) and interacts with cellular structures (e.g., DNA and RNA). Efflux of the probe causes an increase of the total fluorescence and efflux inhibition causes a decrease of the total fluorescence. (C) Intracellular conversion of a probe into a fluorescent product. A non-fluorescent probe (rectangles with a hatched box) is added to the medium surrounding the cells. The probe can penetrate into the cells where it is enzymatically converted (e.g., cleaved or reduced) into a fluorescent product (black squares). The conversion rate of the intact probe into the fluorescent product depends on the intracellular concentration of the intact probe. Efflux of the intact probe slows down the production of the fluorescent product and efflux inhibition increases the rate of fluorescent product generation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495556&req=5

Figure 2: Schematic representation of efflux assay formats. (A) Increase of fluorescence intensity upon internalization of a probe. A probe with low fluorescence intensity in the medium surrounding the cells (open circles) is taken up by cells (double arrow). The fluorescence intensity of the probe increases when the probe is internalized (black circles) and interacts with cellular structures such as DNA, proteins, or membranes. Efflux of the probe (arrow through the box to the outside) causes a decrease of the total fluorescence and efflux inhibition causes an increase of the total fluorescence. (B) Quenching of fluorescence intensity upon internalization of a probe. A fluorescent probe (black circles) is added to the medium surrounding the cells. The fluorescence intensity of the probe decreases when the probe is taken up by the cells (open circles) and interacts with cellular structures (e.g., DNA and RNA). Efflux of the probe causes an increase of the total fluorescence and efflux inhibition causes a decrease of the total fluorescence. (C) Intracellular conversion of a probe into a fluorescent product. A non-fluorescent probe (rectangles with a hatched box) is added to the medium surrounding the cells. The probe can penetrate into the cells where it is enzymatically converted (e.g., cleaved or reduced) into a fluorescent product (black squares). The conversion rate of the intact probe into the fluorescent product depends on the intracellular concentration of the intact probe. Efflux of the intact probe slows down the production of the fluorescent product and efflux inhibition increases the rate of fluorescent product generation.
Mentions: Many protocols use molecules whose fluorescent properties are sensitive to their environment and change upon entry into a cell (Figures 2A,B). A prominent example is ethidium bromide (EtBr) since the quantum yield of the fluorescence increases when ethidium intercalates into DNA (LePecq and Paoletti, 1967; Mine et al., 1999; Morita et al., 2001; Li et al., 2003). Active efflux causes reduced intracellular levels of EtBr and as a consequence a decrease of fluorescence whereas inhibition of efflux pumps is recorded as a signal increase (Figure 2A). The same assay principle (Figure 2A) applies to 1-anilinonaphtalene-8-sulphonate (ANS) accumulation because the fluorescence quantum yield increases when ANS binds to hydrophobic structures (e.g., proteins, membranes) in the cell. ANS is a substrate of MexD and has been used to study this transporter in P. aeruginosa (Walmsley et al., 1994; Mao et al., 2002; Kamal et al., 2013). Another example is 1,2′-dinaphthylamine which has been used to measure AcrB efflux in E. coli because it is a substrate of AcrB and becomes strongly fluorescent when it partitions into a phospholipid bilayer (Bohnert et al., 2011a).

Bottom Line: Subtle differences in physicochemical features of compound-pump and compound-solvent interactions largely determine how compounds are affected by efflux activity.The combination of different high-resolution techniques helps to gain insight into the functioning of these molecular machineries.This review discusses substrate recognition patterns based on experimental evidence and computer simulations with a focus on MexB, the pump subunit of the main RND transporter in P. aeruginosa.

View Article: PubMed Central - PubMed

Affiliation: Basilea Pharmaceutica International Ltd., Basel, Switzerland.

ABSTRACT
Pseudomonas aeruginosa infections are becoming increasingly difficult to treat due to intrinsic antibiotic resistance and the propensity of this pathogen to accumulate diverse resistance mechanisms. Hyperexpression of efflux pumps of the Resistance-Nodulation-Cell Division (RND)-type multidrug efflux pumps (e.g., MexAB-OprM), chromosomally encoded by mexAB-oprM, mexCD-oprJ, mexEF-oprN, and mexXY (-oprA) is often detected in clinical isolates and contributes to worrying multi-drug resistance phenotypes. Not all antibiotics are affected to the same extent by the aforementioned RND efflux pumps. The impact of efflux on antibiotic activity varies not only between different classes of antibiotics but also between members of the same family of antibiotics. Subtle differences in physicochemical features of compound-pump and compound-solvent interactions largely determine how compounds are affected by efflux activity. The combination of different high-resolution techniques helps to gain insight into the functioning of these molecular machineries. This review discusses substrate recognition patterns based on experimental evidence and computer simulations with a focus on MexB, the pump subunit of the main RND transporter in P. aeruginosa.

No MeSH data available.


Related in: MedlinePlus