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Real-time monitoring of New Delhi metallo-β-lactamase activity in living bacterial cells by 1H NMR spectroscopy.

Ma J, McLeod S, MacCormack K, Sriram S, Gao N, Breeze AL, Hu J - Angew. Chem. Int. Ed. Engl. (2014)

Bottom Line: Disconnections between in vitro responses and those observed in whole cells confound many attempts to design drugs in areas of serious medical need.NDM-1 activity in cells was also shown to be inhibited by spermine, a porin inhibitor, although in an in vitro assay, the influence of spermine on the activity of isolated NDM-1 protein is minimal.This new approach may have generic utility for monitoring reactions involving diffusible metabolites in other complex biological matrices and whole-cell settings, including mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Discovery Sciences, AstraZeneca Boston, Waltham, MA 02451 (USA).

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1H NMR spectra of meropenem hydrolysis in the presence of 5 nM purified NDM-1 enzyme (A), E. coli cells (OD600=10.0) without NDM-1 plasmid (B), and E. coli cells (OD600=2.5) expressing NDM-1 (C). All samples were prepared in 50 mM sodium phosphate at pH 7.0 with 10 % deuterated water. The hydrolysis of meropenem (100 μM) at different time points was monitored by focusing on the 1H NMR signals from the nitrogen-attached methyl groups (Scheme 1). The green and red dotted lines denote the signals of substrate and product, respectively.
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fig01: 1H NMR spectra of meropenem hydrolysis in the presence of 5 nM purified NDM-1 enzyme (A), E. coli cells (OD600=10.0) without NDM-1 plasmid (B), and E. coli cells (OD600=2.5) expressing NDM-1 (C). All samples were prepared in 50 mM sodium phosphate at pH 7.0 with 10 % deuterated water. The hydrolysis of meropenem (100 μM) at different time points was monitored by focusing on the 1H NMR signals from the nitrogen-attached methyl groups (Scheme 1). The green and red dotted lines denote the signals of substrate and product, respectively.

Mentions: Carbapenems such as meropenem and imipenem, once trusted as a last resort to treat the most serious bacterial infections, can now be hydrolyzed by various β-lactamases, in particular, NDM-1 (Scheme 1).13 This reaction can be observed by using an in vitro NMR assay in which meropenem is treated with the purified NDM-1 enzyme (Figure 1 A). Meropenem is relatively stable for a long period of time in E. coli cells lacking carbapenemases (Figure 1 B). However, the drug is gradually degraded in the presence of E. coli cells carrying the NDM-1 enzyme (Figure 1 C). Despite the background signals from the cells and sample preparation (Figure S1 in the Supporting Information), the hydrolysis process can be clearly monitored by focusing on the 1H NMR signals from the methyl groups on meropenem. Under the experimental conditions employed (100 μM meropenem and a suspension of NDM-1 E. coli cells with an optical density at 600 nm (OD600) of 2.5 in sodium phosphate buffer), the intensity of the 1H signals from the methyl groups is about fivefold higher than from cells alone (Figure 1 B and C). In addition, the background 1H signals from the aromatic region are negligible (Figure S2), thus making this method generally applicable to common drugs and compounds in chemical libraries, of which approximately 80 % have aromatic groups.14 It is also a sensitive assay: even the terminal −N(CH3)2 protons (1H chemical shifts: δ=3.07 and 2.99 ppm, Figure 1) yield resolvable changes in chemical shift on ring opening. The viability of the NDM-1 E. coli cells was checked before and after the NMR experiments. The plating colony test shows that one hour of NMR measurements did not lead to any change in cell viability (Figure S3). To confirm that the enzymatic activity is from NDM-1 in the cells and to rule out the possibility that meropenem induces cell lysis and subsequent NDM-1 leakage into the medium, NDM-1 E. coli cells treated with meropenem were spun down and fresh meropenem was added to the supernatant to monitor the change in the meropenem 1H signals. Hydrolysis of meropenem was not observed (Figure S4), which demonstrates that the reaction occurs inside the cells. By contrast, when periplasmic proteins were released by treating NDM-1 E. coli cells with chloroform,15 hydrolysis of meropenem in the supernatant was observed by NMR spectroscopy (Figure S5). This result strongly supports the conclusion that the enzymatic reaction catalyzed by NDM-1 indeed occurs in the periplasmic space where most β-lactamases are known to reside.12


Real-time monitoring of New Delhi metallo-β-lactamase activity in living bacterial cells by 1H NMR spectroscopy.

Ma J, McLeod S, MacCormack K, Sriram S, Gao N, Breeze AL, Hu J - Angew. Chem. Int. Ed. Engl. (2014)

1H NMR spectra of meropenem hydrolysis in the presence of 5 nM purified NDM-1 enzyme (A), E. coli cells (OD600=10.0) without NDM-1 plasmid (B), and E. coli cells (OD600=2.5) expressing NDM-1 (C). All samples were prepared in 50 mM sodium phosphate at pH 7.0 with 10 % deuterated water. The hydrolysis of meropenem (100 μM) at different time points was monitored by focusing on the 1H NMR signals from the nitrogen-attached methyl groups (Scheme 1). The green and red dotted lines denote the signals of substrate and product, respectively.
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Related In: Results  -  Collection

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fig01: 1H NMR spectra of meropenem hydrolysis in the presence of 5 nM purified NDM-1 enzyme (A), E. coli cells (OD600=10.0) without NDM-1 plasmid (B), and E. coli cells (OD600=2.5) expressing NDM-1 (C). All samples were prepared in 50 mM sodium phosphate at pH 7.0 with 10 % deuterated water. The hydrolysis of meropenem (100 μM) at different time points was monitored by focusing on the 1H NMR signals from the nitrogen-attached methyl groups (Scheme 1). The green and red dotted lines denote the signals of substrate and product, respectively.
Mentions: Carbapenems such as meropenem and imipenem, once trusted as a last resort to treat the most serious bacterial infections, can now be hydrolyzed by various β-lactamases, in particular, NDM-1 (Scheme 1).13 This reaction can be observed by using an in vitro NMR assay in which meropenem is treated with the purified NDM-1 enzyme (Figure 1 A). Meropenem is relatively stable for a long period of time in E. coli cells lacking carbapenemases (Figure 1 B). However, the drug is gradually degraded in the presence of E. coli cells carrying the NDM-1 enzyme (Figure 1 C). Despite the background signals from the cells and sample preparation (Figure S1 in the Supporting Information), the hydrolysis process can be clearly monitored by focusing on the 1H NMR signals from the methyl groups on meropenem. Under the experimental conditions employed (100 μM meropenem and a suspension of NDM-1 E. coli cells with an optical density at 600 nm (OD600) of 2.5 in sodium phosphate buffer), the intensity of the 1H signals from the methyl groups is about fivefold higher than from cells alone (Figure 1 B and C). In addition, the background 1H signals from the aromatic region are negligible (Figure S2), thus making this method generally applicable to common drugs and compounds in chemical libraries, of which approximately 80 % have aromatic groups.14 It is also a sensitive assay: even the terminal −N(CH3)2 protons (1H chemical shifts: δ=3.07 and 2.99 ppm, Figure 1) yield resolvable changes in chemical shift on ring opening. The viability of the NDM-1 E. coli cells was checked before and after the NMR experiments. The plating colony test shows that one hour of NMR measurements did not lead to any change in cell viability (Figure S3). To confirm that the enzymatic activity is from NDM-1 in the cells and to rule out the possibility that meropenem induces cell lysis and subsequent NDM-1 leakage into the medium, NDM-1 E. coli cells treated with meropenem were spun down and fresh meropenem was added to the supernatant to monitor the change in the meropenem 1H signals. Hydrolysis of meropenem was not observed (Figure S4), which demonstrates that the reaction occurs inside the cells. By contrast, when periplasmic proteins were released by treating NDM-1 E. coli cells with chloroform,15 hydrolysis of meropenem in the supernatant was observed by NMR spectroscopy (Figure S5). This result strongly supports the conclusion that the enzymatic reaction catalyzed by NDM-1 indeed occurs in the periplasmic space where most β-lactamases are known to reside.12

Bottom Line: Disconnections between in vitro responses and those observed in whole cells confound many attempts to design drugs in areas of serious medical need.NDM-1 activity in cells was also shown to be inhibited by spermine, a porin inhibitor, although in an in vitro assay, the influence of spermine on the activity of isolated NDM-1 protein is minimal.This new approach may have generic utility for monitoring reactions involving diffusible metabolites in other complex biological matrices and whole-cell settings, including mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Discovery Sciences, AstraZeneca Boston, Waltham, MA 02451 (USA).

Show MeSH
Related in: MedlinePlus