Download Protein expression by a Beijing strain differs from that of another

Microbiology (2005), 151, 1139–1150
DOI 10.1099/mic.0.27518-0
Protein expression by a Beijing strain differs from
that of another clinical isolate and Mycobacterium
tuberculosis H37Rv
Carmen Pheiffer,1 Joanna C. Betts,2 Helen R. Flynn,2 Pauline T. Lukey2
and Paul van Helden1
1
MRC Centre for Molecular and Cellular Biology, Department of Medical Biochemistry,
University of Stellenbosch Medical School, PO Box 19063, Tygerberg, 7505, South Africa
Correspondence
Paul van Helden
2
[email protected]
GlaxoSmithKline Research and Development, Stevenage, Hertfordshire SG1 2NY, UK
Received 27 July 2004
Revised
10 December 2004
Accepted 23 December 2004
The Beijing strain family has often been associated with tuberculosis (TB) outbreaks and drug
resistance worldwide. In this study the authors have compared the protein expression and antigen
recognition profiles of a local Beijing strain with a less prevalent clinical isolate belonging to the
family 23 strain lineage, and the laboratory strain Mycobacterium tuberculosis H37Rv. Using
two-dimensional electrophoresis, liquid chromatography tandem mass spectrometry and Western
blot analysis several proteins were identified as quantitatively increased or decreased in both
clinical strains compared to H37Rv. Remarkably, the Beijing strain showed increased expression
of a-crystallin and decreased expression of Hsp65, PstS1, and the 47 kDa protein compared
to the other clinical strain and H37Rv. One- and two-dimensional Western blot analysis of antigens
expressed by the three strains, using plasma from TB patients, confirmed differential antigen
expression by strains and patient-to-patient variation in humoral immunity. These observed protein
differences could aid the elucidation of mechanisms underlying the success of the Beijing strain
family, measured by global dissemination, compared to other M. tuberculosis strains.
INTRODUCTION
The Beijing strain family is a family of Mycobacterium
tuberculosis strains, speculated to have originated in Asia
(van Soolingen et al., 1995), of which the strain W and strain
W-like families responsible for many cases of drug resistance
are a subset (Bifani et al., 2002). Molecular epidemiological
studies have shown that these strains (both drug-susceptible
and -resistant) are distributed worldwide and are able to
spread in large clonal clusters (Glynn et al., 2002). The
worldwide occurrence of Beijing strains and their frequent
association with outbreaks and drug resistance makes study
of this strain family important in the understanding of
M. tuberculosis pathogenesis. It is speculated that strains
belonging to the Beijing family have a genetic advantage to
cause disease and that the wide dispersion of this family
compared to other less prevalent clinical isolates may be
related to differential protein expression (Bifani et al., 2002).
Proteomics, the study of the protein complement of the
genome, has been used extensively to identify differences
Abbreviations: 1D, one-dimensional; 2D, two-dimensional; BCG,
Mycobacterium bovis bacille Calmette–Guérin; BCIP, 5-bromo-4chloro-indolyl-phosphatase; CF, culture filtrate; F11, family 11; F23,
family 23; ICD, isocitrate dehydrogenase; IPG, immobilized pH gradient;
LC/MS/MS, liquid chromatography tandem mass spectrometry; NBT,
nitro blue tetrazolium; TB, tuberculosis; WCL, whole-cell lysate.
0002-7518 G 2005 SGM
with respect to virulence and pathogenesis between mycobacterial strains. Comparative proteome analysis has revealed
numerous differences in the cellular protein composition of
the laboratory strains M. tuberculosis H37Rv and Erdman
to that of the vaccine strain Mycobacterium bovis bacille
Calmette–Guérin (BCG) (Jungblut et al., 1999; Mattow
et al., 2001). In addition, a number of differences have also
been observed in the expression of proteins in culture
supernatants of H37Rv and BCG (Mattow et al., 2003), and
H37Rv and the attenuated strain H37Ra (He et al., 2003).
Analysis of the proteome of M. tuberculosis H37Rv and a
clinical strain CDC1551, believed to elicit a more vigorous
host immune response than H37Rv (Manca et al., 1999),
identified several quantitative differences in the cellular
protein composition of these strains (Betts et al., 2000).
In a previous study we compared protein expression of
two M. tuberculosis clinical strains, originally isolated from
patients from a community in Cape Town, South Africa,
with a very high tuberculosis (TB) incidence (Warren et al.,
2000), to the laboratory strain H37Rv using one-dimensional
(1D) PAGE, ELISA and Western blotting (Pheiffer et al.,
2002). Based on the number of IS6110 insertions and
spoligotyping, the two clinical strains were classified as
belonging to the Beijing family and family 23 (F23) (Pheiffer
et al., 2002). Results from that study (Pheiffer et al., 2002)
showed that protein expression by M. tuberculosis strains
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 04:26:35
Printed in Great Britain
1139
C. Pheiffer and others
was mainly growth phase dependent, although some differences between the strains were observed. However, due to
the low resolving power of 1D gel electrophoresis, the
identity of the differentially expressed proteins could not
be ascertained. Here, we have extended our previous study
through the use of two-dimensional (2D) PAGE coupled
with identification of protein spot differences by MS and
Western blot analysis of the expression of 14 M. tuberculosis
antigens. Furthermore we have used plasma from patients
infected with Beijing, F23 and family 11 (F11) strains to
investigate differences in antigen recognition. F11 strains
constitute another highly prevalent strain family in the
study community.
This study has revealed a number of differences in cellular
and culture supernatant protein composition between M.
tuberculosis H37Rv, the Beijing strain and F23, particularly
between the two clinical strains compared to H37Rv.
Careful analysis of these differences and the differences in
the antibody recognition profiles could possibly explain the
different frequencies of the clinical strains in our study
population, where the Beijing strain is at least tenfold more
frequent than F23 strains (Warren et al., 2000). In addition
to providing clues as to the differences in pathogenicity
and prevalence of strains, identification of protein expression differences between strains will aid the development
of vaccines, serodiagnostic tests and the choice of drug
targets. This is believed to be the first study to profile
expression patterns of cellular and culture supernatant
proteins of a Beijing strain by 2D gel electrophoresis and the
first study attempting to correlate strain prevalence with
protein expression.
METHODS
M. tuberculosis strains and culture. M. tuberculosis H37Rv
(ATCC 25618) and two local clinical strains, Beijing (SAWC 1524)
and F23 (SAWC 1296), were grown in modified Sauton medium
(Andersen et al., 1991). Briefly, mycobacterial strains at late exponential growth (OD600>0?6) were diluted 1 : 10 into 200 ml Sauton
medium and grown at 37 uC without stirring. After 4 weeks, cultures
were harvested by centrifugation (1900 g, 20 min) and mycobacterial
pellets were washed twice with PBS containing 1 % (v/v) Tween-20.
Proteins used in Western blots with plasma from TB patients were
prepared from strains during exponential growth in Middlebrook
7H9 medium (Becton Dickinson) supplemented with 10 % (v/v)
albumin-dextrose-catalase and 0?05 % (v/v) Tween-80 as previously
described (Pheiffer et al., 2002). Morphology and acid-fastness
were checked by Ziehl–Nielsen staining (Heifets & Good, 1994). All
strains were sensitive to isoniazid, rifampicin and ethambutol.
Isocitrate dehydrogenase (ICD) assay. The amount of ICD in
culture supernatants was measured using the Sigma Diagnostics ICD
kit. In this procedure, ICD catalyses the oxidative decarboxylation of
L-isocitrate to 2-oxoglutarate, and the reduced NADPH produced is
measured at 340 nm.
Protein extraction. Whole-cell lysate (WCL) proteins were
extracted by bead disruption of the mycobacterial pellet in lysis
buffer [0?3 % (w/v) SDS, 200 mM DTT, 50 mM Tris/HCl pH 7?0,
1 mM PMSF and complete protease inhibitor cocktail] (Roche
Molecular Biochemicals) as previously described (Pheiffer et al.,
1140
2002). Culture filtrate (CF) proteins were prepared from culture
supernatants sterilized by sequential filtration of the culture supernatant through 0?45 mm and 0?22 mm filter units (Corning).
Filtrates were then concentrated using Centricon Plus-80 filter units
(Biomax-PB membrane, 5000 MWCO), followed by Centricon units
(YM membrane, 3000 MWCO) (Millipore). WCL and CF protein
concentrations were estimated using the Bradford assay (Bradford,
1976).
2D gel electrophoresis. Proteins were separated by 2D gel electrophoresis as previously described (Betts et al., 2000), with minor
modifications. Briefly, 20 mg (60 mg for Western blotting) protein
was resuspended in rehydration buffer [8 M urea, 2 % (w/v)
CHAPS, 10 mM DTT, 2 % (v/v) immobilized pH gradient (IPG)
buffer (pH 4–7), trace bromophenol blue] and applied to pH 4–7
IPG strips (Amersham Biosciences) for overnight rehydration. IEF
was performed using a Multiphor II system (Amersham Biosciences)
as follows: 100 V for 2 h, 300 V for 2 h, 1000 V for 1 h, 3500 V for
20 h (3500 V for 27?5 h for Western blotting). Second-dimension
separation was carried out by placing equilibrated IPG strips
(Bjellqvist et al., 1993, http://www.expasy.ch/ch2d/protocols/) onto
12 % PAGE gels and sealing with 0?5 % (w/v) agarose in cathode
buffer (defined below), containing a trace amount of bromophenol
blue. Anode (375 mM Tris/HCl, pH 8?8) and cathode (192 M
glycine, 0?1 %, w/v, SDS, pH 8?3) buffers as described by Herbert
et al. (1998) were used. Proteins were visualized by silver staining
using an ammoniacal stain (Bjellqvist et al., 1993, http://www.
expasy.ch/ch2d/protocols/). Gels were air-dried (Bio-Rad GelAir
Dryer) and compared by visualization on a light box. When protein
spot differences were noted, gels were rerun, stained with a silver
stain compatible with MS (Betts & Smith, 2001) and spots of
interest excised from the gel.
Sample preparation for MS. Excised gel pieces were washed with
double-distilled H2O for 10 min, followed by 100 % acetonitrile for
5 min, centrifuged and then dehydrated by vacuum centrifugation.
Automated robotic digestion was carried out using a MassPREP
station (Micromass), equipped with four probes for aspirating and
dispensing reagent and washing solutions, and a heated incubation
platform. Samples were destained with 50 mM ammonium bicarbonate/
acetonitrile (1 : 1, v/v), then reduced and alkylated with 10 mM DTT
and 55 mM iodoacetamide respectively. This was followed by in-gel
digestion with porcine trypsin (Promega) (6 ng ml21) in 50 mM
ammonium bicarbonate (25 ml) for 5 h at 37 uC. The resulting
peptides were then extracted with 1 % (v/v) formic acid/acetonitrile
(98 : 2, v/v).
Nanoscale liquid chromatography tandem mass spectrometry
(LC/MS/MS) and database searching. Samples were introduced
using the Micromass CapLC system (Micromass), comprising a low
flow capillary HPLC pump and autosampler. A 10 port valve was
configured with a pre-concentration column (300 mm ID65 mm
C18 PepMap, LC Packings) and a nanoscale analytical column
(75 mm ID615 cm C18 PepMap, LC Packings). Peptides were eluted
using a reverse-phase gradient of 5–50 % buffer B over 30 min
[A=5 % (v/v) acetonitrile, 0?1 % (v/v) formic acid; B=95 % (v/v)
acetonitrile, 0?1 % (v/v) formic acid] at a flow rate of approximately
200 nl min21. All data were acquired using a Q-Tof Ultima API
(Micromass) hybrid quadrupole orthogonal acceleration time of flight
mass spectrometer equipped with a nanospray source. Up to eight
precursor ions were automatically selected from the time of flight
(TOF)/MS survey scan for MS/MS per cycle, and collision energies
were chosen automatically based on the m/z value and the charge
state of the selected precursor ions. Peptide sequence data generated
were searched against the non-redundant protein database using
MASCOT (http://www.matrixscience.com). Search parameters included
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 04:26:35
Microbiology 151
Mycobacterium tuberculosis protein expression
Table 1. Mouse mAbs
mAb
IT-4
IT-7
IT-13
IT-19
IT-23
IT-38
IT-42
IT-43
IT-49
IT-51
IT-52
IT-53
IT-58
HYB76-8
Corresponding antigen*
Dilution
of mAbD
16 kDa (a-crystallin)
40 kDa (L-alanine dehydrogenase)
65 kDa (Hsp65)
19 kDa
38 kDa (PstS1)
20 kDa
82 kDa (KatG)
56 kDa
32–33 kDa (Ag85 complex)
17 kDa
25 kDa (MPT51)
96 kDa
47 kDa
ESAT6
1 : 1000
1 : 2500
1 : 500
1 : 100
1 : 100
1 : 100
1 : 100
1 : 100
1 : 100
1 : 100
1 : 100
1 : 100
1 : 100
1 : 100
Table 1) and plasma (1 : 100 and 1 : 500) and screened for antibody
binding with a horseradish-peroxidase-conjugated goat anti-mouse
IgG (1 : 10 000, CALTAG laboratories) as the secondary antibody for
mAbs and an alkaline-phosphatase-conjugated goat anti-human
IgG (1 : 2000, Kirkegaard & Perry Laboratories) for plasma. Bound
antigens were detected using chemiluminescence detection reagents
(ECL Western blotting detection reagents, Amersham Biosciences)
or with the 5-bromo-4-chloro-indolyl-phosphatase (BCIP)/nitro
blue tetrazolium (NBT) substrate (Kirkegaard & Perry Laboratories)
for plasma samples.
2D Western blotting. For Western blotting, 2D gels were transferred to nitrocellulose membranes (S&S Protran BA85, Schleicher &
Schuell) using semi-dry blotting. Blots obtained from 2D gels were
probed with mAbs IT-4 and IT-23 using the same conditions as for
1D Western blotting. For blots using plasma from TB patients,
plasma was diluted 1 : 150 and screened for antibody binding using
alkaline-phosphatase-conjugated goat anti-human IgG (Kirkegaard
& Perry Laboratories) as described above. Immunoreactive antigens
were detected using the BCIP/NBT substrate (Kirkegaard & Perry
Laboratories) as described above.
Patients. Patients were recruited from a high TB incidence com-
*Antigen against which the monoclonal antibody was raised.
DDilution of mAb used in Western blots.
the fixed modification carboamidomethyl, due to the alkylation of
cysteine residues by iodoacetamide and the variable modification,
oxidation of methionine residues.
Monoclonal antibodies (mAbs). Mouse mAbs against a range of
M. tuberculosis proteins (Table 1) (Engers et al., 1986, KhanolkarYoung et al., 1992; Sonnenberg & Belisle, 1997) were made available
by the Department of Microbiology, Colorado State University,
through funds from the National Institutes of Health, National
Institute of Allergy and Infectious Disease, Contract NO1-AI-75320.
HYB76-8 directed to ESAT6 (Sorensen et al., 1995) was received
from Karin Weldingh (Statens Serum Institut, Copenhagen, Denmark).
1D SDS-PAGE and Western blotting. Proteins were separated
by SDS-PAGE using a 4 % stack over a 12 % resolving gel and
transferred to PVDF membranes (Amersham Biosciences) by tank
blotting. Blots were incubated with mAbs (dilutions are listed in
munity and were homogeneous with respect to social class and
ethnicity. All patients were culture-positive for M. tuberculosis
drug-sensitive organisms. The smear status, disease episode, antituberculosis chemotherapy status and IS6110 genotype of the infecting M. tuberculosis strain were documented for all patient samples
(Table 2). Blood was collected by clinical staff; plasma was collected
by centrifugation at 2500 g for 5 min, and stored at 220 uC. Ethical
approval for this study was obtained from the University of
Stellenbosch Faculty of Health Sciences ethics committee and
samples were only taken after informed consent was given.
RESULTS
2D PAGE and protein identification
Mycobacterial strains were cultured in modified Sauton
medium, a synthetic medium without protein enrichment
(Andersen et al., 1991), for 4 weeks. This enabled CF
proteins to be analysed free of BSA contamination. Pilot
Table 2. TB patient data
Patient no.
97
484
522
672
726
880
881
941
Strain family*
Smear statusD
Anti-TB treatmentd
Disease episode§
F11
F11
F11
F11
F23
F29/Beijing
F29/Beijing
F29/Beijing
positive
negative
negative
positive
negative
positive
positive
positive
yes (6 months)
no
no
no
yes (4 days)
no
no
no
second
first
first
first
first
first
first
first
*M. tuberculosis strains are grouped into families based on the number of IS6110 insertion elements identified by DNA fingerprinting. The strain
family of the M. tuberculosis strain isolated from the patient sputum is shown.
DSmear status refers to whether a patient’s sputum stained positive or negative for acid-fast bacilli.
dAnti-TB treatment refers to the 6-month TB treatment regimen prescribed by DOTS (directly observed therapy shortcourse: isoniazid,
pyrazinamide, rifampicin, streptomycin). The time blood was taken after the commencement of treatment is shown.
§Disease episode refers to the TB episode the patient was experiencing.
http://mic.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 04:26:35
1141
C. Pheiffer and others
studies showed that after 4 weeks growth all three strains
were in late exponential growth, thereby minimizing
growth-related differences between strains. Whole-cell
lysate and CF proteins were separated by 2D PAGE using
pH 4–7 IPG strips and 12 % PAGE gels. Pilot experiments
demonstrated that many M. tuberculosis proteins focus
between pH 4 and 7, and the best resolution was achieved
using this pH range. The 2D gels of WCL and CF proteins
derived from M. tuberculosis H37Rv, the Beijing strain and
F23 are shown in Fig. 1. Some overlap between the proteins
detected in the WCL and CF was observed, suggesting
leakage of cell wall proteins or limited bacterial lysis.
Fig. 1. 2D gels of WCL and CF proteins extracted from the three M. tuberculosis strains. (a) H37Rv, WCL; (b) H37Rv, CF;
(c) Beijing strain, WCL; (d) Beijing strain, CF; (e) F23, WCL; (f) F23, CF. Protein spots with increased intensity in clinical
strains are ringed and those with increased intensity in H37Rv are boxed. Their identities are listed in Table 3.
1142
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 04:26:35
Microbiology 151
Mycobacterium tuberculosis protein expression
Quantification of ICD in culture supernatants showed no
significant difference in the extent of autolysis in the three
strains (data not shown).
The protein expression profiles were highly similar between
the strains, enabling visual comparison. Spot differences
consistent between quadruplicate gels, and obvious by
visual examination, were analysed further. Protein spots
of interest were analysed by LC/MS/MS. All proteins
identified had their counterparts in all three strains,
although quantitative expression differences were observed.
Protein spots identified by LC/MS/MS are indicated in
Fig. 1 and their identities listed in Table 3. Eight protein
spots were increased in the WCLs of both clinical isolates
relative to H37Rv. Seven of these were identified as acrystallin, and two proteins, Rv2005c and the 35 kDa
antigen Rv2744c, were identified within the eighth spot.
Previous studies have also reported the appearance of
multiple a-crystallin species with different molecular masses
and isoelectric points (Betts et al., 2000; Sonnenberg &
Belisle, 1997). One of the factors contributing to mobility
variants of a-crystallin is the oxidation of methionine
residues, as recently demonstrated by Abulimiti et al. (2003).
Three spots, identified as Ag85A, had decreased intensity in
the CFs of both clinical strains compared to H37Rv. Spot
9 also contained Rv1096 and spot 10 three other proteins:
Ag85C, Rv0831c and CysA3. Spot 12, identified as containing both PstS1 and Ag85B, was decreased in CFs of the
Beijing strain compared to both H37Rv and F23. Two of
the protein spots identified by LC/MS/MS contained
novel proteins, Rv1096, Rv0831c and Rv3117, not previously identified by proteomic studies (Jungblut et al.,
1999; Rosenkrands et al., 2000a, b; http://www.mpiib-berlin.
mpg.de/2D-PAGE).
Reactivity of mAbs with proteins extracted from
M. tuberculosis strains
In some cases, spots excised from 2D gels were identified
as containing two or more proteins by LC/MS/MS. This
included proteins of the Ag85 complex and PstS1. Western
blot analysis was therefore employed to identify the proteins
responsible for the differences in spot intensities observed
on 2D gels and to investigate the expression of 12 other
antigens, selected on the basis of mAb availability.
1D and 2D Western blotting identified PstS1 as the protein
responsible for the decreased expression of spot 12 (Fig. 1,
Table 3. Protein spot differences observed for whole-cell lysate and culture filtrate protein fractions of M. tuberculosis H37Rv,
the Beijing and the F23 strain
Spot
Fraction
ORF
1–7
WCL/CF
Rv2031c
8
WCL
Rv2005c
Rv2744c
9
CF
Rv1096D
Rv3804c
10
CF
Rv0129c
Rv0831cD
Rv3117D
Rv3804c
11
CF
Rv3804c
12
CF
Rv0934
Rv1886c
Gene name
Protein
Matched peptides
Increased expression in both clinical strains compared to H37Rv
hspX
Heat-shock protein, HspX
SEFAYGSFVR
TVSLPVGADEDDIK
–
Conserved hypothetical protein
YANAIGSAELAESSVQGR
35kd_ag
Conserved 35 kDa alanine-rich protein
GLLGSVSSSLVR
Decreased expression in both clinical strains compared to H37Rv
–
Possible glycosyl hydrolase
ANDVIAAATGR
LVTVSELLGPR
fbpA
Antigen 85A
FLEGFVR
ASDMWGPK
NDPLLNVGK
ALGATPNTGPAPQGA
fbpC
Antigen 85C
NDPMVQIPR
EMPAWLQANK
–
Conserved hypothetical protein
NQAIVVETTAYR
cysA3
Probable thiosulphate sulphurtransferase
DFVDAQQFSK
fbpA
Antigen 85A
NDPLLNVGK
ALGATPNTGPAPQGA
fbpA
Antigen 85A
FLEGFVR
NDPLLNVGK
ALGATPNTGPAPQGA
Decreased expression in Beijing strain only
pstS1
Periplasmic phosphate-binding lipoprotein
VLAAMYQGTIK
fbpB
Antigen 85B
AADMWGPSSDPAWER
Mr*
pI*
16 096
5?00
30 985
29 257
5?53
5?71
31 109
6?52
35 686
6?08
36 771
5?92
33 885
31 014
35 686
5?88
5?14
6?08
35 686
6?08
38 243
34 580
5?14
5?62
*Predicted Mr and pI values calculated using the Expasy compute pI/Mw tool (http://www.expasy.ch/tools/pi_tool.html).
DNovel proteins not previously identified by proteomics (Jungblut et al., 1999; Rosenkrands et al., 2000a, b; http://www.mpiib-berlin.mpg.de/
2D-PAGE).
http://mic.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 04:26:35
1143
C. Pheiffer and others
Fig. 2. Protein expression in M. tuberculosis strains. Ten micrograms of protein extracted from WCL (lanes 1, 3, 5) and CF
(lanes 2, 4, 6) of M. tuberculosis H37Rv (lanes 1, 2), the Beijing strain (lanes 3, 4) and F23 (lanes 5, 6) was separated by
12 % SDS-PAGE, transferred onto PVDF membranes and probed with mAbs directed to (a) a-crystallin, (b) L-alanine
dehydrogenase, (c) Hsp65, (d) PstS1, (e) KatG, (f) Ag85 complex, (g) 96 kDa protein, (h) 47 kDa protein, (i) ESAT6,
( j ) 19 kDa protein, (k) 17 kDa protein, (l) 20 kDa protein, (m) 56 kDa protein and (n) 25 kDa protein.
Table 3) in the Beijing strain compared to the F23 strain
and H37Rv (Fig. 2d, Fig. 3b). 2D Western blotting showed
the existence of more than one species of PstS1, which has
been previously reported by Sonnenberg & Belisle (1997)
and is probably due to post-translational modifications
such as glycosylation, phosphorylation or acetylation. The
(a) a-Crystallin
M. tuberculosis H37Rv
WCL
CF
Beijing
WCL
F23
CF
WCL
CF
WCL
CF
(b) PstS1
M. tuberculosis H37Rv
WCL
CF
Beijing
WCL
F23
CF
Fig. 3. a-Crystallin and PstS1 expression in M. tuberculosis strains. Sixty micrograms of protein extracted from M.
tuberculosis H37Rv WCL (1, 7) and CF (2, 8), the Beijing strain WCL (3, 9) and CF (4, 10) and the F23 strain WCL (5, 11)
and CF (6, 12) was separated by 12 % 2D PAGE, transferred to nitrocellulose membranes and probed with mAbs directed to
(a) a-crystallin and (b) PstS1.
1144
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 04:26:35
Microbiology 151
Mycobacterium tuberculosis protein expression
absence of PstS1 on 2D blots of CFs of the F23 strain
(Fig. 3b) could possibly be due to decreased expression
of PstS1 in CFs compared to WCLs, as observed on 1D
Western blots (Fig. 2d). Additionally, proteins may be
more concentrated in bands on 1D gels than in spots on
2D gels and proteins may transfer more efficiently from
1D gels than from 2D gels. Several species corresponding
to a-crystallin were observed and were expressed more
highly in the Beijing strain compared to F23 and H37Rv
(Fig. 2a and 3a). Consistent with the 2D results, expression
of a-crystallin was increased in the WCL compared to the
CF in all the strains. Expression of the 47 kDa protein was
decreased in the Beijing strain compared to H37Rv and
F23 (Fig. 2h). Two proteins, Hsp65 and the Ag85 complex, were downregulated in both clinical strains relative
to H37Rv (Fig. 2c, f), while Hsp65 was also downregulated
in the Beijing strain compared to F23. Multiple bands of
Hsp65, possibly different isoforms or degradation products,
were observed in both WCLs and CFs. Hsp65 observed in
CFs is possibly due to bacterial lysis. L-Alanine dehydrogenase and ESAT6 (Fig. 2b, 2i) showed decreased expression
in H37Rv compared to both clinical strains.
The expression of the other proteins was approximately
similar in all the strains analysed, although some quantitative differences between their expression levels in WCLs
and CFs were observed. For example, expression of KatG
was increased in the CF of H37Rv compared to the clinical
strains, but decreased in the WCL of H37Rv compared to
the clinical strains (Fig. 2e). The mAb directed to KatG
(IT42) reacted with two bands in both WCLs and CFs, with
the lower band corresponding to the correct molecular
mass of 82 kDa. Sonnenberg & Belisle (1997) have also
reported that IT42 reacts with more than one protein. The
upregulation of a-crystallin, L-alanine dehydrogenase and
ESAT6, and the downregulation of Hsp65 and Ag85 in
the clinical strains compared to H37Rv, as well as the
decreased expression of PstS1 in the Beijing strain compared
to H37Rv and F23, were confirmed by ELISA (data not
shown).
Reactivity of plasma samples to proteins
extracted from the three M. tuberculosis strains
To establish whether there was differential antigen expression by the M. tuberculosis strains, plasma-derived antibodies of TB patients were tested against WCL proteins
extracted from M. tuberculosis H37Rv, the Beijing strain and
F23. After 1D PAGE separation and transfer to PVDF
membranes, proteins were probed with plasma from TB
patients infected with Beijing, F23 or F11 strains (Table 2,
Fig. 4). As expected, plasma from TB patients reacted with
a wide variety of M. tuberculosis proteins, ranging from
10 kDa to over 105 kDa (Fig. 4). Plasma samples were
tested on antigens fractionated on different gels; therefore
figures were aligned according to molecular mass markers,
which were included on each gel, and mobility of immunodominant antigens. As noted previously by others, patientto-patient variation, where each patient had a characteristic
http://mic.sgmjournals.org
banding pattern (Fig. 4), was evident. However, some prominent differences between the antigens expressed by the
strains were observed; these are circled in Fig. 4 and listed
in Table 4. A band of approximately 60 kDa (circle 1) was
recognized by patient 880 in H37Rv but not in F23 or the
Beijing strain (Fig. 4f). Patient 880 also reacted with a
protein of about 40 kDa (circle 2) in protein extracts of
F23 and the Beijing strain only (Fig. 4f), with more
prominent expression in the Beijing strain. Similarly, a
band of approximately 15 kDa (circle 5) was recognized
more strongly in the Beijing strain compared to the other
strains by patient 880. Proteins of approximately 28 and
25 kDa which were recognized strongly by patient 97 in
H37Rv are indicated by circles 3 and 4 (Fig. 4a).
2D Western blotting of patient plasma samples 97, 726,
880 and 941 against WCL proteins extracted from M.
tuberculosis H37Rv, the Beijing strain and F23 was used
to further investigate the differences seen using the 1D
approach through the increased resolution obtainable
(Fig. 5). Many differences in antigen expression levels
between the strains were identified, extending the findings
using 1D Western blotting (Table 4). Again, each patient
showed a characteristic antigen recognition pattern. Patients
97, 880 and 941 reacted with fewer proteins in the Beijing
strain compared to the other strains, indicated by boxes 1,
4 and 7, respectively, in Fig. 5, perhaps suggesting lower
levels of expression of these antigens in the Beijing strain.
Interestingly, patients 880 and 941 were infected with
strains belonging to the Beijing family. Patient 97 also
recognized a spot at approximately 10 kDa more strongly
in the clinical strains than H37Rv (Fig. 5, spot 2). Many
of the other differences observed in antigen recognition
between the strains for patient 97 (Fig. 4a) were obscured
by a broad, diffuse smear on 2D blots (Fig. 5). The smear
is probably lipoarabinomannan (LAM), an immunodominant glycolipid which migrates between 30 and
40 kDa, as demonstrated previously (Hunter et al., 1986).
Patient 726 recognized more proteins in the Beijing strain
compared to H37Rv and F23, which is surprising as this
patient is infected with an F23 strain (Fig. 5, box 3). The
40 kDa and 60 kDa changes observed on 1D blots using
plasma from patient 880 (Fig. 4f) are in a region containing
many spots (Fig. 5, box 4) on the 2D blots and therefore
cannot be uniquely identified. However, notable differences in protein expression within this region, particularly
between the clinical strains and H37Rv, were evident.
Similarly, patient 941 recognized a spot migrating at about
32 kDa more strongly in the clinical strains (Fig. 5, spot
9). In contrast, patient 880 showed decreased recognition
of a spot at approximately 30 kDa (Fig. 5, spot 5) in the
Beijing strain, as did patient 941 with a spot at 35 kDa
(Fig. 5, spot 8).
Although it is difficult to speculate which proteins these
spot differences represent, comparison with silver-stained
2D gels suggests that spot number 6, which is recognized
by patient 880 only, represents a-crystallin. Supporting this
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 04:26:35
1145
C. Pheiffer and others
Fig. 4. Antigen expression by M. tuberculosis strains. Plasma from TB patients (a) 97, (b) 484, (c) 522, (d) 672, (e) 726, (f)
880, (g) 881, (h) 941 was probed against whole-cell lysate proteins extracted from M. tuberculosis H37Rv (lane 1, 1 : 100
plasma dilution; lane 2, 1 : 500 plasma dilution), the Beijing strain (lane 3, 1 : 100 plasma dilution; lane 4, 1 : 500 plasma
dilution) and the F23 strain (lane 5, 1 : 100 plasma dilution; lane 6, 1 : 500 plasma dilution). Proteins were separated by 12 %
SDS-PAGE under reducing conditions and then transferred onto PVDF membranes. Molecular mass markers are indicated on
the left. Prominent differences between the strains are circled.
Table 4. Antigen expression differences detected between strains
Band/spot
1D Western blots
1
2
3
4
5
2D Western blots
1
2
3
4
5
6
7
8
9
Patient
Approx. mol. mass (kDa)
H37Rv*
Beijing
F23
880
880
97
97
880
60
40
28
25
14
+
2
++
++
+
2
++
2
+
++
2
+
2
+
+
97
97
726
880
880
880
941
941
941
35250
10
15225
35250
30
14
35250
35
32
+++
2
+
+++
++
+++
++
+
2
+
+
+++
+
+
++
+
2
+
++
+
++
++
++
++
++
+
+
*Relative expression levels are indicated by +. Protein bands/spots absent are indicated by 2.
1146
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 04:26:35
Microbiology 151
Mycobacterium tuberculosis protein expression
Fig. 5. Antigen expression by M. tuberculosis strains. WCL proteins extracted from cultures of M. tuberculosis H37Rv, the
Beijing strain and F23 were separated by 12 % 2D PAGE, transferred to nitrocellulose membranes and probed with a 1 : 150
dilution of plasma samples from TB patients 97, 726, 880 and 941. Molecular mass markers are indicated on the right.
Differential antigen recognition is indicated by circles and boxes.
speculation is the fact that a band migrating at approximately the molecular mass of a-crystallin was prominently
recognized by patient 880 on 1D blots (Fig. 4f, circle 5).
Analysis of a-crystallin expression across the three strains
using mAbs (Fig. 3a) showed increased expression of different protein species in the Beijing strain, not reflected using
patient anti-sera. This may suggest that these forms are
not expressed in vivo or are not recognized by patient
antibodies. Several of the proteins for which expression
differences were observed using serology have similar
molecular masses to proteins identified as different between
the strains on the silver-stained 2D gels. However, the
serology approach has identified several other antigens
not apparent from comparison of the silver-stained gels,
possibly due to low abundance compared to other proteins. As such, the serology has proved a complementary
approach in identifying antigens differentially expressed
http://mic.sgmjournals.org
between the strains and further investigation of methods to
identify these proteins would be warranted.
DISCUSSION
The availability of the genome sequence of M. tuberculosis
H37Rv (Cole et al., 1998) has facilitated the study of the
proteome of the bacillus. Here, we describe a comparison
of the proteome of M. tuberculosis H37Rv and two local
clinical strains, one belonging to the Beijing strain family
and ten-fold more prevalent than the other clinical strain
(F23) in the study community. The Beijing strain family is
responsible for at least 18?7 % of all TB cases in the study
community (Warren et al., 2000). It was hoped that comparison of protein expression and antigen recognition
patterns of these strains would give some clues as to why the
Beijing strain is more prevalent than the other clinical strain.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 04:26:35
1147
C. Pheiffer and others
The high prevalence of the Beijing genotype worldwide is
indicative of the success of this M. tuberculosis strain type
as a human pathogen (Glynn et al., 2002). It has been suggested that the global dissemination of this strain genotype
may be linked to an altered phenotype, thereby conferring
an advantage over M. tuberculosis strains belonging to other
genotypes (Lopez et al., 2003). This is supported by the
observation that Beijing strains are more virulent and elicit a
non-protective immune response compared to other genotypes, during experimental disease in mice (Lopez et al.,
2003). Infection with Beijing genotypes has also been
linked to the development of fever during the early phase
of treatment (van Crevel et al., 2001). These findings
highlight the need to elucidate the mechanisms underlying
the success of Beijing strains compared to other M. tuberculosis strains. It has been suggested that the wide dispersion of the Beijing strain family compared to other less
prevalent clinical isolates may be related to differential
protein expression (Bifani et al., 2002).
In agreement with previous studies of protein expression
across different M. tuberculosis strains (Betts et al., 2000;
Jungblut et al., 1999; Mattow et al., 2001), this study
revealed that a large portion of the proteome is similarly
expressed. Despite this, we have been able to detect several
differences in protein expression and shown distinct differences in antigen expression levels across the three strains,
with several changes being particular to the Beijing strain.
Several species of a-crystallin, a M. tuberculosis virulence
factor (Monahan et al., 2001; Sherman et al., 2001; Yuan
et al., 1998), were more highly expressed in the Beijing
strain compared to H37Rv and F23. Altered expression of
this protein may be a factor contributing to the virulence
of Beijing strains. The success of the Beijing family as a
human pathogen may in part be due to decreased expression
of Hsp65, PstS1 and the 47kDa protein as it has previously
been suggested that reduced expression of certain major
antigens may allow strains to evade the host immune
response (Stewart et al., 2001). Moreover, differences in
antigen expression levels between the strains, as observed
from 1D and 2D Western blots with patient plasma, may be
significant and could assist the Beijing strain to minimize
recognition by the host immune response, thereby facilitating the increased prevalence of Beijing strains. In order to
further assess the importance of the antigens identified here
in general strain prevalence or whether these differences are
unique to the Beijing family, expression levels could be
determined in other prevalent strains. Importantly, certain
antigen expression differences between strains may only
become evident when the bacteria are growing within
the host environment. Therefore, it will be necessary to
analyse proteins extracted from mycobacteria isolated
from conditions that mimic the host environment, for
example macrophages or animal models. In addition, to
further validate the antigens found to be differentially
expressed here and determine whether they also show
expression differences in vivo, sera from patients infected
with the Beijing strain could be used to probe against
1148
purified recombinant proteins of the antigens of interest,
with the caveat that patient variation and extent of disease
may influence antigen recognition.
Differential protein expression may explain the heterogeneous host humoral immune response and why no
serodiagnostic test for TB has yet been developed
(Lyashchenko et al., 1998). The most marked difference
in protein expression between the Beijing and F23 was
observed for PstS1. PstS1 is a 38 kDa M. tuberculosis
complex-specific phosphate-binding lipoprotein, and a
known B- and T-cell stimulant (Harboe & Wiker, 1992).
To date, PstS1 has shown promise for serological diagnosis of TB, with sensitivities of 70 % and 73 % for smearnegative pulmonary TB and extrapulmonary TB patients,
respectively (Wilkins & Ivanyi, 1990). However, in a recent
evaluation of commercially available tests the highest sensitivity achieved with PstS1 was 55 % (Pottumarthy et al.,
2000). The absence of PstS1 antibodies in some TB patients
may be due to decreased expression of PstS1 in some clinical strains, as observed for the Beijing strain in this study.
These findings suggest that the development of a serodiagnostic test for TB may be hindered by variable protein
expression by M. tuberculosis strains, and support the
development of a test measuring the levels of antibodies
to a panel of antigens that are common to different M.
tuberculosis genotypes (Al Zahrani et al., 2000; Amicosante
et al., 1999). Furthermore, differential expression of PstS1
by different M. tuberculosis genotypes suggests that this
antigen may not be a good choice for a vaccine, and may
partially explain the discrepant results obtained when using
PstS1 as a subunit or DNA vaccine candidate (Falero-Diaz
et al., 2000).
This study has demonstrated extensive humoral heterogeneity between patients, even though they were homogeneous with respect to social class and ethnicity. Humoral
heterogeneity has previously been shown (Lyashchenko
et al., 1998) and attributed, in part, to differential protein expression by M. tuberculosis genotypes. Variation in
antigen recognition was observed even between patients
infected with the same genotype, highlighting the importance of other factors, in addition to differential protein
expression, in dictating the host humoral immune response. These factors could be host factors (Bothamley et al.,
1989), extent of disease (Jackett et al., 1988), or even
multiple infections with different M. tuberculosis genotypes (Warren et al., 2004). Taken together, these results
suggest that detection of mycobacterial antigens (Wallis
et al., 1998) may be more valuable than antibody detection, since antigen detection will not be influenced by the
significant humoral heterogeneity between patients.
In conclusion, this study has shown that proteome analysis of M. tuberculosis genotypes may contribute to our
understanding of the pathogenesis of tuberculosis by
identifying differentially expressed proteins, and potentially helping us to understand why certain strain families,
such as the Beijing family, are more successful than others.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 04:26:35
Microbiology 151
Mycobacterium tuberculosis protein expression
In addition to aiding an understanding of pathogenicity
and strain prevalence, identification of antigens differentially expressed between strains provides important information for consideration in the design of serodiagnostic
tests, vaccines and even drug target selection.
Falero-Diaz, G., Challacombe, S., Banerjee, D., Douce, G., Boyd, A.
& Ivanyi, J. (2000). Intranasal vaccination of mice against infection
with Mycobacterium tuberculosis. Vaccine 18, 3223–3229.
Glynn, J. R., Whiteley, J., Bifani, P. J., Kremer, K. & van Soolingen, D.
(2002). Worldwide occurrence of Beijing/W strains of Mycobacterium
tuberculosis: a systematic review. Emerg Infect Dis 8, 843–849.
Harboe, M. & Wiker, H. G. (1992). The 38-kDa protein of Myco-
bacterium tuberculosis: a review. J Infect Dis 166, 874–884.
ACKNOWLEDGEMENTS
He, X. Y., Zhuang, Y. H., Zhang, X. G. & Li, G. L. (2003). Com-
We thank the National Institutes of Health and the National Institute
of Allergy and Infectious Diseases for mAbs made available through the
Department of Microbiology at Colorado State University. We would
also like to thank Karin Weldingh of the Statens Serum Institut,
Denmark, for kindly providing us with HYB76-8. We are grateful to
Eileen Hoal van Helden for kindly supplying TB plasma samples. This
work was funded by the GlaxoSmithKline Action TB initiative and the
National Research Foundation, South Africa.
parative proteome analysis of culture supernatant proteins of
Mycobacterium tuberculosis H37Rv and H37Ra. Microbes Infect 5,
851–856.
Heifets, L. B. & Good, R. C. (1994). Current laboratory methods for
the diagnosis of tuberculosis. In Tuberculosis: Pathogenesis, Protection,
and Control, pp. 85–110. Edited by B. R. Bloom. Washington, DC:
American Society for Microbiology.
Herbert, B. R., Molloy, M. P., Gooley, A. A., Walsh, B. J., Bryson,
W. G. & Williams, K. L. (1998). Improved protein solubility in two-
dimensional electrophoresis using tributyl phosphine as reducing
agent. Electrophoresis 19, 845–851.
REFERENCES
Hunter, S. W., Gaylord, H. & Brennan, P. J. (1986). Structure and
Abulimiti, A., Qiu, X., Chen, J., Liu, Y. & Chang, Z. (2003). Reversible
antigenicity of the phosphorylated lipopolysaccharide antigens from
the leprosy and tubercle bacilli. J Biol Chem 261, 12345–12351.
methionine sulfoxidation of Mycobacterium tuberculosis small heat
shock protein Hsp16.3 and its possible role in scavenging oxidants.
Biochem Biophys Res Commun 305, 87–93.
Jackett, P. S., Bothamley, G. H., Batra, H. V., Mistry, A., Young, D. B.
& Ivanyi, J. (1988). Specificity of antibodies to immunodominant
Al Zahrani, K., Al Jahdali, H., Poirier, L., Rene, P., Gennaro, M. L. &
Menzies, D. (2000). Accuracy and utility of commercially available
mycobacterial antigens in pulmonary tuberculosis. J Clin Microbiol
26, 2313–2318.
amplification and serologic tests for the diagnosis of minimal
pulmonary tuberculosis. Am J Respir Crit Care Med 162, 1323–1329.
Jungblut, P. R., Schaible, U. E., Mollenkopf, H. J. & 7 other authors
(1999). Comparative proteome analysis of Mycobacterium tubercu-
Amicosante, M., Houde, M., Guaraldi, G. & Saltini, C. (1999). Sensi-
losis and Mycobacterium bovis BCG strains: towards functional
genomics of microbial pathogens. Mol Microbiol 33, 1103–1117.
tivity and specificity of a multi-antigen ELISA test for the serological
diagnosis of tuberculosis. Int J Tuberc Lung Dis 3, 736–740.
Andersen, P., Askgaard, D., Ljungqvist, L., Bennedsen, J. & Heron, I.
(1991). Proteins released from Mycobacterium tuberculosis during
Khanolkar-Young, S., Kolk, A. H. J., Andersen, A. B. & 11 other
authors (1992). Results of the third immunology of leprosy/
growth. Infect Immun 59, 1905–1910.
immunology of tuberculosis antimycobacterial monoclonal antibody
workshop. Infect Immun 60, 3925–3927.
Betts, J. C. & Smith, M. A. (2001). Proteomics. Methods Microbiol 54,
Lopez, B., Aguilar, D., Orozco, H. & 8 other authors (2003). A
315–334.
Betts, J. C., Dodson, P., Quan, S., Lewis, A. P., Thomas, P. J.,
Duncan, K. & McAdam, R. A. (2000). Comparison of the proteome of
marked difference in pathogenesis and immune response induced by
different Mycobacterium tuberculosis genotypes. Clin Exp Immunol
133, 30–37.
Mycobacterium tuberculosis strain H37Rv with clinical isolate CDC
1551. Microbiology 146, 3205–3216.
Lyashchenko, K., Colangeli, R., Houde, M., Al Jahdali, H.,
Menzies, D. & Gennaro, M. L. (1998). Heterogeneous antibody
Bifani, P. J., Mathema, B., Kurepina, N. E. & Kreiswirth, B. N. (2002).
responses in tuberculosis. Infect Immun 66, 3936–3940.
Global dissemination of the Mycobacterium tuberculosis W-Beijing
family strains. Trends Microbiol 10, 45–52.
Manca, C., Tsenova, L., Barry, C. E., III, Bergtold, A., Freeman, S.,
Haslett, P. A., Musser, J. M., Freedman, V. H. & Kaplan, G. (1999).
Bjellqvist, B., Pasquali, C., Ravier, F., Sanchez, J. C. &
Hochstrasser, D. (1993). A nonlinear wide-range immobilized pH
Mycobacterium tuberculosis CDC1551 induces a more vigorous host
response in vivo and in vitro, but is not more virulent than other
clinical isolates. J Immunol 162, 6740–6746.
gradient for two-dimensional electrophoresis and its definition in a
relevant pH scale. Electrophoresis 14, 1357–1365.
Bothamley, G. H., Beck, J. S., Schreuder, G. M., D’Amaro, J.,
de Vries, R. R., Kardjito, T. & Ivanyi, J. (1989). Association of
Mattow, J., Jungblut, P. R., Schaible, U. E., Mollenkopf, H. J.,
Lamer, S., Zimny-Arndt, U., Hagens, K., Muller, E. C. & Kaufmann,
S. H. (2001). Identification of proteins from Mycobacterium
tuberculosis and M. tuberculosis-specific antibody levels with HLA.
J Infect Dis 159, 549–555.
tuberculosis missing in attenuated Mycobacterium bovis BCG strains.
Electrophoresis 22, 2936–2946.
Bradford, M. M. (1976). A rapid and sensitive method for the
Mattow, J., Schaible, U. E., Schmidt, F. & 7 other authors (2003).
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal Biochem 72, 248–254.
Comparative proteome analysis of culture supernatant proteins from
virulent Mycobacterium tuberculosis H37Rv and attenuated M. bovis
BCG Copenhagen. Electrophoresis 24, 3405–3420.
Cole, S. T. R., Brosch, J., Parkhill, T. & 39 other authors (1998).
Deciphering the biology of Mycobacterium tuberculosis from the
complete genome sequence. Nature 393, 537–544.
Engers, H. D., Houba, V., Bennedsen, J. & 19 other authors
(1986). Results of a World Health Organization-sponsored workshop
to characterize antigens recognized by Mycobacterium-specific
monoclonal antibodies. Infect Immun 51, 718–720.
http://mic.sgmjournals.org
Monahan, I. M., Betts, J., Banerjee, D. K. & Butcher, P. D. (2001).
Differential expression of mycobacterial proteins following phagocytosis by macrophages. Microbiology 147, 459–471.
Pheiffer, C., Betts, J., Lukey, P. & van Helden, P. (2002). Protein
expression in Mycobacterium tuberculosis differs with growth stage
and strain type. Clin Chem Lab Med 40, 869–875.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 04:26:35
1149
C. Pheiffer and others
Pottumarthy, S., Wells, V. C. & Morris, A. J. (2000). A comparison of
seven tests for serological diagnosis of tuberculosis. J Clin Microbiol
38, 2227–2231.
van Crevel, R., Nelwan, R. H., de Lenne, W., Veeraragu, Y.,
van der Zanden, A. G., Amin, Z., van der Meer, J. W. &
van Soolingen, D. (2001). Mycobacterium tuberculosis Beijing
Rosenkrands, I., Weldingh, K., Jacobsen, S., Hansen, C. V.,
Florio, W., Gianetri, I. & Andersen, P. (2000a). Mapping and
genotype strains associated with febrile response to treatment.
Emerg Infect Dis 7, 880–883.
identification of Mycobacterium tuberculosis proteins by twodimensional gel electrophoresis, microsequencing and immunodetection. Electrophoresis 21, 935–948.
van Soolingen, D., Qian, L., de Haas, P. E. & 7 other authors (1995).
Rosenkrands, I., King, A., Weldingh, K., Moniatte, M., Moertz, E. &
Andersen, P. (2000b). Towards the proteome of Mycobacterium
Wallis, R. S., Perkins, M., Phillips, M. & 11 other authors (1998).
tuberculosis. Electrophoresis 21, 3740–3756.
Sherman, D. R., Voskuil, M., Schnappinger, D., Liao, R., Harrell, M. I.
& Schoolnik, G. K. (2001). Regulation of the Mycobacterium
tuberculosis hypoxic response gene encoding alpha-crystallin. Proc
Natl Acad Sci U S A 98, 7534–7539.
Sonnenberg, M. G. & Belisle, J. T. (1997). Definition of Myco-
Predominance of a single genotype of Mycobacterium tuberculosis in
countries of east Asia. J Clin Microbiol 33, 3234–3238.
Induction of the antigen 85 complex of Mycobacterium tuberculosis
in sputum: a determinant of outcome in pulmonary tuberculosis
treatment. J Infect Dis 178, 1115–1121.
Warren, R. M., Sampson, S. L., Richardson, M., Van Der Spuy, G. D.,
Lombard, C. J., Victor, T. C. & van Helden, P. D. (2000). Mapping
of IS6110 flanking regions in clinical isolates of Mycobacterium
tuberculosis demonstrates genome plasticity. Mol Microbiol 37,
1405–1416.
bacterium tuberculosis culture filtrate proteins by two-dimensional
polyacrylamide gel electrophoresis, N-terminal amino acid sequencing,
and electrospray mass spectrometry. Infect Immun 65, 4515–4524.
Warren, R. M., Victor, T. C., Streicher, E. M., Richardson, M.,
Beyers, N., van Pittius, N. C. & van Helden, P. D. (2004). Patients
Sorensen, A. L., Nagai, S., Houen, G., Andersen, P. & Andersen,
A. B. (1995). Purification and characterization of a low-molecular-mass
with active tuberculosis often have different strains in the same
sputum specimen. Am J Respir Crit Care Med 169, 610–614.
T-cell antigen secreted by Mycobacterium tuberculosis. Infect Immun
63, 1710–1717.
Wilkins, E. G. & Ivanyi, J. (1990). Potential value of serology for
Stewart, G. R., Snewin, V. A., Walzl, G. & 7 other authors
(2001). Overexpression of heat-shock proteins reduces survival of
Yuan, Y., Crane, D. D., Simpson, R. M., Zhu, Y. Q., Hickey, M. J.,
Sherman, D. R. & Barry, C. E., III (1998). The 16-kDa alpha-crystallin
Mycobacterium tuberculosis in the chronic phase of infection. Nat
Med 7, 732–737.
(Acr) protein of Mycobacterium tuberculosis is required for growth
in macrophages. Proc Natl Acad Sci U S A 95, 9578–9583.
1150
diagnosis of extrapulmonary tuberculosis. Lancet 336, 641–644.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 12 Aug 2017 04:26:35
Microbiology 151