Professional Documents
Culture Documents
Institute of Medical Microbiology & Infection Control, University Hospital of Frankfurt, Paul-Ehrlich Str. 40,
D-60596 Frankfurt/Main, Germany
b
Institute of Pharmaceutical Chemistry, Johann-Wolfgang-Goethe University, Frankfurt/Main, Germany
Accepted 7 December 2007
Abstract
The numerous genes and proteins encoded in the borrelial genome have been shown to undergo differential
expression in response to environmental cues. To gain a better understanding of possible interactions between
antimicrobial agents and Borrelia, we investigated here the effects of increasing concentrations of penicillin G and
doxycycline on the protein expression of the Borrelia burgdorferi s.s. isolate LW2 after 24 and 48 h of incubation. For
14 protein spots in Borrelia exposed to penicillin G at 0.25 and 0.5 mg/ml and for 5 protein spots in Borrelia exposed to
doxycycline at 0.5 and 1 mg/ml, differences in spot intensity were identied by use of high resolution two-dimensional
electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-ight mass spectrometry (MALDITOF-MS). At concentrations of both antimicrobial agents around the median minimal inhibitory concentration
(0.5 mg/ml), all but one of the detected spots showed a considerable down-regulation as revealed by a X50% decrease
of spot intensity in comparison to untreated controls. Most of the spots identied thus far belong to proteins that are
encoded by genes localized on the borrelial chromosome and are known to be involved in the different pathways of
bacterial cell metabolism. Interestingly, one spot, identied as triosephosphate isomerase, was clearly up-regulated in
the presence of doxycycline. Our data provide for the rst time scientic evidence that B. burgdorferi s.l., although it
possesses a small genome and extremely limited biosynthetic capabilities, shows a variable but distinct physiological
response to exposure with penicillin G and doxycycline.
r 2008 Elsevier GmbH. All rights reserved.
Keywords: Borrelia burgdorferi; MALDI-TOF MS; Antimicrobial susceptibility; Penicillin; Doxycycline; 2D electrophoresis; Protein
expression
Introduction
Borrelia burgdorferi s.l., the causative agent of Lyme
borreliosis, is used to adapt to rapidly changing
Corresponding author. Tel.: +49 69 6301 6441;
fax: +49 69 6301 5767.
E-mail address: K.Hunfeld@em.uni-frankfurt.de (K.-P. Hunfeld).
1438-4221/$ - see front matter r 2008 Elsevier GmbH. All rights reserved.
doi:10.1016/j.ijmm.2007.12.001
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directly for 10% Tris/tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis or stored at 80 1C.
The focused strips used for the second dimension were
rst reduced in 50 mM Tris (pH 8.8) equilibration buffer
containing 6 M urea, 30% glycerol, 2% SDS, 1%
dithiothreitol, and traces of bromophenol blue for
15 min and then alkylated in the same equilibration
buffer containing 260 mM iodoacetamide. The complemented rst-dimension strips were separated on a 10%
Tris/tricine-sodium dodecyl sulfate-polyacrylamide gel
electrophoresis in a Protean II Xi chamber (Bio-Rad)
together with a broad-range protein marker (Precision
Protein standard, Bio-Rad). Running conditions were
0.5 h at 40 V and 17.5 h at 85 V. Protein spots were
detected by silver-staining (analytical gels, Fig. 1) or
Coomassie-staining (preparative gels).
80 1C until use. To determine the protein concentrations of the different samples, a modied Bradford
protein assay (Bio-Rad) was used.
SDS-PAGE
IEF
P-4802 P-4803 P-4804
P-3703
P-2702
75
D/P-4703
D/P-4705
P-5702
P-7602
50
P-7603
D-0504
37
Mr [kD]
P-4702
P-4706
25
P-7103
D-4101
D/P-7001
15
3
10
pH
Fig. 1. Representative two-dimensional gel analysis of Borrelia burgdorferi s.s. isolate LW2. Twenty micrograms of protein derived
from a culture in the log-phase of growth were focused in a pH-gradient ranging between pH 3 and 10. After isoelectric focusing
(IEF), the sample was separated through a 10% Tris/tricine SDS-PAGE and subsequently silver stained. Location and molecular
mass of differentially expressed proteins in borrelial samples exposed to penicillin G and doxycycline at concentrations around the
MIC are indicated. Such protein spots were then cored from preparative gels and further analyzed by MALDI-TOF MS (see Table 1
and Fig. 2). The positions of molecular mass standard in kDa are indicated on the right. The linear pH gradient ranging from pH 3
to 10 is indicated at the bottom of the photograph.
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2500
2500
2500
250
2000
2000
2000
200
1500
1500
1500
150
1000
1000
1000
100
500
500
500
50
0
P-3703
P-2702
750
1500
1250
1000
750
500
250
0
600
450
300
150
0
P-4706
1500
600
500
400
300
200
100
0
P-4803
P-4802
3000
2400
1800
1200
600
0
0
P-4702
900
600
300
0
P-5702
P-4804
2000
800
1600
600
1200
1200
400
800
200
400
P-7603
P-7602
P-7103
200
350
600
150
280
450
210
100
300
140
50
150
70
0
D/P-4703
D/P-4705
800
1600
600
1200
400
800
200
400
D/P-7001
0
D-0504
D-4101
Fig. 2. Comparative analyses of protein synthesis in antibiotic-exposed Borrelia. Protein preparations of Borrelia exposed to 0.25
and 0.5 mg/ml of penicillin G or to 0.5 and 1 mg/ml of doxycyline for 24 and 48 h and untreated controls were subjected to 2 DE and
subsequent software-assisted densitometry. Bars display the differences in spot intensities of the proteins affected by penicillin G (P-)
and doxycycline (D-) as measured by densitometry and are expressed as OD values (black bars indicate untreated controls; white
bars indicate antibiotic-exposed samples). Proteins affected by both penicillin G and doxycyline (D/P-4703, D/P-4705, and D/P7001) are summarized in a single diagram where bars on the right indicate penicillin-treated samples and bars on the left represent
doxycycline-treated samples. The error bars represent the standard error of the mean (SEM) calculated for the means of spot
intensity obtained from three independent gel runs for antibiotic-exposed samples and untreated controls.
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MALDI-TOF-MS
Delayed extractionTM (DE) MALDI time of ight
mass spectra were recorded on a Voyager-DE STR
instrument (ABI) using a nitrogen laser (l 336 nm,
repetition rate 20 Hz) for desorption and ionisation
with an acquisition mass range from 600 to 5000m/z and
the low mass gate set to 550m/z. The total acceleration
voltage was 20 kV with 68.5% grid voltage on the rst
grid, 0.02% guide wire voltage, 150 ns delay and a
mirror voltage ratio of 1.12. Spectra were externally
calibrated with SequazymeTM Peptide Mass Standards
Kit (ABI). Between 1000 and 2000 laser shots were
accumulated for each mass spectrum. All spectra were
smoothed, noise-ltered and deisotoped using Data
Explorer (V. 4.3, ABI). Deisotoped peaks were labeled
by the software and the 100 most intense peaks were
used for database searching. Autolytic tryptic peptides
or peptides resulting from the identied protein were
used for internal calibration.
Results
In vitro susceptibility testing results
MICs of penicillin G and doxycycline for B. burgdorferi s.s. isolate LW2 ranged from 0.5 to 1 mg/ml (median
MIC: 0.5 mg/ml for both substances), as determined by a
colorimetric assay in independent experiments performed on 3 different days (data not shown).
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Table 1. Protein spots and corresponding proteins showing up- or down-regulation after exposure of Borrelia burgdorferi s.s. strain
LW2 to increasing concentrations of penicillin G, doxycycline, or both
Spot
pI
(estimated
from gel)
Mol. mass
(estimated
kDa from gel)
Up- or
downregulation
Mol. mass
(calculated kDa
from database)
P-2702
P-3703
P-4702
P-4706
P-4802
P-4803
5.9
6.2
6.4
6.7
6.4
6.5
65
65
63
63
75
75
k
k
k
k
k
k
68.13
68.13
63.496
NA
NA
80.298
P-4804
P-5702
6.6
7.0
75
57
k
k
NA
50.061
P-7103
P-7602
P-7603
D/P-4703
D/P-4705
D/P-7001
D-0504
D-4101
8.1
7.8
7.9
6.5
6.6
7.9
5.2
6.4
25
50
49
65
63
23
42
26
k
k
k
k
k
k
k
m
30.417
55.856
53.227
NA
NA
26.569
42.372
27.984
Proteins were identied by MALDI-TOF MS after preparative isolation from 2-DE gels.
pI: isoelectric point; P: proteins affected by penicillin G exposure; D: proteins affected by doxycycline exposure; NA: not available.
Discussion
The occurrence of changes both in the morphology
and the protein expression pattern of B. burgdorferi s.l.
have been reported in response to a variety of adverse
environmental conditions (Barbour and Hayes, 1986;
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References
Alban, P.S., Johnson, P.W., Nelson, D.R., 2000. Serumstarvation-induced changes in protein synthesis and morphology of Borrelia burgdorferi. Microbiology 146, 119127.
Bandow, J.E., Brotz, H., Leichert, L.I., Labischinski, H.,
Hecker, M., 2003. Proteomic approach to understanding
antibiotic action. Antimicrob. Agents Chemother. 47,
948955.
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