Real-time monitoring of D-Ala-D-Ala dipeptidase activity of VanX in living bacteria by isothermal titration calorimetry

Article abstract of DOI:10.1016/j.ab.2019.05.002

The D,D-dipeptidase enzyme VanX is the main cause of vancomycin resistance in gram-positive bacteria because of hydrolysis of the D-Ala-D-Ala dipeptide used in cell-wall biosynthesis. Continuous assay of VanX has proven challenging due to lack of a chromophoric substrate. Here, we report a direct approach for continuous assay of VanX in vitro and in vivo from hydrolysis of D-Ala-D-Ala, based on the heat-rate changes measured with isothermal titration calorimetry (ITC). With the ITC approach, determination of kinetic parameters of VanX hydrolyzing D-Ala-D-Ala and the inhibition constant of D-cysteine inhibitor yielded K_M of 0.10 mM, k_cat of 11.5 s^?1, and K_i of 18.8 μM, which are consistent with the data from ninhydrin/Cd(II)assays. Cell-based ITC studies demonstrated that the VanX expressed in E. coli and in clinical strain VRE was inhibited by D-cysteine with IC₅₀ values of 29.8 and 28.6 μM, respectively. Also, the total heat from D-Ala-D-Ala (4 mM)hydrolysis decreases strongly (in absolute value)from 1.26 mJ for VRE to 0.031 mJ for E. faecalis, which is consistent with the large MIC value of vancomycin of 512 μg/mL for VRE and the much smaller value of 4 μg/mL for E. faecalis. The ITC approach proposed here could be applied to screen and evaluate small molecule inhibitors of VanX or to identify drug resistant bacteria.

Full text of DOI:10.1016/j.ab.2019.05.002

Accepted Manuscript
Real-time monitoring of D-Ala-D-Ala dipeptidase activity of VanX in living bacteria by
isothermal titration calorimetry
Miao Lv, Yue-Juan Zhang, Fan Zhou, Ying Ge, Mu-Han Zhao, Ya Liu, Ke-Wu Yang
PII:
S0003-2697(18)31215-6
https://doi.org/10.1016/j.ab.2019.05.002
YABIO 13313
DOI:
Reference:
To appear in: Analytical Biochemistry
Received Date: 5 December 2018
Revised Date: 17 April 2019
Accepted Date: 3 May 2019
Please cite this article as: M. Lv, Y.-J. Zhang, F. Zhou, Y. Ge, M.-H. Zhao, Y. Liu, K.-W. Yang, Real-
time monitoring of D-Ala-D-Ala dipeptidase activity of VanX in living bacteria by isothermal titration
calorimetry, Analytical Biochemistry (2019), doi: https://doi.org/10.1016/j.ab.2019.05.002.
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ACCEPTED MANUSCRIPT
Real-time monitoring of D-Ala-D-Ala dipeptidase activity of
VanX in living bacteria by isothermal titration calorimetry
A novel ITC approach for continuous assay of the activity and inhibition of VanX enzyme in vitro
and in living bacterial by using D-Ala-D-Ala as the substrate.

ACCEPTED MANUSCRIPT
Real-time monitoring of D-Ala-D-Ala dipeptidase activity of
VanX in living bacteria by isothermal titration calorimetry
Miao Lv, Yue-Juan Zhang, Fan Zhou, Ying Ge, Mu-Han Zhao, Ya Liu, 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.
*Corresponding author: E-mail: kwyang@nwu.edu.cn; Tel: +8629-8153-5035.
Abstract
The D,D-dipeptidase enzyme VanX is the main cause of vancomycin resistance in
gram-positive bacteria because of hydrolysis of the D-Ala-D-Ala dipeptide used in cell-wall
biosynthesis. Continuous assay of VanX has proven challenging due to lack of a chromophoric
substrate. Here, we report a direct approach for continuous assay of VanX in vitro and in vivo
from hydrolysis of D-Ala-D-Ala, based on the heat-rate changes measured with isothermal
titration calorimetry (ITC). With the ITC approach, determination of kinetic parameters of VanX
hydrolyzing D-Ala-D-Ala and the inhibition constant of D-cysteine inhibitor yielded KM of 0.10 mM,
kcat of 11.5 s-1, and Ki of 18.8 μM, which are consistent with the data from ninhydrin/Cd(II) assays.
Cell-based ITC studies demonstrated that the VanX expressed in E. coli and in clinical strain VRE
was inhibited by D-cysteine with IC50 values of 29.8 and 28.6 μM, respectively. Also, the total
heat from D-Ala-D-Ala (4 mM) hydrolysis decreases strongly (in absolute value) from 1.26 mJ for
VRE to 0.031 mJ for E. faecalis, which is consistent with the large MIC value of vancomycin of 512
μg/mL for VRE and the much smaller value of 4 μg/mL for E. faecalis. The ITC approach proposed
here could be applied to screen and evaluate small molecule inhibitors of VanX or to identify
drug resistant bacteria.
Keywords: vancomycin; VanX; continuous assay; inhibition; isothermal titration calorimetry
1. Introduction
Vancomycin, a glycopeptide antibiotic, is widely used for the treatment of gram-positive
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bacterial infections [1]. It inhibits the cell wall biosynthesis of gram-positive bacteria by binding
specifically to cell wall peptidoglycans that terminate in D-Ala-D-Ala [2]. This blockade of the
transpeptidation of uncrosslinked D-Ala-D-Ala containing cell wall components ultimately leads
to osmotic lysis of bacteria [3]. However, the overuse of this drug has caused rapid emergence of
vancomycin-resistant bacteria, such as vancomycin-resistant Staphylococcus aureus (VRSA) and
vancomycin-resistant Enterococci (VRE) [4,5].
Vancomycin resistance in clinical VRSA and VRE strains require expression of five genes for
high-level resistance, four of the gene products, VanS, VanR, VanH, and VanA, produce a
modified peptidoglycan unit in which the terminal D-Ala is replaced by D-lactate [6-8]. The fifth
gene product, VanX, a Zn(II)-dependent dipeptidase, hydrolyzes D-Ala-D-Ala as shown in Scheme
1, allowing the D,D-depsipeptide to accumulate and incorporate into the growing peptidoglycan
termini mediated by the VanH and VanA enzymes [9]. Vancomycin binds to the modified
peptidoglycan termini D-Ala-D-Lac 1000-fold less tightly than to D-Ala-D-Ala peptidoglycan due
to the loss of one hydrogen bond, and unimpeded peptide chain cross-linking, thus yielding a
mechanically strong cell wall and resistance to lysis and cell death in the presence of vancomycin
[8,10-12]. Clearly, VanX is a potential drug target, and screening inhibitors of VanX is an effective
way to combat vancomycin resistance [9,12,13].
Scheme 1. D-Ala-D-Ala hydrolysis catalysed by VanX
Given the enormous biomedical importance of VanX, many attempts have been made to
develop assays for VanX activity in searching for effective compounds for inhibition of VanX
[13,14]. The current methods for assaying VanX activity, such as ninhydrin/Cd(II)-based
measurement and capillary electrophoresis (CE) [15,16], are noncontinuous and are limited
because of lack of chromophoric a substrate. The ninhydrin/Cd(II)-based assay is time-consuming,
while the disadvantages of capillary electrophoresis are poor reproducibility, narrow linear range
and low sensitivity [17]. In addition, both assays are performed in vitro without the complete
context of the physiological environment. Clearly, there is a general need for a straightforward
and feasible approach that enables real-time activity assay and inhibition of VanX.
Isothermal titration calorimetry (ITC) is an ideal approach to measure the change in thermal
power generated during enzyme catalyzed reactions. ITC is sensitive, has a fast response time,
does not require any labelling chemistry, and provides kinetic parameters (KM, kcat, Ki) as well as
thermodynamic information on the enzyme reaction [18,19]. The approach has been applied to
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assess the activity and binding mode of enzyme inhibitors [19-21]. The experimental basis of ITC
is to monitor the heat power produced or absorbed during a titration of one solution into
another [22]. Processing of the data requires a model accounting for the observations [22].Here,
we report an ITC method for continuous assay of activity and inhibition of VanX in vitro as well as
the VanX harbored in bacteria through using D-Ala-D-Ala as substrate.
2. Materials and methods.
2.1. Materials and reagents
The substrate, D-Ala-D-Ala dipeptide and D-cysteine were purchased from Sigma Chemical Co.
(St. Louis, MO, USA). The clinical bacterial strains Enterococcus faecalis and VRE were obtained
from Clinical Laboratory, Xijing Hospital, Air Force Medical University (Xi'an, China), and E. coli
BL21 (DE3) cells were purchased from Wolsen Co. Ltd (Xi'an, China). All other chemicals were
analytical grade.
2.2. Overexpression and purification of VanX
The overexpression and purification of VanX were carried out as previously reported [23]. The
BL21 (DE3) E. coli cells containing the maltose-binding protein-VanX (MBP-VanX) in pIADL14
plasmid was cultured overnight in 4 × 1 liter of lysogeny broth (LB) containing 25 μg/mL
kanamycin. The inocula were shaken at 30 °C until reaching an OD600 of 1.8, and then were
induced with 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 75 min, harvested by
centrifugation, and ruptured by ultrasound. The cell lysate was loaded onto an amylose affinity
column and eluted with 30 mM Tris, pH 7.6, containing 10 mM maltose and 200 mM NaCl. The
fractions containing MBP-VanX were identified by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE), and the pure protein fractions were concentrated. MBP was
removed from MBP-VanX by thrombin digestion, the cleaved protein solution was further
purified by a Q-Sepharose column eluted with a linear gradient of 0-500 mM NaCl in 30 mM Tris,
pH 8.0 to offer the desired VanX. The purity of VanX was analyzed by SDS-PAGE and the
concentration was determined using Beer’s law with the extinction coefficient of 51,200 M-1 cm-1
at 280 nm.
2.3. Ninhydrin/Cd(II) assays
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The activity of VanX and its inhibition studies by D-cysteine were quantitatively assayed with
D-Ala-D-Ala as substrate in 50 mM HEPES pH 8.0 at 37 °C as previously reported [12,15]. The
absorbance was detected at 505 nm (Agilent UV-Vis 8453 spectrophotometer) and quantified
with free amino acid as standard (Fig. S1). Kinetic experiments were carried out using 50 nM
VanX with 0.1-0.5 mM D-Ala-D-Ala as a substrate. Rates were also determined in the presence of
10-90 μM inhibitor by pre-incubating the enzyme with D-cysteine for 30 min at room
temperature before starting the kinetic experiments. The kinetic parameters were determined
using the SigmaPlot v.12.0 by the equation for competitive inhibition Eq. (1), where v is the rate
of catalysis, kcat is the catalytic constant, [E]tot is the enzyme concentration, [S] is the initial
substrate concentration, KM is the Michaelis constant, [I] is the inhibitor concentration, Ki
inhibition constant. The experiments were performed in triplicate using different enzyme
preparations and average values were reported.
ꢅ ꢇ ꢅ ꢇ
ꢁꢂꢃꢄ
ꢆ
ꢉ
ꢄꢈꢄ
ꢀ =
(1)
ꢅ ꢇ
ꢏ
ꢅ ꢇ
ꢉ ꢊꢋꢌꢍꢎꢊꢐꢑ
ꢒ
2.4. Determination of MIC
The minimum inhibitory concentration (MIC) of antibiotics against bacteria was determined
using the broth micro-dilution method in 96-well microliter plate format [24,25]. Single colonies
of VRE and Enterococcus faecalis in LB agar plates were transferred into 5 mL of Mueller-Hinton
(MH) liquid medium. Strains were grown in MH medium to OD600= 0.4 at 37 °C. The final
concentrations of bacteria were diluted to 5×105 by Mueller Hinton Broth (MHB) and then added
to different concentrations of Vancomycin ranging from 4 to 4096 µg/mL in a series of two fold
dilutions. The mixture was incubated at 37 °C for 16 h. The MIC results were taken as the lowest
concentration that completely inhibited visible growth. Each measurement was performed in
duplicate.
2.5. Calorimetric assays
2.5.1. Calorimetric assays with purified VanX
In a single injection experiment, the substrate solution was injected into the cell to cause a
continuous change in thermal power which eventually returned to baseline, representing full
conversion of the substrate into product within a certain period of time. For the experiment with
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purified VanX protein, 30 μL of substrate D-Ala-D-Ala (1, 2, 4, and 6 mM) in syringe was injected
into the sample cell filled with 210 μL of 1.3 μM purified VanX prepared in the same buffer. The
reference cell was loaded with deionized water and the experiments were set at 37 °C with a
stirring speed of 750 rpm. After collecting initial heat rate measurements for 1 min (establishing
a base line), the substrate solution was injected into the instrument sample cell. Heat rate
(microJoules/second) was recorded as a function of time. Data were collected every 1 s until the
signal returned to the baseline and the apparent enthalpy change (ΔHapp) of the reaction was
calculated by dividing the integrated heat (Qtotal) by the n moles of substrates converted
according to Eq. (2), where dQ/dt is the heat rate, Vinj is the volume of the injection, and [S]t=0 is
the substrate concentration.
ꢗ∞
ꢘꢙ
ꢘꢄ
ꢜꢝ
ꢡꢄꢈꢄꢃꢢ
ꢣ
ꢄꢚꢛ
ꢓꢔꢕꢖꢖ
=
=
(2)
ꢅ ꢇ
ꢉ
ꢞꢑꢟꢠ
ꢄꢚꢛ
The reaction rate (v) is estimated from the decay heat curve of the substrate using Eq. (3), and
instantaneous substrate concentrations at time t are calculated from Eq. (4):
ꢅ ꢇ
ꢎ
ꢜꢡ
ꢀ = ꢜ ꢖ
=
(3)
(4)
ꢜꢝ
ꢞꢂꢤꢢꢢꢥꢦꢃꢧꢧ ꢜꢝ
ꢗꢫꢘꢄ
ꢛ
ꢘꢙ
ꢅ ꢇ
ꢨ
= ꢅꢨꢇꢝꢩꢪ −
ꢝ
ꢞꢂꢤꢢꢢꢥꢦꢃꢧꢧ
where Vcell is the volume of the sample cell. Data were fit to the Michaelis-Menten equation (Eq.
5) using non-linear least squares regression to get the kinetic constants KM and kcat for enzymatic
hydrolysis of each substrate:
ꢅ
ꢇ
ꢅ ꢇ
ꢉ
ꢄꢈꢄ
ꢆ
ꢀ = ꢁ
(5)
ꢂꢃꢄ
ꢅ ꢇ
ꢋꢌꢊ ꢉ
The single injection experiments were also carried out in the presence of inhibitor by
pre-incubating VanX enzyme with 10-80 μM D-cysteine prepared in the same buffer, and the
inhibition constant of inhibitor were calculated by global fitting of the data to the general
inhibition equation (Eq. 1) using Origin software. All experiments were performed in triplicate
using different enzyme preparations and average values and standard deviations of ΔHapp, KM,
kcat, and Ki were determined.
2.5.2. Calorimetric assays with living bacterial cells
Substrate hydrolysis within bacterial cells was studied by monitoring the change in thermal
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power after the injection of 30 μL of 1-6 mM D-Ala-D-Ala in 30 mM Tris, pH 8.0 into the cell
loaded with 210 μL bacterial cell suspensions. The bacterial strains used in this study were
recombinant E. coli-VanX cells, E. coli BL21 (DE3) (negative control without VanX), Enterococcus
faecalis and positive clinical isolate VRE (producing VanX). All cells were grown on LB agar plates.
A single colony was picked from these plates and was incubated overnight in LB medium at 37 °C.
The cultures of recombinant E. coli-VanX cells and of clinical VRE were then centrifuged and
adjusted to an OD600 of 2.0 with 30 mM Tris, pH 8.0 before cell suspensions were loaded for ITC
studies. To determine the IC50 values of inhibitor against VanX using the cell-based ITC method,
we pre-incubated D-cysteine at various concentrations (0-320 µM) with E. coli cells expressing
VanX and VRE for 30 min and then placed the solution in the sample cell (210 µL). After collecting
initial heat rate measurements for 1 min (establishing a base line), 30 μL of 4 mM substrate
solution was injected into the sample cell and the heat rate was recorded every 1 s. In addition,
to verify the measured heat rate was only due to enzymatic hydrolysis of substrate, control
experiments were performed by injecting substrate into buffer, substrate into the same amount
of a reference strain and buffer into cell suspensions. All experiments were carried out at 37°C
with a stirring speed of 750 rpm.
3. Results and discussion
3.1. Overexpression and purification of VanX
VanX, a Zn(II) ion-containing dipeptidase, catalyzes hydrolysis of dipeptide D-Ala-D-Ala. As
previously reported, VanX was overexpressed successfully as MBP-VanX fusion protein in E. coli
BL21 (DE3) cells [23]. The MBP tag was removed by thrombin cleavage and the target protein
VanX was separated by Q-Sepharose anion exchange column. Fig. 1 shows the results of
overexpression and purification of VanX from E. coli. The obtained VanX protein had a purity of
95% (Lane 7).
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Fig. 1. SDS-PAGE of VanX purification. Lane 1: protein molecular weight marker. Lane 2: total cell lysate of E. coli
BL21 cells transformed with expression vector encoding the MBP-VanX fusion protein before IPTG induction. Lane
3: total cell lysate of E. coli BL21 cells transformed with expression vector encoding the MBP-VanX fusion protein
after 1mM IPTG induction. Lane 4: supernatant crude protein after crushing and centrifugation. Lane 5:
MBP-VanX protein eluted from amylose column. Lane 6: MBP-VanX protein cleaved by thrombin. Lane 7: purified
VanX protein eluted from Q-Sepharose column.
3.2. Activity assay of VanX by ITC
The hydrolysis of D-Ala-D-Ala by VanX (Scheme 1) involves a series of heat effects [26]. The
change of thermal power (dQ/dt) generated during the reaction was monitored by Malvern
MicroCal ITC200 in a multiple or single injection mode. The substrate is titrated into a solution of
enzyme in the sample cell, and the dQ/dt value is registered. Since dQ/dt is proportional to the
reaction rate, the analysis can provide the kinetic parameters (KM, kcat, Ki) [27,28].
ITC was employed to monitor the hydrolysis of D-Ala-D-Ala by VanX in a single injection
mode. Successive titration of 30 μL of 4 mM D-Ala-D-Ala into the sample cell filled with 1.3 μM
VanX and injection of substrate to buffer was performed as a control. The typical thermograms
are shown in Fig. 2A. A decrease of thermal power (dQ/dt) following injection of the substrate is
observed, indicating that the hydrolysis is an exothermic reaction. The thermal power generated
(dQ/dt) reached the maximum at ~140 s, and heat rate returned to baseline within 220 s,
suggesting that the substrate was hydrolysed completely. The power (dQ/dt) at each substrate
concentration, obtained by measuring baseline displacement, was converted to enzyme turnover
rates according to Eq. (3). The reaction rate data were then analyzed by the Origin program
provided by Malvern company and were further fitted to the Michaelis-Menten equation (Eq. 5)
using non-linear least squares regression (Fig. 2B) to give the kinetic constants KM (0.10 mM) and
kcat (11.5 s-1) (Table 1).
To examine the reliability of the ITC approach, the ninhydrin/Cd(II)-based assay was also
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employed to evaluate the substrate hydrolysis as previously reported [15]. The resulting KM and
kcat values (0.14 mM and 43.8 s-1) are in agreement with the data from ITC assay (considering the
difference in the two sets of assays and that different enzyme concentrations were used). KM and
kcat are also consistent with the data (0.11 mM and 46 s-1) reported by Anissimova et al. [17],
indicating that the ITC method is a viable approach for monitoring VanX activity.
Fig. 2. Enzyme kinetics of VanX catalyzed D-Ala-D-Ala hydrolysis using ITC assay in a single injection method. (A)
Raw thermal power observed in the calorimetric assay, performed at 37 °C with 30 μL injections of 4 mM
D-Ala-D-Ala in the syringe into 1.3 μM VanX (black) and buffer (red) in the sample cell. (B) Global fitting of VanX
enzymatic activity using data from the single injection ITC assay. Baseline displacement of the thermal power
after injection of the substrate was converted to reaction rate and was plotted according to the
Michaelis-Menten equation to give the values of KM and kcat.
3.3 Inhibition assay of VanX by ITC
To investigate whether the method could be used in inhibition assay of VanX, we assessed
D-cysteine, a previously reported inhibitor of VanX [13], in the single injection mode of ITC. The
inhibition of D-Ala-D-Ala hydrolysis with VanX by D-cysteine is shown in Fig. 3A. It clearly shows
that the compound inhibits VanX effectively, a high concentration of inhibitor leads to a low
initial hydrolysis rate of D-Ala-D-Ala. Through fitting the initial velocity versus substrate
concentration at each inhibitor concentration, we determined the Ki value of D-cysteine to be
18.8 μM, which is consistent with the value (16 μM) reported [13]. Also, using the
ninhydrin/Cd(II)-based assay (Fig. 3B), we determined the Ki value to be 23.2 μM, which is in
agreement with the data from the ITC assay. These results suggest that the ITC method could be
used for screening inhibitors of VanX.
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Fig. 3. Inhibition of D-Ala-D-Ala hydrolysis with VanX by D-cysteine. (A) Reaction rate of hydrolysis substrate at
different substrate concentrations in the absence and presence of D-cysteine calculated from the single injection
ITC assay. The data was fitted according to the Michaelis-Menten equation to obtain the value of Ki. The baseline
controls without VanX were subtracted from the experimental data to offer the overlaid heat flow curves. (B)
Michaelis-Menten plots to determine the kinetic parameters of D-cysteine using the ninhydrin/Cd(II)-based
method.
Table 1. The determined kinetic data of VanX hydrolyzing D-Ala-D-Ala and inhibition of VanX
by D-cysteine through ITC and Ninhydrin/Cd(II)-based method
Method
KM (mM)
0.10 ± 0.01
0.14 ± 0.03
kcat (s-1)
11.5 ± 0.3
43.8 ± 0.2
kcat/Km (s-1/mM-1)
115.0
Ki (μM)
18.8 ± 0.2
23.2 ± 0.3
ITC
Ninhydrin/Cd(II)
312.9
3.4 Inhibition assays of VanX in living bacterial cells
Given the enormous biomedical importance of VanX, it is necessary to develop a
straightforward and feasible approach to monitor small molecules such as drugs as inhibitors or
substrates of VanX in living cells. In a single injection mode of ITC, a typical heat rate curve of
VanX catalyzed hydrolysis of D-Ala-D-Ala is shown in Fig. S2. As the concentration of the substrate
decreased, the heat rate signal returns gradually to baseline, which indicates the end of the
reaction.
The single injection mode of ITC was employed to monitor hydrolysis of D-Ala-D-Ala by the
VanX enzyme and E. coli BL21 (DE3) that produce VanX, while the E. coli cells without VanX were
monitored as a control. The progress of D-Ala-D-Ala hydrolysis monitored by ITC is showed in Fig.
4. It clearly shows that the dipeptide at different concentration is quickly hydrolyzed when it is
treated with the purified VanX enzyme (Fig. 4A), and an increase of substrate concentration
results in an increase of dQ/dt values. Under the in vivo test conditions employed (6 mM
D-Ala-D-Ala and a suspension of E. coli cells with an optical density at 600 nm (OD600) of 2.0 in 30
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mM Tris-HCl, pH 8.0), D-Ala-D-Ala is quite stable, if the E. coli do not express VanX (Fig. 4B). The
weak thermal power (< 2.1 μJ/s) observed in response to various concentrations of D-Ala-D-Ala is
due to the heat of dilution and/or intrinsic bacteria metabolic heat [29,30]. However, the
D-Ala-D-Ala is hydrolyzed in the presence of E. coli producing VanX (Fig. 4C).
To test whether the VanX enzyme remained inside the cells or leaked into the medium during
the ITC experiment, the E. coli cells expressing VanX and treated with D-Ala-D-Ala were removed
by centrifugation and the resulting cell-free supernatant was supplemented with fresh dipeptide.
No significant thermal power caused by dipeptide hydrolysis was observed in the cell-free
supernatant (Fig. S3), indicating that VanX enzyme had not leaked from E. coli cells into the
medium, and confirming that the thermal power was contributed by VanX expressed in E.coli (Fig.
4C). The thermograms of substrate hydrolysis by the purified VanX enzyme and by E. coli
producing VanX are extremely similar, both reaching maximal hydrolysis rates at ~140 s. However,
the maximum thermal power reached during the conversion of 6 mM D-Ala-D-Ala with bacteria is
~18.8 μJ/s, which is significantly smaller than ~20.9 μJ/s for purified VanX. This may be due to the
different amount of VanX expressed in bacteria as compared to that used with purified enzyme.
To assess the bacteria metabolic heat and/or dilution heat, titration of 30 μL of D-Ala-D-Ala
(4 mM) into the VanX free E. coli cells and 30 μL Tris buffer into the E. coli cells with and without
VanX with different OD600 values, and the results (Fig.S4 and S5) show that the thermal power
changed very little from -0.2 to -1.0 µJ/sec with the increase of OD600 value from 0.5 to 6,
suggesting that the residual heat is mainly from the dilution of enzyme substrate or buffer during
the assays, because the metabolic heat derived from cell growth often took place over a much
longer period of time (hours to days) [31]. To further check if the viability of the E. coli cells
expressing VanX was affected by the ITC procedure, a plating colony test was carried out before
and after the ITC experiment. The results showed that ITC experiments for one hour did not lead
to any change in cell viability (Fig. S6).
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Fig. 4. Overlaid heat flow curves of D-Ala-D-Ala hydrolysis by purified VanX (A), E. coli BL21 cells (B) and E. coli
BL21 expressing VanX (C), (OD600 = 2). All samples were prepared in 30 mM Tris-HCl, pH 8.0. The heat rate during
hydrolysis of D-Ala-D-Ala (1-6 mM in syringe) was measured every 1 s.
To verify whether the cell-based ITC method could be applied to clinical strain assays, we
examined the degradation of D-Ala-D-Ala by two clinical isolates: VRE which expresses VanX, and
Enterococcus faecalis which does not express VanX. The progress of D-Ala-D-Ala hydrolysis
monitored by ITC is shown in Fig. 5. The substrate at different concentrations was quickly
hydrolysed by VRE, increased substrate concentration resulted in increased dQ/dt values, but not
by Enterococcus faecalis. Given the consistency of total heat (Qtotal) obtained by ITC and the
susceptibility of vancomycin [32], we determined the minimum inhibitory concentration (MIC) of
vancomycin against above two clinical isolates. The MIC values of VRE and E. faecalis are 512 and
4 μg/mL, respectively (Table 2), which are consistent with the Qtotal values (1.26 mJ for VRE and
0.031 mJ for E. faecalis at 4 mM), suggesting that the ITC method can be used to screen clinical
strains for VanX by monitoring the heat generated during the hydrolysis of substrate.
Fig. 5. Overlaid heat rate curves of D-Ala-D-Ala hydrolysis by VRE (A) and Enterococcus faecalis (B) (OD600 = 2). All
samples were prepared in 30 mM Tris-HCl, pH 8.0. The heat rate during hydrolysis of D-Ala-D-Ala (1-6 mM in
syringe) was measured every 1 s.
Table 2. MIC and total heat of vancomycin against clinical isolates VRE expressing VanX and
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Enterococcus faecalis
Bacteria
MIC (μg/mL)
Susceptibility
Qtotal (mJ)
1.26 ± 0.07
0.031 ± 0.002
VRE (VanX)
512
4
resistance
sensitive
Enterococcus faecalis
Further, the ITC approach was employed to assess the inhibition action of D-cysteine to E.
coli and VRE cells that express VanX. 30 μL of 4 mM D-Ala-D-Ala was injected into the sample cell
filled with 210 μL of pre-incubated inhibitor at different concentrations with VanX protein
solution at a concentration of 1.30 μM. The same experiments were performed with E. coli cells
expressing VanX or VRE at an OD600 of 2.0. To rule out the dilution effects, the control
experiments were performed with the test buffer lacking VanX, E. coli and Enterococcus faecalis
cells that do not express VanX. ITC data monitoring inhibition (corrected for the almost negligible
control data, Fig. S7A, S7B, and S7C) is shown in Fig. 6. The higher the inhibitor concentrations,
the lower the hydrolysis rates of substrate (Fig. 6A, 6B, and 6C). In the absence of inhibitor, the
(dQ/dt)max value corresponding to the rate of uninhibited enzyme-catalyzed D-Ala-D-Ala
hydrolysis was registered as 100% enzyme activity [33]. We determined the fifty percent
inhibitory concentration (IC50) values of D-cysteine to be 25.1, 29.8 and 28.6 μM, respectively (Fig.
6D, 6E and 6F), which are in agreement with Ki values (23.2) that we determined and the data (16
μM) reported from ninhydrin/Cd(II)-based assay in vitro [13], indicating that this approach could
be used for assaying inhibition of VanX in vitro and in vivo.
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Fig. 6. Overlaid heat rate curves of D-Ala-D-Ala hydrolysis by purified VanX enzyme (A), E. coli cells expressing
VanX (B) and VRE (C) in the presence of D-cysteine at various inhibitor concentrations, and IC50 measurements for
D-cysteine to purified VanX enzyme (D), E. coli cells expressing VanX (E) and VRE (F). The insets in A-C show that
total heat Qtotal calculated by integrating the different power curves obtained with inhibitor at various
concentrations is constant, as expected. The baseline controls without VanX expression (Fig. S6) were subtracted
from the experimental data to offer the overlaid heat flow curves. 30 μL of 4 mM substrate was injected to 210
μL of VanX solution at a concentration of 1.30 μM or E. coli cells expressing VanX or VRE at an OD600 of 2.0.
4. Conclusions
Based on the heat absorbed or released during the specific reaction of an enzyme with its
substrate, we report a simple and straightforward ITC method using D-Ala-D-Ala as substrate for
continuous real-time activity and inhibition assay of VanX enzyme, VanX harboured in E. coli cells,
and clinical isolate VRE expressing VanX. This method allowed the determination of kinetic
parameters of VanX hydrolyzing the dipeptide in vitro and determination of IC50 values of
D-cysteine inhibiting VanX harboured in clinical isolate. The cell-based ITC method proposed here
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could be applied to screen and evaluate small molecule inhibitors of VanX in whole cells or to
identify drug resistant bacteria.
Acknowledgments
This work was supported by the National Natural Science Foundation of China to K.W.Y (Grant
Number: 81361138018 and 21572179) and the Science Foundation of Shaanxi Academy of
Sciences (Grant Number: 2017K-08).
Abbreviations
ITC, isothermal titration calorimetry; VRSA, vancomycin-resistant Staphylococcus aureus; VRE
vancomycin-resistant Enterococci; MBP, maltose-binding protein; LB, lysogeny broth; E. coli,
Escherichia coli; IC50, fifty percent inhibitory concentrations; MIC, minimal inhibitory
concentration.
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Research highlights
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ò
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Continuous assay of VanX activity by isothermal titration calorimetry (ITC) is described.
The inhibition constant of VanX enzyme inhibitor, D-Ala-D-Ala, is given.
The method can be applied to evaluate the activity of VanX inside living bacterial.