Rescue of the streptomycin antibiotic activity by using streptidine as a "decoy acceptor" for the aminoglycoside-inactivating enzyme adenyl transferase

Article abstract of DOI:10.1039/b704785a

The use of streptidine as a "decoy acceptor" allows the antibiotic activity of streptomycin to recover against the Escherichia coli strain overexpressing the aminoglycoside-modifying enzyme 6-O-adenyl transferase. The Royal Society of Chemistry.

Full text of DOI:10.1039/b704785a

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Rescue of the streptomycin antibiotic activity by using streptidine as a
‘decoy acceptor’’ for the aminoglycoside-inactivating enzyme adenyl
transferase
‘
Montserrat Latorre, Pablo Pe n˜ alver, Julia Revuelta, Juan Luis Asensio, Eduardo Garc ´ı a-Junceda* and
Agatha Bastida*
Received (in Cambridge, UK) 29th March 2007, Accepted 21st May 2007
First published as an Advance Article on the web 13th June 2007
DOI: 10.1039/b704785a
The use of streptidine as a ‘‘decoy acceptor’’ allows the
antibiotic activity of streptomycin to recover against the
Escherichia coli strain overexpressing the aminoglycoside-
modifying enzyme 6-O-adenyl transferase.
from Bacillus subtilis by cloning it in the pET28-b(+) vector. The
vector pET-aadk-his was transformed in the E. coli BL21 (DE3)
strain and the recombinant protein purified using a Ni affinity
6
2+
11
column. The activity of the pure ANT(6) (Scheme 1) was
followed by HPLC and the identity of the adenylated product 2
12
1
was confirmed by H-NMR spectroscopy.
Emergence of bacterial resistance for all classes of antipathogenic
1
agents has become a serious problem over recent years.
Here, we report the use of streptidine 3 as a ‘‘decoy acceptor’’ of
ANT(6) to rescue the antibiotic activity of streptomycin 1 against
bacterial strains overexpressing this aminoglycoside-modifying
enzyme.
Aminoglycosides were one of the first groups of antibiotics to
2
meet the challenge of resistance. Acquired resistance to amino-
3
glycoside antibiotics can occur via three different mechanisms:
12
mutation of the ribosomal target; reduced permeability for the
antibiotics; and enzymatic modification of the drugs leading to
inactivation. The most prevalent source of clinically relevant
resistance is conferred by the third mechanism, the enzymatic
In our previous paper, the antibiotic recognition epitope was
determined by STD-NMR (saturation transfer spectroscopy)
experiments. The data obtained indicate that positions 1 and 6
in the streptidine moiety are in close contact with the protein
binding site. In order to investigate if streptidine 3 could be
O-adenylated by the recombinant enzyme, we obtained streptidine
3 by acid methanolysis of the streptomycin 1 with H SO –
4
inactivation of the drugs. There are well over 50 aminoglycoside-
modifying enzymes (AME) that have been characterized at the
5
gene level. Genes encoding for these enzymes can be found on the
2
4
13
bacterial chromosome, on broad-host-range plasmids or integrated
into transposons. These characteristics facilitate the quick
6
dissemination of these genes. The AME can be classified as
MeOH (Scheme 2). The streptidine was subjected to enzymatic
adenylation with ATP and recombinant ANT(6). Formation of
1
the adenylated product (AMP–streptidine) was confirmed by H-
1
3
14
NMR, C-NMR and (ES+) m/z spectroscopies. The most
N-acetyltransferases (AACs), O-adenyl transferases (ANTs) and
O-phosphotransferases (APHs). In each of these families are
several enzymes that catalyze the reactions with different
regioselectivity and substrate specificity. There is strong evidence
that inhibitors of AME have the potential to reverse resistance and
7
rescue antibiotic activity if they are administered together with
existing aminoglycosides, following a methodology well established
8
for b-lactam antibiotics. However, a deeper knowledge of the
molecular mechanism of the AME and of their structures and
interactions with the drugs, is needed to facilitate the design either
of effective and potent inhibitors, or of novel aminoglycosides, not
9
susceptible to modification by these enzymes.
The ANT family is the smallest of the three groups, with only
10 enzymes identified to date, including enzymes that regioselec-
tively adenylate the 6 and 30 positions of streptomycin 1 and the 9
7
and 30 positions of spectinomycin. Up to now, the 3D structure of
10
only the Staphylococcus aureus ANT(49), has been determined.
In our group, we are involved in the overexpression and
physicochemical characterization of aminoglycoside modifying
enzymes. For this reason, we have overexpressed the aadk gene
Departamento de Qu ´ı mica Org a´ nica Biol o´ gica, Instituto de Qu ´ı mica
Org a´ nica General, CSIC, Madrid 28006, Spain.
E-mail: eduardo.junceda@iqog.csic.es; agatha.bastida@iqog.csic.es;
Fax: +34-915 644 853; Tel: +34-915 622 900
Scheme 1 Recombinant ANT(6) catalyzes adenylation of streptomycin
. Typical reaction conditions are ATP (10 mM), streptomycin (10 mM),
1
MgCl
2
(10 mM), ANT(6) (10 mM) in Tris-HCl buffer (50 mM, pH 7.5).
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a
Table 2 In vivo activity of streptomycin 1 alone or in combination
with streptidine 3 against E. coli strains expressing, or not, ANT(6)
Strain
1
3
1 + 3
BL21(DE3)
BL21(DE3)/pET-aadk-his
5
.200
.400
.400
5
50 (10)
b
6
(ANT(6))
a
Activity of the aminoglycoside antibiotics is expressed by the MIC
value (mg ml ). MIC values for streptomycin in the presence of 50
2
1
b
2
or 400) mg ml of streptidine.
1
(
Scheme 2 Acid methanolysis of streptomycin 1 gives streptidine 3 and
methyl dihydrostreptobiosaminidine (4). (a) 0.5 g of 1 was dissolved in 4 ml
2 4
of MeOH containing 0.15 ml of H SO . After 48–72 h at room
This work was supported by the Spanish Ministerio de
temperature, streptidine 3 appears as a white precipitate.
Educaci o´ n y Ciencia (Grant CTQ2004-03523/BQU). ML thanks
the regional government of Madrid for the award of a pre-doctoral
2+
relevant kinetic parameters for the recombinant ANT(6) are
summarized in Table 1. These data strongly suggest that the
enzyme presents higher affinity for streptomycin than for
streptidine (the two KM values differ by one order of magnitude)
which was expected.
fellowship. The authors acknowledge Dr P. Armisen for the Ni -
IDA-agarose gift.
Notes and references
In view of this result, we decided to explore if streptidine could
act in vivo as a ‘‘decoy acceptor’’ of ANT(6), allowing the
streptomycin activity to recover (Table 2).
{ Minimal inhibitory concentrations (MIC) were measured according to a
15
published method. E. coli BL21(DE3) was grown in 1 ml of Mueller–
Hinton broth to an optical density (OD600) of 0.5 units. The desired
concentrations of antibiotic were added from stock solutions. After
incubation at 37 uC for 24 h, the OD600 of each sample was read. In the
21
Streptomycin is a powerful antibiotic with an MIC{ of 5 mg ml
6
case of the recombinant strain BL21(DE3)/pET-aadk-his , the described
for the E. coli BL21(DE3) strain. However, as expected, when the
aminoglycoside-modifying enzyme ANT(6) was overexpressed
streptomycin completely lost its antibiotic activity (MIC .
protocol was slightly modified. The bacteria were grown in 1 ml of
Mueller–Hinton broth to an OD600 of 0.3 units. Then, temperature was
switched to 30 uC, and the culture was induced with 0.5 mM isopropyl b-D-
2
1
1-thiogalactopyranoside (IPTG). When the OD600 reached 0.5 units, the
2
00 mg ml ). On the other hand, streptidine did not show any
desired concentrations of antibiotic were added. The samples were
incubated at 30 uC for 24 h. In both cases, the MIC was taken as the
lowest antibiotic concentration inhibiting bacterial growth by greater than
90%.
antibiotic activity either in presence or absence of ANT(6) (MIC .
2
1
4
00 mg ml ). When streptomycin and streptidine were co-
administered to the BL21(DE3)/pET-aadk-his strain, a significant
decrease in the streptomycin MIC value was observed (Table 2).
6
21
1 C. A. Smith and E. N. Baker, Curr. Drug Targets: Infect. Disord., 2002,
2, 143.
G. D. Wright, Curr. Opin. Microbiol., 1999, 2, 499.
Thus, when the antibiotic was administered with 50 mg ml of
21
streptidine, its MIC value dropped to 50 mg ml , but if the
2
21
concentration of streptidine was increased to 400 mg ml the MIC
3 J. D. Hayes and C. R. Wolf, Biochem. J., 1990, 272, 281; G. A. Jacoby
and G. C. Archer, N. Engl. J. Med., 1991, 324, 601; H. C. Neu, Science,
21
for streptomycin lowered to 10 mg ml , recovering its antibiotic
1992, 257, 1064.
activity to a great extent. The streptidine : streptomycin ratio of
4
J. Davies and G. D. Wright, Trends Microbiol., 1997, 5, 234;
G. D. Wright, in Bacterial Resistance to Antimicrobials, ed. K. Lewis,
A. A. Salyers, H. W. Taber and R. G. Wax, Marcel Dekker, New York,
40 : 1 needed to recover the antibiotic activity of the streptomycin
in vivo, is lower than the difference between the kcat/KM of ANT(6)
with both substrates. This could be due to the fact that the
simultaneous presence of streptidine and/or AMP–streptidine can
limit the overall rate of the reaction with streptomycin.
2
002, pp. 91–121.
D. M. Daigle, G. A. McKay and G. D. Wright, J. Biol. Chem., 1997,
72, 24755.
6 K. J. Shaw, P. N. Rather, R. S. Hare and G. H. Miller, Microbiol. Rev.,
993, 57, 138.
5
2
1
In conclusion, we have shown that the streptidine moiety of
streptomycin is a substrate for the aminoglycoside inactivating
enzyme ANT(6). The addition of this molecule in cell culture
restores the activity of the streptomycin antibiotic normally
inactivated by the ANT(6) enzyme, because the streptidine
competes with the streptomycin acting as a ‘‘decoy acceptor’’ of
the ANT(6). Thus, streptidine could be a good starting compound
for the design of more efficient ‘‘decoy acceptors’’ of aminoglyco-
side-modifying enzymes.
7
8
S. Magnet and J. S. Blanchard, Chem. Rev., 2005, 105, 477.
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M. Torrado, F. Corzana, J. M. Ca n˜ adillas, P. Groves, E. Garc ´ı a-
Junceda, J. Jimenez-Barbero and J. L. Asensio, J. Am. Chem. Soc.,
9
2006, 128, 100.
1
0 J. Sakon, H. H. Liao, A. M. Kanikula, M. M. Benning, I. Rayment and
H. M. Holden, Biochemistry, 1993, 32, 11977; L. C. Perdersen,
M. M. Benning and H. M. Holden, Biochemistry, 1995, 34, 13305.
Table 1 Kinetic parameters of ANT(6) from Bacillus subtilis
1
1 A complete account of the cloning, overexpression and biochemical
characterization of the recombinant ANT(6), will be published
elsewhere.
2
1
21
21
21
M
Substrate
V
max/mmol min mg
K
M
/mM (kcat/K
M
)/s
2
Streptomycin 0.06
Streptidine 0.0012
0.04
0.6
9.2 6 10
1.2
12 F. Corzana, I. Cuesta, A. Bastida, A. Hidalgo, M. Latorre, C. Gonzalez,
E. Garc ´ı a-Junceda, J. Jim e´ nez-Barbero and J. L. Asensio, Chem.–Eur.
J., 2005, 11, 5102.
a
All enzymatic reactions were carried out at: pH = 7.5; 25 uC and
0 mM of enzyme. The concentrations of ATP and MgCl were fixed
13 R. L. Peck, R. P. Graber, A. Walti, E. W. Peel, C. E. Hoffhine and
1
2
1
K. Folkers, J. Am. Chem. Soc., 1946, 68, 29. Streptidine: H-NMR
500 MHz, D O, pH = 3.0) d = 3.25–3.45 (m, 6H); MS (ES+) m/z 263
at 10 mM when the concentration of streptomycin and streptidine
was modified.
(
[
2
+
M + H] .
2
830 | Chem. Commun., 2007, 2829–2831
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1
1
4 AMP–streptidine: H-NMR (D
H, J = 7.1 Hz), 3.45 (m, 5H), 3.90 (dd, 1H, J = 9.3, 18.8 Hz), 4.09 (m,
H), 4.18 (d, 1H, J = 1.6 Hz), 4.27 (m, 1H), 4.31 (s, 1H), 4.39 (t, 1H, J =
.56 Hz), 4.45 (t, 1H, J = 5.4 Hz), 6.07 (d, 1H, J = 5.2 Hz), 8.34 (d, 1H,
2
O at 500 MHz, pH = 3.0) d = 1.06 (t,
d = 15.3, 58.2, 59.0, 65.5, 70.2, 70.6, 71.6, 74.3, 77.1, 84.3, 88.2, 145.3,
158.0, 158.3; MS (ES+) m/z (%): 592.2 [M + H] .
15 P. M. Waterworth, in Laboratory Methods in Antimicrobial
Chemotherapy, ed. L. Garrod, Churchill Livingstone Press,
Edinburgh, 1978, pp. 31–40.
+
1
1
4
13
2
J = 4.5 Hz), 8.52 (t, 1H, J = 5.7 Hz); C-NMR (D O at 125 Hz)
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Chem. Commun., 2007, 2829–2831 | 2831