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Introduction
Methicillin-resistant Staphylococcus
aureus (MRSA) remains a major cause of nosocomial disease in the world,
causing 50% or more of hospital-acquired
S aureus infections in several
countries[1]. In addition, recent reports have
indicated that the epidemiology of MRSA may be
undergoing a change through the emergence of community-acquired
MRSA (CA-MRSA)[2,3]. CA-MRSA is capable of causing
infections in otherwise healthy people and may have a
serious or even fatal outcome[4,5]. According to the World Health
Organization, more than 60% of S aureus strains worldwide
are now resistant to methicillin. The mortality from severe
MRSA infection is reported to be as high as
10%_34%[6,7].
Infections caused by MRSA are not effectively treated
by most antibacterial agents and are a major challenge for
chemotherapy because these bacteria show resistance to all
β-lactam antibiotics. MRSA has traditionally been treated
with glycopeptides, such as vancomycin. However, the
emergence of vancomycin-intermediate or -resistant
Staphylococci has spurred renewed efforts in the discovery of new
targets in MRSA for novel antibacterial agents with
mechanisms radically different from existing
compounds[8].
S aureus resist the attack by β-lactam antibiotics in 2
ways: by producing β-lactamase, which inactivates the
β-lactam antibiotics, and by expressing new transpeptidases,
which are impervious to antibiotic activity. Two genes,
blaZ encoding β-lactamase and mecA encoding the
penicillin-binding protein PBP2a, render S
aureus resistant to antibiotics,
respectively[9]. The transcription of these genes is
regulated by transmembrane signal sensor/transducer proteins
(BlaR1 and MecR1) and their partner repressor proteins (BlaI
and MecI). When a β-lactam antibiotic binds to the
extracellular sensor domain of BlaR1, a conformational change within
BlaR1 leads to autocatalytic activation of the
integral-membrane metalloprotease domain. The active protease facing
the cytosol specifically cleaves BlaI, directly or indirectly,
which subsequently initiates blaZ expression. The genes
for signal transducers and repressors are contained in a gene
operon (blaR1-blaI) that is divergently transcribed from its
regulated gene blaZ[10]. This system highlights the key role
of the methicillin sensor-transducer as the eventual
transcriptional regulator of MRSA
response[11,12]. Therefore, blocking the sensor/transducer pathway may represent a
novel approach to reverse antibiotic resistance in MRSA.
In recent years, catalytic nucleic acids were composed
entirely of DNA, which have been generated by in
vitro selection strategies. These molecules ideally combine the
catalytic activity of ribozymes with the stability of
oligo-deoxynucleotides. The typical deoxyribozyme or DNAzyme,
known as the "10_23" model, is
capable of cleaving a specific phosphodiester linkage between an unpaired purine (A,
G) and a paired pyrimidine (C, U) under
simulated physiological conditions. DNAzymes have considerable
advantages over ribozymes in that they are easier to synthesize
and less sensitive to chemical and enzymatic degradation
than RNA-based reagents. They also exhibit greater
catalytic efficiency than conventional hairpin and hammerhead
ribozymes[13,14], so it is considered as an easily "drugable"
tool to knock down target genes. In this study, we explored
the use of a synthetic deoxyribozyme targeting
blaR1 transcripts as a potential tool in inhibiting the expression of
blaR1, blocking the signal pathway of
blaR1-blaI-blaZ and thereafter leading to the reduction of BlaZ expression. The
PS-DRz602 (anti-blaR1 phosphorothioate deoxyribozyme) was
found to reduce β-lactamase mRNA expression concomitantly
and led to the restoration of the susceptibility of MRSA to
oxacillin.
Materials and methods
Bacterial strain and electrocompetent S
aureus preparation The strain of MRSA, WHO-2, obtained from Chinese
National Center for Surveillance of Antimicrobial Resistance
(Beijing, China) was used in the study. WHO-2 exhibited a
moderate level of resistance to oxacillin (minimum inhibitory
concentration [MIC]=32 μg/mL), in which the
mecR1-mecA[15] and
blaR1-blaZ[16] genes were detected by PCR. A
methicillin-susceptible S aureus strain, ATCC (American Type
Culture Collection) 29213, was used as a positive control.
Competent S aureus was prepared according to a
previously published paper[17]. Briefly, 2 mL of
overnight-cultured WHO-2 was transferred to 200 mL broth medium and
incubated at 37 oC with moderate agitation until the optical
density (OD)600 reached 0.55_0.65. The cells were
centrifuged at 2817×g for 10 min at
4 oC and the supernatant was removed. The pellet was washed by resuspending in
ice-cold deionized water in the same volume of culture medium
and the suspension was centrifuged. The supernatant was
carefully removed and the pellet was washed a second time
using the same procedure. The pellet was then washed 4
times with 40, 10, 2, and 1 mL of 10% cold glycerol,
respec-tively, with the procedure mentioned above. Finally, the cell
pellet was resuspended in 1 mL of cold 10% glycerol and 50
μL aliquots and stored at -80 oC.
Modified DNAzyme The sequence of most active
DNAzyme in this study was PS-DRz602:
5'-GCTTGA-GTTGAGGGCTAGCTACAACGACGCAGTA
-3', which is complementary to the sequence of nt 1378_1359 of
blaR1 in S aureus; the internucleoside linkages in 2 arms of DNAzyme
were phosphorothioated to increase nuclease resistance
(modified bases are underlined). PS-DRz341 (control
mismatched sequence) had been randomly aligned with the same
number of bases as 5'-CAGTATGCATGCACGCTTG
TAA-CCGTAAGTACGC-3', in which the underlined bases were
also phosphorothioated. PS-DRz602 and PS-DRz341 were
synthesized by Shanghai Sangon Biological Engineering
Technology and Services (Shanghai, China).
DNAzyme delivery Different concentrations of
phos-phorothioated PS-DRz602 (5, 10, and 15 mg/L) were
introduced into the S aureus strain WHO-2 by the Electroporator
(JY2000-1B electroporation apparatus, Ningbo Scientz
Biotechnology, Ningbo, China) at conditions of 25 µf, 900 V,
200 Ω, and time constant 3.6_4.2 ms. Briefly, the cells of
bacteria and PS-DRz602 were mixed and transferred into
prechilled cuvettes. After the pulses were applied, the
cuvettes were removed and 1 mL of SOC (Super Optimal
Catabolite) medium (0.5% yeast extract, 2% tryptone, 10
mmol/L NaCl, 2.5 mmol/L KCl, 10 mmol/L
MgCl2, 20 mmol/L MgSO4, and 20 mmol/L glucose [pH 7.0]) was added immediately.
The cells were allowed to recover by incubating for 1 h at
37 oC with shaking. Transformation efficiencies should be
approximately 9×108 transformants/µg of DNAzyme.
Bacterial susceptibility assay The growth
determination of the cells receiving different concentrations of
PS-DRz602 by electroporation was carried out as follows: the
cells were diluted 106 times and 50 µL dilution was then spread
onto the Mueller-Hinton agar that contained 6 mg/L oxacillin;
the plates were incubated for 48 h at 35
oC. The number of colonies were counted for plates with >10 and <500 colonies,
and the total colony-forming unit (CFU) per sample was
determined by correcting the colony count for the dilution.
The MIC of oxacillin for PS-DRz602-treated MRSA and
MSSA (methicillin sensitive S aureus) were determined by
the 2-fold microdilution method with the Mueller-Hinton
broth supplemented with oxacillin in the range of 0.25_128
µg/mL. 100 µL of 5×105 CFU/mL test bacteria was added to
96-well microtitre plates and grown at 35 °C for 20 h. 10 µL of
1% triphenyl tetrazolium chloride (TTC), a colorimetric
indicator, was added to each well of the microtitre plates and
incubated for 1.5 h at 35 °C. The TTC-based MIC was
determined as being the lowest concentration of oxacillin that
showed no red color changes and indicated the complete
growth inhibition.
RNA extraction The culture of the S aureus
strain WHO-2 was centrifuged at
1957×g for 10 min at 4 oC and the
supernatant was decanted. The cell pellet was suspended in
100 µL lysis solution (50 mmol/L Tris-HCl [pH 7.5], 5 mmol/L
EDTA [pH 8], and 50 mmol/L NaCl) with 300 µg lysozyme
(Sigma-Aldrich, St Louis, MO, USA) and 5 µg lysostaphin
(Sigma-Aldrich, USA) for 30 min at room temperature. The
total RNA was extracted from the bacterial lysis with Trizol
reagent (Invitrogen, Carlsbad, CA, USA) following the
manufacture's instructions, and the RNA samples were
treated with DNase I to remove any genomic DNA
contamina-tion.
Reverse transcription reaction The cDNA of
blaR1 and blaZ was synthesized, respectively, by reverse
transcription (RT) from 1 µg of each RNA sample using SuperScriptIII
reverse transcriptase (Invitrogen, USA). The 14 µL mixture
(1 µg RNA, 0.1 µg random primer, 4 µL of 2.5 mmol/L dNTP
mix, and sterile water) was heated at 65 °C for 5 min and
incubated on ice for at least 1 min. 1 µL of 0.1 mol/L DTT
(dithio-threitol), 4 µL of 5× RT buffer, 20 U RNase inhibitor,
and 100 U SuperScriptIII reverse transcriptase was added to
the mixture in a final volume of 20 µL. The RT conditions
were 5 min at 25 °C, 45 min at 50 °C, and 15 min at 70 °C.
Real-time PCR detection The nucleotide sequences for
the various primers are listed in Table 1. All the primers were
synthesized commercially (Shanghai Sangon Biological
Engineering Technology and Services, China).
The PCR was run in a DNA Engine Opticon (MJ Research,
Waltham, MA, USA) with SYBR Green I. The PCR reagents
consisted of: 12.5 µL SYBR Premix Ex Taq (DRR041S, TaKaRa,
Otsu, Shiga, Japan), 0.5 µL of 50×ROX reference dye
(DRR041S, TaKaRa, Japan), 0.75 µL of each primer (10
µmol/L), and 1 µL of sample cDNA in a final volume of 25 µL. Each
plate included its own negative controls: no template
controls (where all the reaction reagents except for cDNA were
used). The thermal cycling conditions were: an initial
denaturation step at 95 °C for 5 min, then 50 cycles at 95 °C for
10 s, 55 °C for 20 s, and 72 °C for 20 s. The melting curves of
the PCR products were acquired by the stepwise increase of
the temperature from 60 to 90 °C (temperature transition
0.5 °C/s). The standard deviation of the fluorescence values
recorded from cycles 3 to 15 was multiplied by 10 to define
the cycle threshold line. The specificity of the amplified
products was verified by analysis of the dissociation curves
as well as by ethidium bromide-stained 1% agarose gels.
The DNA Engine Opticon system detects and plots the
increase of each PCR product in fluorescence versus the
PCR cycle number to produce a continuous measurement of
PCR amplification. To provide the precise quantification of
the initial target in each PCR reaction, the amplification plot
was examined at a point during the early log phase of
product accumulation. This was accomplished by assigning a
fluorescence threshold above the background and
determining the time-point at which each sample's amplification plot
reached the threshold (defined as the threshold cycle
number or Ct). Differences in the threshold cycle number were
used to quantify the relative amount of PCR target contained
within each tube.
Construction of standard curves for
blaR1, blaZ, and 16SrRNA
genes The cDNA of control group was 10 fold series diluted and the analysis was performed as follows:
For each sample, a difference in
Ct values (ΔCt ) was
calculated for target genes (blaR1/blaZ) by taking the mean
Ct of duplicate tubes and subtracting the mean
Ct of the duplicate tubes for the reference RNA
(16SrRNA) measured on an aliquot from the same RT reaction.
ΔCt =Ct (target
gene)_Ct
(16SrRNA).
Comparative calculation and determination of the
relative expression levels of blaR1 and blaZ
in the differently treated groups The relative expression of
blaR1 or blaZ mRNA was calculated using the comparative
Ct method. Real-time relative quantitations of
blaR1 and blaZ expressions were performed as previously
described[18]. The 16SrRNA gene, which was expressed at relatively the same
level throughout the developmental cycle in WHO-2, was
used as the control to normalize the quantity of a cDNA
target to determine differences in the amount of total cDNA
in a reaction[18,19]. The
ΔΔCt values were calculated as the
following equation: ΔΔCt
=ΔCt
(treatment)_ΔCt (control).
The ΔCt for the treated sample was then subtracted from the
ΔCt for the untreated control sample to generate
ΔΔCt .
The mean of these ΔΔCt measurements was then used to
calculate the expression of the test gene
(2-ΔΔCt ) relative to the reference gene and normalized to the untreated control
as follows: Relative
expression=2-ΔΔCt. The evaluation of
2-ΔΔCt indicates the fold change in gene expression relative to
the untreated control.
Statistical analysis Values are expressed as mean±SD
and one-way ANOVA analysis followed by SNK
(Student-Newman-Keuls) t-test was performed.
P<0.05 was considered statistically significant.
Results
Effects of PS-DRz602 on colony forming of the WHO-2
strain The number of WHO-2 colonies on the Mueller-Hinton
agar containing oxacillin (6 mg/L) was significantly decreased
to 78.2%, 56.7%, and 37.8% of the control value in all
anti-BlaR1 PS-DRz602-treated groups concentration-dependently.
However, the growth of WHO-2 was neither influenced in
the mismatched PS-DRz341-treated group nor affected in the
control group without PS-DRz602 treatment (Figure 1).
Partial restoration of antibiotic susceptibility in the
MRSA strain WHO-2 We found that the down-regulation
of blaR1 by the introduction of
anti-blaR1 PS-DRz602 can partially increase the susceptibility of WHO-2 to oxacillin
(Table 2). The MIC of oxacillin was reduced from 32 to 8
µg/mL in the presence of 5, 10, and 15 µg/mL
anti-blaR1
PS-DRz. In contrast, the MIC of oxacillin for mismatched
PS-DRz341 not targeting blaR1 remained unchang-ed.
Competence or electroporation alone did not affect the MIC of
oxacillin on WHO-2 (Table 2).
Real-time quantitation assays for
blaR1 and blaZ transcription We next determined whether the conversion of
antibiotic resistance to antibiotic susceptibility in MRSA
strain WHO-2 was accompanied by the inhibition of
blaR1 and blaZ mRNA expression through
anti-blaR1 PS-DRz602.
An analysis of the melting curves of the PCR products
for blaR1, blaZ, or 16SrRNA showed a single-peak graph for
all amplifications, indicating that a single PCR product was
formed. This was confirmed by running 10 µL of each
product on an ethidium bromide-stained 1% agarose gel.
The standard curves for blaR1 or
blaZ and 16SrRNA were generated using cDNA from WHO-2 (Figures 2, 3). To
demonstrate that the PCR efficiencies for the target and the
control genes were approximately equal, the values of the 2
standard curves were used to determine the absolute value
of the slope of the log of cDNA for each dilution versus
ΔCt (difference in the cycle threshold obtained for the 2 PCR
systems for the same cDNA dilution) for the respective
dilution. This validation experiment involved pairwise
comparisons between blaR1 and
16SrRNA or blaZ and 16SsRNA.
The slope was 0.012 and 0.010 for each experiment,
respec-tively, indicating approximately equal amplification
efficiencies between blaR1 or blaZ and
16SrRNA (Figures 2, 3).
Comparative transcription of a single gene for
blaR1 and blaZ The change in the transcription of the target genes
(blaR1 and blaZ) normalized to
16SrRNA was monitored in the different concentration groups. As expected, the
transcriptional change of blaR1 in the differently treated groups
was coincidental to that of blaZ. Compared with
the control group, the relative transcription of
blaR1 in 3 groups (5, 10, 15 mg/L
PS-DRz602) was decreased to 81%, 51%, and 31% of
the control values, respectively, in a
concentration-dependent manner (Table 3). Interestingly, the blockade of the
transcription of blaR1, a sensor transducer gene, led to the
concentration-dependent repression of its downstream gene
blaZ to 82%, 57%, and 41% of the control values,
respectively(Table 4).
Discussion
Because of the emergence and spread of resistance
genes[9_12,20], MRSA has been the cause of major outbreaks
and epidemics among hospitalized patients, with high
mortality and morbidity rates. Recently, some schemes have
shown both a dramatic rise in the total numbers of cases of
S aureus bacteremia reported annually and an increase in the
proportion of such cases that involve MRSA (from 2% in
1990 to >40% in the early 2000s)[21]. However, the
emergence of vancomycin-resistant MRSA and treatment failure
of MRSA infections has led to the urgent need for
alternative anti-MRSA therapies.
The different antisense approaches have demonstrated
the feasibility of using antisense oligonucleotides or
oligonucleotide analogs in the treatment of bacterial infections,
and the promising results have been observed by
researchers in vitro and in
vivo[22,23]. Sarno et al demonstrated that
selectively-designed antisense oligonucleotides could bind
to AAC(6')-I-type acetyltransferase (aminoglycoside
6'-N-acetyltransferase type Ib) mRNA, mediate RNase H digestion,
and thereafter decrease the level of resistance to amikacin in
Escherichia coli[24]. The application of peptide-PNA (peptide
nucleic acid) conjugates, antisense phosphorodiamidate
morpholino oligomer, or antisense DNA analogs showed a
significant inhibition of gene expression in E
Coli, respec-tively, and effectively reversed the multiple drug resistance
of E coli[25_27]. When PS-ODN (phosphorothioate
oligo-deoxynucleotide), targeting a specific region of the
mycobacterium aspartokinase (ask) gene in
Mycobacterium smegmatis were utilized in combination with ethambutol, the
results showed a significant inhibition of growth of
drug-resistant strains of M
smegmatis[28]. The inhibition of glutamine synthetase activity with antisense
oligonucleotides to glutamine synthetase mRNA also held back the
replication of Mycobacterium
tuberculosis[29]. Resistance to β-lactam was reversed by the blockade of the expression
of bla in the presence of antisense peptide nucleic acid in
E coli[30] and resistance to vancomycin was fully reversed
when an Enterococcus faecalis isolator harbored
recombinant shuttle vectors containing a
vanH promoter-vanA antisense gene
cassette[31]. The peptide nucleic acid
treatment was effective in rescuing 100% of infected
animals[23]. Antisense antibiotics have been proposed as a new hope
for bacteria infection therapy through targeting specific genes
in bacteria[32].
Our previous study indicated that a phosphorothioate
oligodeoxynucleotide ODN6087 could inhibit
mecR1 and mecA expression and partially restore the susceptibility of a
MRSA strain[33]. The current study demonstrated that
PS-DRz602 targeted to the sequence of blaR1 mRNA
significantly inhibited the resistance of MRSA strain WHO-2 to
oxacillin, together with the reduction of
blaZ mRNA expression. The colony forming of WHO-2 was decreased
to 78.2% (5 mg/L), 56.7% (10 mg/L), and 37.8% (15 mg/L) of
the control values, respectively, in a
concentration-dependent manner after PS-DRz602 treatment. However,
anti-blaR1 PS-DRz602 had limited activity to reverse the susceptibility
of MRSA strain WHO-2 in the present study. The reason for
the insufficient restoration of susceptibility is that we
delivered the PS-DRz to competent S aureus only once by
electro-poration. Although the delivery efficiency was estimated at
around 9×108 transformants/micrograms in all groups,
PS-DRz was not retained in the culture medium constantly and
bacterial proliferation diluted the PS-DRz. As demonstrated,
the MIC of oxacillin to WHO-2 were reduced from 32 to 8
µg/mL, which was still 2-folds higher than the margin value (2
µg/mL) of oxacillin sensitivity to S
aureus and the inhibitory efficiency of the
anti-blaR1 PS-DRz602 at 78.2%_37.8% of control values. However, if the highly-efficient delivery
system is applied and the antisense PS-DRz is sustainable
throughout proliferation, then a high efficiency of
anti-blaR1 PS-DRz will be achieved.
Since BlaR1 is an upstream regulatory element for the
initiation of BlaZ expression via the inactivation of BlaI, it
was found that BlaZ expression was consequently greatly
inhibited after the blockade of BlaR1. Although there is no
direct evidence to prove the cleaving activity of PS-DRz602
to the mRNA substrates of BlaR1 precisely in WHO-2, the
mismatched anti-blaR1 PS-DRz341 had no effects on the
growing of WHO-2, as well as no influence on the expression of
BlaR1, and β-lactam mRNA would provide indirect evidence
to support the selectivity of PS-DRz602. In addition, in the
cytoplasm of cells, a full length of mRNA often has a
secondary structure. If the target sites of DNAzymes are within
the secondary structure of the RNA, DNAzymes may still be
ineffective[34,35]. Some studies have shown that DNAzymes
work well in the in vitro system, but do not work in the whole
cell[35,36]. Mitchell et al and Patzel
et al found that without any prior screening, the initially synthesized DNAzyme
sequence specifically reduced the target gene
expression[34,37].
In conclusion, the results of our present study suggested
that the blockade of the blaR1-blaZ signaling pathway via a
DNAzyme was an alternative strategy to the reverse
phenotype of antibiotic resistance of MRSA, and BlaR1 may be an
attractive target for antimicrobial agent development.
Acknowledgement
We are grateful to Prof Yue MA (Chinese National
Center for Surveillance of Antimicrobial Resistance, Beijing,
China) for providing the WHO-2 strain.
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