Bacterial 2-component signal transduction systems: a fluorescence polarization screen for response regulator-protein binding.
ABSTRACT Two-component signal transduction systems are the primary means by which bacteria sense environmental change and integrate an adaptive response. In pathogenic bacteria, 2-component signal transduction (TCST) kinases are involved in the expression of virulence and antibiotic resistance. This makes bacterial TCST systems attractive targets for pharmacologic intervention. This paper describes a fluorescence polarization assay that quantifies the binding between bacterial DNA promoter segments and their cognate response regulator proteins. Using the Van RSTCST system from Enterococcus faecium, which encodes vancomycin resistance, the authors demonstrate inhibition of response regulator protein/promoter segment binding with a known inhibitor. Observed binding constants were comparable to those reported in surface plasmon resonance measurements and gel shift measurements.
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ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects approximately 2 billion people worldwide and is the leading cause of mortality due to infectious disease. Current TB therapy involves a regimen of four antibiotics taken over a six month period. Patient compliance, cost of drugs and increasing incidence of drug resistant M. tuberculosis strains have added urgency to the development of novel TB therapies. Eradication of TB is affected by the ability of the bacterium to survive up to decades in a dormant state primarily in hypoxic granulomas in the lung and to cause recurrent infections. The availability of M. tuberculosis genome-wide DNA microarrays has lead to the publication of several gene expression studies under simulated dormancy conditions. However, no single model best replicates the conditions of human pathogenicity. In order to identify novel TB drug targets, we performed a meta-analysis of multiple published datasets from gene expression DNA microarray experiments that modeled infection leading to and including the dormant state, along with data from genome-wide insertional mutagenesis that examined gene essentiality. Based on the analysis of these data sets following normalization, several genome wide trends were identified and used to guide the selection of targets for therapeutic development. The trends included the significant up-regulation of genes controlled by devR, down-regulation of protein and ATP synthesis, and the adaptation of two-carbon metabolism to the hypoxic and nutrient limited environment of the granuloma. Promising targets for drug discovery were several regulatory elements (devR/devS, relA, mprAB), enzymes involved in redox balance and respiration, sulfur transport and fixation, pantothenate, isoprene, and NAD biosynthesis. The advantages and liabilities of each target are discussed in the context of enzymology, bacterial pathways, target tractability, and drug development. Based on our bioinformatics analysis and additional discussion of in-depth biological rationale, several novel anti-TB targets have been proposed as potential opportunities to improve present therapeutic treatments for this disease.BMC Infectious Diseases 02/2007; 7:84. · 3.03 Impact Factor
Journal of Biomolecular Screening
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2005 10: 270J Biomol Screen
Matthew G. Erickson, Andrew T. Ulijasz and Bernard Weisblum
Bacterial 2-Component Signal Transduction Systems: A Fluorescence Polarization Screen for Response
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10.1177/1087057104273930 Erickson et al.Bacterial 2-Component Signal Transduction Systems
Bacterial 2-Component Signal Transduction Systems:
A Fluorescence Polarization Screen for
Response Regulator-Protein Binding
MATTHEW G. ERICKSON, ANDREW T. ULIJASZ, and BERNARD WEISBLUM
grate an adaptive response. In pathogenic bacteria, 2-component signal transduction (TCST) kinases are involved in the ex-
vention.Thispaperdescribesafluorescencepolarization assay thatquantifiesthebinding between bacterialDNApromoter
segments and their cognate response regulator proteins. Using the VanRSTCST system from Enterococcus faecium, which
encodesvancomycin resistance,theauthorsdemonstrateinhibition ofresponseregulatorprotein/promotersegmentbinding
with a known inhibitor. Observed binding constants were comparable to those reported in surface plasmon resonance
measurements and gel shift measurements. (Journal of Biomolecular Screening 2005:270-274)
Key words: fluorescence polarization, response regulator, bacterial signal transduction systems, antibiotic resistance
by Hoch and Silhavy,1Stock et al.,2and West and Stock.3
Genetic studies have shown that several TCST systems are es-
sential for virulence or infectivity, suggesting that TCST systems
may be suitable targets for screening compounds with anti-infec-
tive activity. Examples include 1) Streptococcus pneumoniae
CiaRH and RR489, which control cephalosporin resistance and
lung infectivity, respectively4,5; 2) Mycobacterium tuberculosis
CesRK, which regulates tolerance to β-lactam antibiotics7; and 4)
Enterococcus faecium VanRS, which specifies inducible
Thus far, only TCST sensor kinases have been tested as possi-
WO-COMPONENT SIGNAL TRANSDUCTION (TCST) systems
mediate adaptive changes in bacteria. The key participants
anti-infective leads.10-12TCST kinases may be less suitable targets
than their cognate response regulators owing to phosphorylation
by a promiscuous kinase from another system or to nonspecific
phosphorylation from small phosphoryl group donors in the
Conservation of genetic sequences is another issue; the N ter-
minal signal domain of sensor kinases from a given TCST family
domain displays considerable variation. In contrast, the region of
the cognate response regulator that interacts with the phopshoryl
pharmacological intervention aimed at response regulators would
be more difficult to circumvent.
Previously, we used VanRS as a test system to characterize re-
sponse regulator activity and its inhibition based on mobility shift
drug discovery. FP assays are solution based and homogeneous,
desirable attributes for a high-throughput assay.
32P labeled promoter DNA fragments.14In this
When a fluorescent ligand is excited with polarized light, it
emits fluorescence with polarization inversely proportional to its
270www.sbsonline.org © 2005 The Society for Biomolecular Screening
Pharmacology Department, University of Wisconsin Medical School, Madison
Received Jan 23, 2004, and in revised form Sep 23, 2004. Accepted for publica-
tion Oct 29, 2004.
Journal of Biomolecular Screening 10(3); 2005
molecular rotation. When the ligand binds to a target, molecular
rotation decreases and polarization increases. This suggests that
the best results in an FP assay will be realized when the labeled
ligand (probe) is small with respect to the target. Nonetheless, ac-
ceptable results may be obtained when the two are of comparable
In this work, we chose a fluorescein-labeled DNA promoter
segment as probe molecule, whereas a relatively large response
regulator protein served as the target. Use of the smaller promoter
segment as a probe instead of the entire promoter sequence re-
the earlier surface plasmon resonance (SPR) study.15Previously,
a bacterial repressor protein, the tryptophan repressor, with a la-
beled oligonucleotide containing one of its operator sequences.
promoter interactions involved in the regulation of antimicrobial
assay, 5′ double-stranded 18 mer DNA promoter segments were
used. Selective labeling of the 5′ base is readily accomplished on
the DNA synthesizer; oligonucleotides labeled in this fashion are
sufficiently pure for FP “off the column.”
Although we report an assay based on DNA-protein interac-
tion, some caveats applicable to TCST FP assays using labeled
peptides or proteins should be mentioned. The probe molecule
should be labeled at only 1 site if reliable results are expected. In-
variably, the labeling reagents used on peptides and proteins pro-
trolled somewhat by adjusting the relative concentration of
labeling reagent and substrate. A postlabeling purification step is
or thin layer chromatography. The last method is effective for
many peptides and offers both economy and simplicity.17
to use high-purity solvents and reagents: Impurities with fluores-
cence emission falling inside the passband of the instrument’s de-
tector can give spurious results. Any suspect reagents should be
checked against a blank charged with high-purity distilled and
deionized water. Total fluorescence should be comparable if the
reagent is sufficiently pure.
est change in polarization will be seen when the percentage of
bound probe changes from a few percent or less to greater than
realized by fixing the total probe concentration at 0.1 Kdor less
while varying the concentration of receptor from ≤0.05 Kdto ≈10
to 50 Kd.17In most cases, the probe condition will be satisfied by
probes will give a good signal-to-noise ratio on the Wallac instru-
ment at a concentration of about 12 to 15 nM. If an appreciably
efficiency may be required. Fluorescein is a popular choice be-
cause it offers acceptable performance in most situations at low
used in both apreviously reported gelshiftmobility study and the
previously reported SPR study.15
MATERIALS AND METHODS
5′ fluorescein tagged 18-mer oligonucleotide promoter seg-
ments based on the vanH promoter studies of Holman et al. were
used as probe-fluorophores in all FP experiments.8This choice
was made because site-specific dye tagging of oligonucleotides is
S-transferase (GST) fusion protein. The following
oligonucleotides were synthesized at the University of Wisconsin
Biotechnology Center using an ABI DNA synthesizer.
H2 Forward: 5′Z-TTT TCT TAG GAA ATT AAC
H2 Reverse: 5′ GTT AAT TTC CTA AGA AAA
Z = Fluorescein
After synthesis, each strand was dissolved in deionized water to a
concentration of 8 µMand stored at–20 °C. Prior to experiments,
oligos were annealed by mixing equimolar amounts of forward
and reverse strands and heating in a 80 °C bath for 10 min, fol-
lowed by slow cooling to room temperature.
Response regulator protein
The VanR response regulator protein was expressed as a GST
pGEX-2TK (Amersham Pharmacia Biotech) in Escherichia coli
tein was stored at –60 °C prior to use.
(2,3,4)-trifluorphenylisothiazolone (Maybridge Chemical
Company) was stored at –20 °C at a concentration of 1.0 mM. It
was used without further purification. This compound was re-
hibitors of signal transduction that would target alginate
Bacterial 2-Component Signal Transduction Systems
Journal of Biomolecular Screening 10(3); 2005www.sbsonline.org 271
Asp phosphorelay systems.18
In a previous study, this lab showed that compound A inhibits
VanR to accept phosphoryl groups rather than by interfering with
the ability of VanS to donate phosphoryl groups.19
AllFP experiments wereperformed on aWallacVictor Instru-
sion wavelength was 515 nM. All experiments were run at room
temperature, which was about 22 °C at our facility.
nM fluorescein-labeled PvanH2 and increasing amounts of GST-
VanR in 1X gel shift buffer (GSB), as indicated on the abscissa.
GSB consists of 20 mM HEPES, 5 mM MgCl2, 0.1 mM EDTA,
384-well microplates (Corning) were used in all experiments.
First, thewellswerecharged with 30 µLof 1X GSB.Next, 45 µL
of VanR-GST was added to the 1st well and mixed with the 1X
lution to 1X GSB buffer in a plastic trough and mixing by gentle
side-to-side agitation. After mixing, 10 µL oligo solution was
added to each well with a multichannel pipette. Mixing was
achieved by multiple cycles of up and down pipetting. Each plate
1X GSB was used for correction of raw-intensity data.
Experiments using compound A or VanScwere carried out by
adding a solution containing the required amount of inhibitor and
the required amount of oligos to each well with a multichannel
ular components of fluorescence from an 8 nM solution of
fluorescein in ddH2O. For these measurements, a 45-µL well of
ddH2O was used as a blank.
measuring Kiwas generated by running the experiment with dif-
ferent concentrations of compound A, all other parameters being
RESULTS AND DISCUSSION
by binding to multiple promoter binding regions.8,9,20When the
have a markedly lower affinity for the modified cell wall.
Compound A has been shown to inhibit phosphoryl transfer
from VanS~P to VanR by its action on VanR.19As a consequence
of inhibition by compound A, VanS~P accumulates, suggesting
that Asp-55 on the amino terminal regulatory subunit of VanR is
unable to accept phosphate from VanS.
ity retardation studies, suggesting a wider scope of action involv-
tory regions of vanRS and vanHAX contain promoter-binding
regions designated R1, H1, and H2 as shown in Figure 1. A dou-
Erickson et al.
272www.sbsonline.orgJournal of Biomolecular Screening 10(3); 2005
in these studies was taken from the H2 region shown. Coordinates num-
bered upstream with respect to vanRS and vanHAX transcription start
VanR promoter.Expression of vanHAX iscontrolled by bind-
PvanH2complexformation: effect ofCpdA.FPwasusedtomeasure the
complexes formed between fluorescein-labeled PvanH DNA and GST-
VanR. The reaction mixtures contained 13 nM 5′ fluorescein-labeled
Fluorescence polarization (FP) analysis of GST-VanR-
the target sequence for VanR promoter-binding studies.
As shown in Figure 2, compound A inhibits the binding of
This result is comparable to promoter-response regulator interac-
terial TCST. It is also comparable to the value of 40 nM reported
a previously reported gel shift measurement14and to the value of
each of the 3 promoter segments in the previously reported SPR
entKdto 35 nM, 150 nM, and 500 nM. Thiscompareswith the4-
to 5-fold increase reported for the apparent Kdfor the GST-
The structure of compound A is shown in the figure insert.
ited by VanSc, the soluble (cytosolic) domain of the cognate sen-
sor-kinase protein, VanS. The inhibition of the GST-VanR~P and
PvanH by synthetic peptide analogues of VanScwas previously
studied using gel shift measurements. Here, we demonstrate the
of the more laborious gel shift method. As mentioned previously,
80 nM, 150 nM, and 320 nM.
suitable for high-throughput screening of compounds capable of
inhibiting theformation of VanR–PvanH complexes;extension to
forward. In addition, the assay provides for quantification of test
achieved. Because FP uses fluorescence labeling, the use of radio
isotopes is avoided.
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Bacterial 2-Component Signal Transduction Systems
Journal of Biomolecular Screening 10(3); 2005www.sbsonline.org 273
PvanH2 complex formation: inhibition by VanSc, the soluble domain of
instead of gel shift measurements to screen and measure inhibition of re-
sponse regulator–DNA domain interactions by synthetic peptide ana-
values for each concentration of VanSc.
Fluorescence polarization (FP) analysis of GST-VanR-