Subscriber access provided by University of Sunderland
Letter
Real-time monitoring of NDM-1 activity in live bacterial cells by isothermal titration calorimetry: a new approach to measure inhibition of antibiotic-resistant bacteria Yue-Juan Zhang, Wen-Ming Wang, Peter Oelschlaeger, Cheng Chen, Jin-E Lei, Miao Lv, and Kewu Yang ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00147 • Publication Date (Web): 01 Nov 2018 Downloaded from http://pubs.acs.org on November 2, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Infectious Diseases
Real-time monitoring of NDM-1 activity in live bacterial cells by isothermal titration calorimetry: a new approach to measure inhibition of antibiotic-resistant bacteria Yue-Juan Zhang†‖, Wen-Ming Wang†‖, Peter Oelschlaeger‡, Cheng Chen†, Jin-E Lei§, Miao Lv† and Ke-Wu Yang†* †Key
Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Chemical
Biology Innovation Laboratory, College of Chemistry and Materials Science, Northwest University, 1 Xuefu Avenue, Xi'an 710127, P. R. China. ‡Department
of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, 309 East
Second Street, Pomona, California 91766, United States. §The
First Affiliated Hospital of Xi'an Jiaotong University, 277 West Yanta Road, Xi'an 710061, P.R. China.
Corresponding author: E-mail:
[email protected]; Tel: +8629-8153-5035.
1
ACS Paragon Plus Environment
ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 19
The “superbug” infection caused by New Delhi Metallo-β-lactamase (NDM-1) has become an emerging threat. Monitoring NDM-1 has proven challenging due to its shuttling between pathogenic bacteria. Here, we report an isothermal titration calorimetry (ITC) method that can monitor activity and inhibition of NDM-1 in live bacterial cells in real time. This method has been exemplified by monitoring of the activity and inhibition of the target enzyme, and evaluating the breakdown of antibiotics by pathogenic bacteria expressing β-lactamases. Cell-based studies demonstrate that the NDM-1 expressed in bacterial cells was inhibited by four known inhibitors EDTA, D-captopril, ebselen and azolylthioacetamide with IC50 values of 3.8, 48, 0.55 and 17.5 μM, respectively, which are in good agreement with the data from inhibition kinetics using UV-Vis spectroscopy in vivo. This approach could be applied to screen and evaluate small molecule inhibitors of MβLs in whole cells or to identify drug resistant bacteria. KEYWORDS: antibiotic resistance, metallo-β-lactamase, NDM-1, inhibition, isothermal titration calorimetry
2
ACS Paragon Plus Environment
Page 3 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Infectious Diseases
β-Lactam antibiotics are commonly used to treat bacterial infections.1 These antibiotics kill bacteria by inhibiting transpeptidases involved in cell wall biosynthesis.2 However, the frequent use of these antibiotics has produced a large number of bacteria that are resistant to most antibiotics. The major mechanism of resistance to β-lactam antibiotics employed by bacteria is the expression of β-lactamases. These enzymes hydrolyze the amide bond of the β-lactam ring and yield an inactivated product.3 β-Lactamases are grouped into four classes (A-D) based on their amino acid sequence.4-6 Class A, C and D enzymes, known as serine β-lactamases (SβLs), employ an active site serine to carry out a nucleophilic attack on the β-lactam carbonyl, which ultimately results in the cleavage of the β-lactam ring. Class B enzymes, synonymous with metallo-β-lactamases (MβLs), utilize 1 or 2 Zn(II) equivalents for full catalytic activity to hydrolyze β-lactam antibiotics.7 MβLs have further been divided into the B1, B2 and B3 subclasses based on sequence similarity and Zn(II) content.3 Combining β-lactamase inhibitors with existing β-lactam antibiotics is an effective way to overcome antibiotic resistance.8 Currently, several inhibitors of the SβLs are used: clavulanic acid, sulbactam, tazobactam, and avibactam, but no MβL inhibitor is available in the clinic.9 Subclass B1 MβLs, including imipenemase (IMP), Verona Integron-borne Metallo-β-lactamase (VIM), and New Delhi Metallo-β-lactamase (NDM), are spread worldwide and the most clinically prevalent MβLs.10 Among them, NDM-1 can inactivate almost all β-lactam antibiotics, including meropenem and imipenem, often coined “last resort” antibiotics for treating the most serious bacterial infections.11 Gram-negative bacteria harboring the NDM-1 gene are often called “super bugs” and include Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas spp..12 NDM1 is plasmid-encoded, which facilitates its rapid transmission and makes monitoring it in living bacterial cells a daunting challenge. Effective inhibitors of NDM-1 have been sought after extensively.9,
13, 14
However, almost all of these
evaluations of NDM-1 inhibitors were performed in vitro in the absence of the bacterial physiological context. Frequently, these studies are followed by minimum inhibitory concentration (MIC) assays, which inform on what antibiotic and inhibitor concentrations inhibit growth but do not give any details on interactions between MβL, antibiotic, and MβL inhibitor. Clearly, there is a general need for a straightforward and feasible approach that enables real-time monitoring of activity and inhibition of NDM-1 in vivo (inside bacterial cells). NMR and UV-Vis spectroscopy have been employed for in vivo activity monitoring of NDM-1.15, 16 However, both methods have their limitations. The limitation of cell-based NMR technology is its inability to detect the hydrolysis of cephalosporins, such as cefotaxime and ceftazidime by the target enzyme due to interference of the monitoring spectrum by the complicated spectrum of multiple intracellular components.15 Also, the NMR technology is demanding, requiring the use deuterium or a labelling reagent.17 The UV-Vis method is based on direct spectroscopic detection of the substrate (or product) absorbance to 3
ACS Paragon Plus Environment
ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 19
monitor MβL activity. The limitation of this technology is that it requires a substrate (or product) with a characteristic spectroscopic absorption. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) as a potent tool has been used for β-lactamase activity detection, based on the molecular weight detection of hydrolysate of enzymatic substrate.18 However, the presence of false-positive and false-negative results makes it difficult to detect the target molecular peaks,18 especially in the presence of enzyme inhibitors. To our knowledge, MALDI-TOF MS has not been used for real-time activity inhibition evaluation of NDM-1 in vivo. Isothermal titration calorimetry (ITC) is an ideal approach to measure heat change during ligand-target binding. The main principle of ITC is to monitor the time derivative of heat change during a biomolecular reaction or binding event by titrating one solution into another while the source of the heat absorbed or released needs to be identified.19 ITC provides thermodynamic properties, is sensitive, has a fast response time, and does not require any labelling chemistry. It has been applied to assess the activity and binding mode of enzyme inhibitors.20-22 Here, we report a cell-based ITC approach for real-time monitoring of activity and inhibition of MβLs in live bacterial cells.
Scheme 1. Hydrolysis of cephalosporin by New Delhi metallo-β-lactamase 1 (NDM-1)
The hydrolysis of β-lactam by bacterial cells expressing MβLs involves a number of chemical events which result in a series of heat effects.23 Based on the large amount of -lactam injected and the observation time-frame (starting right before addition of substrate and ending after substrate is consumed), the reasonable speculation is that the heat change observed is dominated by the exothermic cleavage reaction of the β-lactam. ITC was employed to monitor the time derivative of heat change. The hydrolysis of cephalosporin by NDM-1 is shown in Scheme 1. The thermal power (dQ/dt) during the reaction is monitored by a MicroCal Auto-ITC200 instrument (Malvern Instruments, Malvern, UK) in a single injection mode. The antibiotic substrate is titrated into a solution of enzyme or enzyme incubated with inhibitor in a sample cell, and the dQ/dt value (change in heat divided by change in time) is registered, which indicates the calorimetric response to substrate hydrolysis. The analysis is straightforward since dQ/dt is proportional to the reaction rate, and the maximum dQ/dt value is proportional to the initial reaction rate.24, 25 A typical heat flow curve of NDM-1catalyzed hydrolysis of cefazolin (Figure S1, Supporting Information) shows that the signal returns gradually to baseline, which indicates the end of the reaction. The total heat release (Qtotal, simply shown here as Q) associated with converting n moles of substrate into product can be obtained by integrating the area under the entire curve.20, 26 With 4
ACS Paragon Plus Environment
Page 5 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Infectious Diseases
the single injection mode that we employed, the time at which maximum heat release occurred varied, because the substrate in the syringe was titrated continuously into the bacterial cell suspension with a set rate of 0.5 μL/s until the substrate was completely added into the sample cell, but the volume added varied. When the last drop of substrate was added, the concentration of substrate in the sample cell and the hydrolysis rate reached their maxima, and then the reflection of thermal power (dQ/dt) reached its maximum. For more details on the background and theory of ITC, see Supporting Information.
Figure 1. Overlaid heat flow curves of cefazolin hydrolysis by the purified NDM-1 enzyme (A), E.coli cells not expressing NDM-1 (B), and E.coli cells expressing NDM-1 (C). All samples were prepared in 30 mM Tris-HCl, pH 8.0. The amount of heat released during hydrolysis of cefazolin was measured every 1 s. The differently colored lines represent curves obtained with the indicated concentrations of antibiotic (total 30 μL) injected to 210 μL of 10 nM NDM-1 (A), cells at OD600 = 0.5 not expressing NDM-1 (B), or cells at OD600 = 0.5 expressing NDM-1 (C).
ITC was employed to monitor the hydrolysis of cefazolin by NDM-1 in vitro as well as NDM-1 expressed by E.coli BL21 (DE3) cells (in-cell), while E.coli cells lacking NDM-1 were monitored as a control. The progress of cefazolin hydrolysis monitored by ITC is shown in Figure 1. The drug at different concentrations is quickly hydrolyzed when treated with purified NDM-1 in vitro (Figure 1A) and an increase of drug results in an increase of both Q and dQ/dt values. Under the in vivo test conditions (1 mM cefazolin and an E. coli suspension with an optical density at 600 nm (OD600) of 0.5 in 30 mM Tris-HCl, pH 8.0), cefazolin is quite stable, if the E. coli cells do not express the MβL (Figure 1B). The weak thermal power (< 0.5 μcal/s) observed in response to various concentrations of cefazolin is likely exerted due to the heat of dilution or intrinsic bacteria metabolic heat.27, 28 However, the -lactam is completely hydrolyzed in the presence of E. coli cells that produce NDM-1 (Figure 1C), although slowly than with purified NDM-1 (Figure 1A). The thermograms of cefazolin hydrolysis by the purified NDM-1 enzyme and the E. coli cells producing NDM-1 are initially similar with both reaching maximal hydrolysis rates at ~100 s. However, with live cells, the signal takes a longer time to return to a value close to baseline, e.g., ~600 s to reach a value
