Expression, Secretion, and Glycosylation of the 45- and 47-kDa Glycoprotein of Mycobacterium tuberculosis in Streptomyces lividans

Abstract

The gene encoding the 45/47 kDa glycoprotein (Rv1860) of Mycobacterium tuberculosis was expressed in Streptomyces lividans under its own promoter and under the thiostrepton-inducible Streptomyces promoter PtipA. The recombinant protein was released into the culture medium and, like the native protein, migrated as a double band at 45 and 47 kDa in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels. However, in contrast to the native protein, only the 47-kDa recombinant protein could be labeled with concanavalin A (ConA). Carbohydrate digestion with jack bean alpha-D-mannosidase resulted in a reduction in the molecular mass of the recombinant protein upper band and completely eliminated ConA binding. Two-dimensional gel electrophoresis revealed only one isoelectric point for the recombinant protein. Comparative fingerprinting analysis of the individually purified upper and lower recombinant protein bands, treated under the same conditions with specific proteases, resulted in similar peptide patterns, and the peptides had the same N-terminal sequence, suggesting that migration of the recombinant protein as two bands in SDS-PAGE gels could be due to differences in glycosylation. Mass spectrometry analysis of the recombinant protein indicated that as in native protein, both the N-terminal and C-terminal domains of the recombinant protein are glycosylated. Furthermore, it was determined that antibodies of human tuberculosis patients reacted mainly against the carbohydrate residues of the glycoprotein. Altogether, these observations show that expression of genes for mycobacterial antigens in S. lividans is very useful for elucidation of the functional role and molecular mechanisms of glycosylation in bacteria.

Full text

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2004, p. 679–685 Vol. 70, No. 2
0099-2240/04/$08.00⫹0 DOI: 10.1128/AEM.70.2.679–685.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Expression, Secretion, and Glycosylation of the 45- and 47-kDa
Glycoprotein of Mycobacterium tuberculosis in
Streptomyces lividans
Martha Lara,
1
Luis Servı´n-Gonza´lez,
2
Mahavir Singh,
3
Carlos Moreno,
4
Ingrid Cohen,
1
Manfred Nimtz,
3
and Clara Espitia
1
*
Departamento de Inmunologı´a
1
and Departamento de Biologı´a Molecular,
2
Instituto de Investigaciones Biome´dicas, Universidad
Nacional Auto´noma de Me´xico, Me´xico D.F., Me´xico; GBF, German National Research Center for Biotechnology,
Braunschweig, 38124 Braunschweig, Germany
3
; and Department of Bacteriology, Royal Free and University
College Medical School, Windeyer Institute, London W1P 6DB, United Kingdom
4
Received 22 May 2003/Accepted 30 October 2003
The gene encoding the 45/47 kDa glycoprotein (Rv1860) of Mycobacterium tuberculosis was expressed in Strep-
tomyces lividans under its own promoter and under the thiostrepton-inducible Streptomyces promoter P
tipA
. The
recombinant protein was released into the culture medium and, like the native protein, migrated as a double
band at 45 and 47 kDa in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels.
However, in contrast to the native protein, only the 47-kDa recombinant protein could be labeled with con-
canavalin A (ConA). Carbohydrate digestion with jack bean ␣-D-mannosidase resulted in a reduction in the
molecular mass of the recombinant protein upper band and completely eliminated ConA binding. Two-
dimensional gel electrophoresis revealed only one isoelectric point for the recombinant protein. Comparative
fingerprinting analysis of the individually purified upper and lower recombinant protein bands, treated under
the same conditions with specific proteases, resulted in similar peptide patterns, and the peptides had the same
N-terminal sequence, suggesting that migration of the recombinant protein as two bands in SDS-PAGE gels
could be due to differences in glycosylation. Mass spectrometry analysis of the recombinant protein indicated
that as in native protein, both the N-terminal and C-terminal domains of the recombinant protein are glyco-
sylated. Furthermore, it was determined that antibodies of human tuberculosis patients reacted mainly against
the carbohydrate residues of the glycoprotein. Altogether, these observations show that expression of genes for
mycobacterial antigens in S. lividans is very useful for elucidation of the functional role and molecular
mechanisms of glycosylation in bacteria.
Glycosylation is an important covalent modification of pro-
teins. While eukaryotic glycoproteins have been characterized
in detail, information about the structure, function, and bio-
synthetic pathways of prokaryotic glycoproteins is scarce. The
list of known bacterial glycoproteins is growing, and the variety
of components and structures observed indicates the impor-
tance of glycosylation in cell processes, as well as the potential
role of glycosylation in pathogenesis (2, 23, 25).
Carbohydrates have been reported to be associated with
antigenic proteins of pathogenic mycobacteria (7, 10, 11). The
importance of carbohydrates attached to proteins in immune
recognition has been demonstrated by the decreased capacity
of the Mycobacterium tuberculosis 45/47 kDa recombinant pro-
tein (r45/47 kDa) to stimulate T-cell lymphocyte responses
when its mannosylation pattern is changed (12, 32).
Until now, glycosylation of M. tuberculosis proteins has been
confirmed only for the 45/47 kDa protein and for the Myco-
bacterium bovis MPB83 protein (Rv2873), in which chemical
linkage between carbohydrate and protein was demonstrated
(5, 24). The function of glycosylation of mycobacterial glyco-
proteins remains unknown. The 45/47 kDa protein corre-
sponds to the Rv1860 sequence, which is encoded by a gene
which has been annotated as modD in the M. tuberculosis
genome sequence (GenBank accession no. X99258). This sug-
gests that the 45/47 kDa protein could be part of a putative
molybdenum transport system.
Proteins homologous to the 45/47 kDa protein have been
found in M. bovis,Mycobacterium avium,Mycobacterium leprae,
and Mycobacterium vaccae. All of these proteins have the abil-
ity to bind to fibronectin (31, 33, 34, 40).
The 45/47 kDa proteins from M. tuberculosis and M. bovis
are immunodominant antigens which are secreted into the
culture medium and migrate as glycosylated double bands in
sodium dodecyl sulfate (SDS)-polyacrylamide gel electro-
phoresis (PAGE) gels (8). In the present study, the M. tuber-
culosis gene encoding the 45/47 kDa protein was expressed in
Streptomyces lividans, a gram-positive, sporulating, mycelial
bacterium which is not pathogenic. Streptomyces strains are a
well-known source of antibiotics and are characterized by their
capacity to produce secreted proteins (26, 30). In addition, like
many other eubacteria, S. lividans has the ability to glycosylate
its own proteins, as well as heterologous proteins (18, 21, 28).
The ability to glycosylate cloned gene products enhances the
usefulness of Streptomyces as a host for the production of
heterologous polypeptides, and this system is a potent tool for
studying glycosylation processes in bacteria. The existence of
vectors for inducible protein expression in S. lividans allows
production of large amounts of proteins suitable for immuno-
* Corresponding author. Mailing address: Instituto de Investigacio-
nes Biome´dicas, Departamento de Inmunologı´a, Apartado Postal 70-
228, 04510 Me´xico, D.F., Me´xico. Phone (525) 6223818. Fax: (525)
6223369. E-mail: espitia@servidor.unam.mx.
679

logical and biochemical characterization of glycoproteins. In
this study we expressed the M. tuberculosis 45/47 kDa protein
in S. lividans in order to assess the potential of the expression
system for obtaining M. tuberculosis glycoproteins with vaccine
and/or diagnostic potential.
MATERIALS AND METHODS
Bacterial strains and plasmids. Escherichia coli XL1-Blue was used as a host
for recombinant plasmids. The laboratory strain M. tuberculosis H37Rv was
obtained from the American Type Culture Collection (Rockville, Md.). Wild-
type S. lividans 1326 and the plasmid vectors pIJ486 and pIJ6021 (4) were
obtained from D. A. Hopwood, John Innes Centre, Norwich, United Kingdom.
Isolation and cloning of the DNA region carrying the 45/47 kDa protein gene.
A cosmid clone carrying the M. tuberculosis gene for the 45/47 kDa protein was
isolated by screening the Tropist3 DNA cosmid library of M. tuberculosis H37Rv
(17); a PCR product corresponding to the amplified gene was used as the probe.
DNA from the positive cosmid colony was enzyme restricted. Fragments were
separated on 8% agarose gels and transferred by blotting onto nylon filters
(Amersham). The filters were then prehybridized and probed at 42°Cin6⫻SSC
(1⫻SSC is 0.15 M NaCl plus 0.015 M sodium citrate, pH 7.0) containing 1 mM
sodium phosphate, 1 mM EDTA, 0.05% skim milk, and 0.5% SDS for 2 and 4 h,
respectively. After this, the filters were washed twice in 2⫻SSC for 15 min each
time and once in 2⫻SSC–0.3% SDS for 15 min and autoradiographed by
exposing the filters to X-ray film (Kodak). A 3.2-kb EcoRI fragment from this
cosmid clone was subcloned into the EcoRI site of pUC18 to obtain pUC18MT-
45. This plasmid carried the complete gene for the 45/47 kDa protein, together
with additional DNA on either side of the gene (1.1 kb upstream and 1.0 kb
downstream).
Cloning and expression of the gene encoding the 45/47 kDa protein in S.
lividans.The 3.2-kb EcoRI fragment obtained from pUC18MT-45 was subcloned
into the EcoRI site of the high-copy-number Streptomyces plasmid vector pIJ486
(4), resulting in plasmid pIJ486MT-45.
PCR amplification of a 983-bp fragment containing the complete 45/47 kDa
protein gene was carried out with oligonucleotides CGGATCCATATGCATC
AGGTGGACCC and GGAATTCAGGCCGGTAAGGTCC. The BamHI and
NdeI restriction sites in the sense primer and the EcoRI site in the reverse primer
(underlined) were included as extensions for further manipulation of the ampli-
fied fragments. In particular, the NdeI site (CATATG) was designed to contain
the ATG start codon of the gene in order to allow cloning into the Streptomyces
expression vector pIJ6021 (4). Amplification was carried out with Taq DNA
polymerase (Perkin-Elmer) as recommended by the manufacturer. The PCR
protocol consisted of an initial denaturation step of 5 min at 95°C, followed by 30
cycles of 1 min of denaturation at 95°C, 1 min of annealing at 50°C, and 1 min
of extension at 72°C and then a 5-min final extension at 72°C. The PCR product
was then digested with BamHI and EcoRI and subcloned into BamHI-EcoRI-cut
pUC18 to obtain plasmid pUC18MT-45.1. To clone this fragment in the Strep-
tomyces expression vector pIJ6021, pUC18MT-45.1 was digested with NdeI and
EcoRI, and the 1-kb insert was subcloned into NdeI-EcoRI-cut pIJ6021 to obtain
pIJ6021MT-45.
Growth of Streptomyces cultures. Spores of S. lividans carrying the different
plasmids were obtained on solid R5 medium (4) with the appropriate antibiotics.
Freshly harvested spores were used to inoculate Luria-Bertani broth modified by
addition of 34% sucrose to obtain dispersed mycelial growth. For growth of S.
lividans harboring pIJ486MT-45, thiostrepton was added at a concentration of 50
␮g/ml, and cultures were grown at 30°C for 72 h with shaking. For cultures
carrying pIJ6021MT-45, kanamycin was added at a concentration of 100 ␮g/ml,
and after 12 h of growth at 30°C, thiostrepton was added at a concentration of 10
␮g/ml and growth was continued for an additional 24 to 36 h. Subsequently,
recombinant culture filtrates (rCF) were obtained by removing the mycelium by
centrifugation at 8,000 ⫻gfor 30 min at 4°C and filtration through Whatman no.
1filter paper disks. Proteins were precipitated from the supernatant with am-
monium sulfate (73% saturation), recovered by centrifugation, dialyzed against
distilled H
2
O, and dried by lyophilization.
Growth of M. tuberculosis cultures. M. tuberculosis H37Rv was cultured on
Proskauer-Beck synthetic medium for 4 to 6 weeks. Culture filtrate (CF) proteins
and a fraction enriched with the 45/47 kDa protein (F4) were obtained from CF
as described elsewhere (6, 7).
Antibodies. Monoclonal antibody 6A3 (MAb 6A3) raised against the 45/47
kDa protein was obtained as described previously (8). Rabbit polyclonal anti-
serum against a synthetic peptide (GEVAPTPTTPTPQRTLPAC) derived from
the C-terminal sequence of the 45/47 kDa protein was produced in New Zealand
rabbits. Animals were immunized subcutaneously on days 0 and 8 with 200 ␮gof
purified peptide in incomplete Freund’s adjuvant. Two weeks later they were
boosted intraperitoneally with 100 ␮g of purified peptide, and then they were
boosted every fortnight for 2 months. Two weeks after the last immunization the
animals were bled, and the resulting antiserum was designated anti-C45.
Human sera. Sera from patients with pulmonary tuberculosis diagnosed by
smear and/or sputum culture were obtained from Hospital General in Mexico.
Healthy control sera were obtained from laboratory workers.
SDS-PAGE and Western blotting. Electrophoresis in 12% polyacrylamide gels
containing SDS and subsequent immunoblotting procedures were carried out by
using the standard methods (19, 38). Five-microgram portions of S. lividans rCF
and M. tuberculosis F4 were electrophoresed in polyacrylamide gels. For antigen
detection, proteins were transferred to polyvinylidene difluoride Immunobilon
nylon membranes (Millipore) and incubated with MAb 6A3 and with anti-C45.
For carbohydrate detection, proteins were stained with concanavalin A (ConA)-
peroxidase (Sigma). Nonspecific binding was blocked by incubating blots with
1% (wt/vol) bovine serum albumin (BSA) in phosphate-buffered saline (PBS)
containing Tween 20 (0.05%, vol/vol); after washes with PBS-Tween 20, the
membranes were incubated for1hatroom temperature (RT) with either MAb
6A3 diluted 1/500, anti-C45 diluted 1/2,000, or ConA (2.5 ␮g/ml). After washes
with PBS-Tween 20, the membranes were incubated with peroxidase conjugates,
anti-mouse immunoglobulin G diluted 1/2,000 (Zymed), or protein A (Sigma)
diluted 1/2,000. After incubation for 30 min at RT, the blots were stained for
peroxidase activity by adding 3,3-diaminobenzidine (Sigma) and hydrogen per-
oxide in PBS.
Two-dimensional PAGE was performed as follows. Urea was added to 10 ␮g
of rCF from S. lividans transformed with pIJ486MT-45 at a final concentration of
9 M, and then lysis buffer containing urea and Nonidet P-40 was added as
described by O’Farrell (27). Samples were separated initially by isoelectric fo-
cusing in tube gels containing 4% ampholytes in the pH range from 3.5 to 5.0
(Pharmacia) and then by SDS-PAGE in the second dimension as described
above. Proteins were transferred to nylon membranes and incubated with MAb
6A3. Reactivity was developed as described above. Labeling of glycopropteins
with biotin-hydrazide and jack bean ␣-D-mannosidase digestion were performed
as described previously (8).
Purification of proteins. The pH of rCF extract from S. lividans transformed
with pIJ6021MT-45 was adjusted to 5 with acetic acid at 4°C, proteins were
recovered by centrifugation at 8,000 ⫻gfor 10 min, and the pellet was resus-
pended in the appropriate buffer. The chromatography procedures were per-
formed with an AKTA-Prime system (Pharmacia Biotech). The ConA-binding
protein (47-kDa protein) was separated from the 45-kDa protein by affinity
chromatography on a ConA-Sepharose column (Pharmacia Biotech); elution
was carried out with a gradient of 0 to 0.05 M ␣-methyl-mannopyranoside
(Sigma) (7). Flowthrough from the ConA column contained the 45-kDa protein,
which was loaded onto a HiTrap Q-Sepharose column equilibrated with 20 mM
Tris-HCl (pH 8). Elution was then carried out with a linear 0 to 1 M NaCl
gradient. Fractions were collected, dialyzed, concentrated, and analyzed by SDS-
PAGE. The r45/47 kDa protein was also purified by anion-exchange chromatog-
raphy from pH 5 rCF supernatant by using the protocol described above. Protein
quantification was carried out with a protein quantification kit (Bio-Rad).
Fingerprinting assay. Digestion of individually purified recombinant 45- and
47-kDa proteins was performed with the protein fingerprint system (Promega)
used according to the manufacturer’s protocol. Briefly, 1 ␮g of purified fractions
or 2.5 ␮g of the r45/47 kDa protein was diluted in electrophoresis cocktail and
heat denatured by incubation at 95°C for 5 min. Then 5 ␮l (0.2 ␮g) of Lys-C and
5␮l (0.2 ␮g) of Glu-C were added to each sample; after digestion, samples were
transferred to polyvinylidene difluoride nylon membranes after SDS–15%
PAGE. Blots were stained with Coomassie blue, and the bands were cut out and
subjected to automated Edman degradation with a gas phase sequencer (PE-
Applied Biosystems, Weiterstadt, Germany).
Protein preparation for mass spectrometric analysis. r45/47 kDa protein pu-
rified by anion-exchange chromatography was separated by SDS-PAGE. Indi-
vidual 45- and 47-kDa bands were excised from the gel after Coomassie blue
staining. The gel slides were washed several times with 200 ␮l of water, dehy-
drated in 50 ␮l of acetonitrile, and dried. Then the gel pieces were washed twice
with 100 mM NH
4
HCO
3
, dehydrated with acetonitrile, and dried. Digestion of
proteins was carried out in 50 mM NH
4
HCO
3
containing 4 ng of trypsin (Pro-
mega Corp.) per ␮lat37°C overnight (⬍15 h). The resulting peptides were
extracted with 25 mM NH
4
HCO
3
–acetonitrile and then with 5% formic acid
(HCOOH)–acetonitrile. After drying, the dried peptides were reconstituted in
20 ␮l of 0.5% HCOOH–5% methanol (MeOH). The peptides were then purified
on reversed-phase C
18
ZipTip pipette tips (Millipore Corp.). Briefly, ZipTips
were washed with 0.5% HCOOH–65% MeOH and equilibrated with 0.5%
680 LARA ET AL. APPL.ENVIRON.MICROBIOL.

HCOOH–5% MeOH. The peptides were applied to the ZipTips and eluted with
1.0% HCOOH–65% MeOH.
Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass
spectrometry (MS) of tryptic peptides. A 0.5- to 1-␮l portion of each concen-
trated peptide solution was mixed with the same volume of a saturated matrix
solution of ␣-cyano-4-hydoxycinnamic acid (Bruker Daltonics) in 0.5%
HCOOH–65%MeOH, spotted onto a 384 MTP target, and dried at RT. The
molecular masses of the tryptic peptides were determined in the positive-ion
mode with a Bruker Ultraflex TOF mass spectrometer (Bruker Daltonics GmbH,
Leipzig, Germany) by using the reflectron and delayed extraction facilities for
enhanced resolution, an N
2
laser (337 nm) operating with a 3-ns pulse width and
10
7
to 10
8
W/cm
2
at the surface of 0.2-mm
2
spots, and an acceleration voltage of
25 kV.
Enzyme-linked immunosorbent assay (ELISA). Polystyrene 96-well microtiter
plates (Costar 3590) were coated with 100 ␮lof2.5␮g of 45-kDa protein per ml,
glycosylated 47-kDa protein, and jack bean ␣-mannosidase-treated 47-kDa pro-
tein in 0.05 M carbonate-bicarbonate buffer (pH 9.6) at 37°C overnight. The
plates were blocked with 2% BSA in PBS–0.05% Tween 20 for1hatRT.Four
human tuberculosis sera previously found to react against native 45/47 kDa
protein and four healthy sera were chosen for the assay. One hundred microliters
of a 1:100 dilution of each serum and a 1:1,000 dilution of MAb 6A3 were added
to each well in triplicate in PBS-Tween 20-BSA and incubated for1hatRT.
After the plates were washed with PBS-Tween 20-BSA, they were incubated with
the appropriate horseradish conjugates diluted in PBS-Tween 20-BSA contain-
ing protein A diluted 1:2,000 and anti-mouse immunoglobulin G diluted 1:2,000.
The plates were incubated for 1 h and then washed. Enzyme activity was assayed
by incubation for 5 min at RT with 50 ␮lof␱-phenylendiamine (Sigma). The
reaction was stopped with 50 ␮l of 3 N HCl, and the optical density at 492 nm was
determined with an automatic microtiter plate reader (Labsystem).
RESULTS
Expression of the 45/47 kDa protein gene of M. tuberculosis
from its own promoter and from the P
tipA
promoter. The M.
tuberculosis gene encoding the 45/47 kDa protein was cloned in
the Streptomyces vector pIJ486, resulting in pIJ486MT-45. To-
tal extracellular protein from S. lividans carrying either pIJ486
or pIJ486MT-45 was electrophoresed on SDS-PAGE gels. The
45/47 kDa protein bands could not be clearly distinguished
from those of S. lividans carrying only the vector (Fig. 1A, lanes
1 and 2). However, Western blot analysis showed that the gene
was expressed in cultures of S. lividans carrying pIJ486MT-45
and that the 45/47 kDa protein was released into the medium
(Fig. 1B, lane 2) (see below). Since in pIJ486MT-45 the cloned
gene is located downstream of a transcriptional terminator
(17), transcription must originate from a promoter present in
the cloned fragment. Overexpression of the 45/47 kDa protein
was achieved by cloning the gene in the expression vector
pIJ6021 under the thiostrepton-inducible promoter P
tipA
,re
-
sulting in plasmid pIJ6021MT-45 (Fig. 1A, lane 4). Total ex-
tracellular protein from S. lividans carrying either pIJ6021 or
pIJ6021MT-45 was analyzed by SDS-PAGE (Fig. 1A, lanes 3
and 4); it was evident that large amounts of the 45/47 kDa
protein were secreted into the culture medium, where it rep-
resented about 25% of the total extracellular protein (Fig. 1A,
lane 4).
Characterization of recombinant proteins by SDS-PAGE
and Western blotting. CF protein extracts from S. lividans
carrying pIJ486MT-45 were prepared and subjected to West-
ern blot analysis with MAb 6A3 and anti-C45 rabbit polyclonal
antibody. MAb 6A3 recognized a double band, which migrated
slightly above the native protein from M. tuberculosis H37Rv in
SDS-PAGE gels (Fig. 1B, lanes 1 and 2). The polyclonal anti-
C45 antibody recognized only the 47-kDa upper band of the
native protein and the 45-kDa lower band of the r45/47 kDa
protein (Fig. 1B, lanes 3 and 4). When the recombinant protein
was analyzed by two-dimensional gel electrophoresis, the up-
per and lower bands had identical acidic isoelectric points (Fig.
1C), in contrast to the native protein bands, which have been
shown to migrate as several spots having different isoelectric
points (8).
To determine whether the recombinant protein produced by
S. lividans was able to bind to ConA, as previously described
for the M. tuberculosis native 45/47 kDa protein (7, 8), rCF
proteins were transferred to a nylon membrane after SDS-
PAGE and incubated with ConA-peroxidase. Only the upper
band showed positive staining with the lectin (Fig. 2A, lane 3).
Accordingly, only this band showed an apparent reduction in
molecular mass of about 0.5 kDa after digestion with ␣-D-
mannosidase, which caused it to migrate at the same level as
the lower band (Fig. 2A, lane 2). In addition, ␣-D-mannosidase
digestion eliminated the ConA reactivity of the upper band
(Fig. 2A, lane 4). To assess whether the recombinant lower
band was glycosylated, recombinant and control S. lividans CF
extracts were labeled with biotin-hydrazide after periodate ox-
idation and then transferred to a nylon membrane and incu-
bated with streptavidine-peroxidase. Although several proteins
in the S. lividans control CF were labeled with biotin-hydra-
zide, the r45/47 kDa bands could be clearly distinguished when
the rCF was used. Differences in the intensity of biotin-hydra-
zide labeling between the bands were observed; the lower band
was most intensively labeled, probably due to differences in
glycosylation between the bands (Fig. 2B, lanes 1 and 2). Con-
trol membranes were developed with MAb 6A3 (Fig. 2B, lanes
3 and 4).
Protein purification. The r45/47 kDa protein was purified
from culture supernatants by anion-exchange chromatography,
and both forms of the protein eluted in one peak at 0.3 M
FIG. 1. Expression of M. tuberculosis 45/47 kDa protein in S. livi-
dans. (A) Coomassie blue-stained SDS-PAGE gel of rCF from S.
lividans carrying different plasmids. Lane 1, pIJ486 control vector with-
out insert; lane 2, pIJ486MT-45 recombinant vector; lane 3, pIJ6021
vector with thiostrepton-inducible Streptomyces promoter P
tipA
; lane 4,
pIJ6021MT-45 recombinant vector. (B) Western blot of native and
rCF proteins. Lanes 1 and 3, M. tuberculosis 45/47 kDa CF-enriched
fraction (F4) as a positive control; lanes 2 and 4, rCF from S. lividans
carrying pIJ486MT-45, which overexpressed the 45/47 kDa protein.
Lanes 1 and 2 were developed with MAb 6A3, while lanes 3 and 4 were
developed with anti-C45 polyclonal antibody. (C) Two-dimensional
PAGE analysis of rCF from S. lividans carrying pIJ486MT-45. The blot
was developed with MAb 6A3.
VOL. 70, 2004 S. LIVIDANS, GLYCOSYLATION, AND M. TUBERCULOSIS 681

NaCl. About 5 mg of protein was obtained from 1 liter of
culture. The protein was further separated into ConA-binding
and nonbinding fractions by ConA affinity chromatography.
The fraction bound to the ConA-Sepharose column was eluted
with ␣-methyl-mannopyranoside and migrated as the 47-kDa
upper band in SDS-PAGE gels. On the other hand, the frac-
tion did not bind to the ConA-Sepharose column and was
recovered from the flowthrough from a HiTrap Q-Sepharose
column, and it migrated as a 45-kDa band in SDS-PAGE gels
(Fig. 3).
Fingerprinting. Endoproteinase digestion of the 47- and 45-
kDa proteins (upper and lower bands, respectively) produced
identical proteolytic patterns. Lys-C digestion released two
peptides with apparent molecular masses of about 20 and 30
kDa, while Glu-C digestion produced two peptides with appar-
ent molecular masses of about 19 and 31 kDa (results not
shown). The N-terminal sequences of the undigested 47- and
45-kDa proteins were determined; the two sequences were
identical, and this showed that the signal peptide was cleaved
off at precisely the same position as in the native protein. This
result agrees with the presence in S. lividans of a signal pepti-
dase that cleaves at the consensus amino acid sequence AXA
in the leader peptide (29). On the other hand, the N-terminal
sequences of the proteolytic fragments generated from diges-
tion of the 47- and 45-kDa proteins were also identical. Figure
4 shows the proteolytic enzyme digestion sites based on the
N-terminal sequences of the different peptides.
The MALDI-TOF MS peptide map of the tryptically di-
gested recombinant 47-kDa protein is shown in Fig. 5. All
major peaks could be assigned. The signal comprising amino
acids 150 to 165 had a molecular ion 76 Da lower than ex-
pected. MS-MS analysis unequivocally demonstrated that
there was a change from Y to S at position 161 (data not
FIG. 2. ␣-D-Mannosidase treatment and biotin-hydrazide labeling
of r45/47 kDa protein. (A) Effects of treatment with ␣-D-mannosidase
of rCF extracts from S. lividans carrying the pIJ486MT-45 vector.
Lanes 1 and 3, rCF extracts not treated with ␣-D-mannosidase; lanes 2
and 4, rCF extracts treated with ␣-D-mannosidase. Lanes 1 and 2 were
developed with MAb 6A3, while lanes 3 and 4 were developed with
ConA-peroxidase. The arrow in lane 4 indicates the position of the
␣-D-mannosidase enzyme which reacted with ConA. (B) Biotin-hydra-
zide labeling. Lane 1, rCF from S. lividans carrying pIJ486; lane 2, rCF
from S. lividans carrying pIJ486MT-45. Blots were developed with
streptavidine-peroxidase. Lanes 3 and 4 were the same as lanes 1 and
2, but the blots were developed with MAb 6A3.
FIG. 3. Purification of r45/47 kDa protein: profile of ConA affinity
chromatography. The left lane of the inset shows the protein that was
retained by the ConA column after elution (47-kDa band), which was
rechromatographed by anion-exchange chromatography and analyzed
by SDS-PAGE. The right lane of the inset shows protein that was not
retained by the ConA column (45-kDa band), which was rechromato-
graphed by anion-exchange chromatography and analyzed by SDS-
PAGE. B in the yaxis is NaCl gradient from 0 to 1 M.
FIG. 4. Cleavage sites of r45/47 kDa protein with proteolytic en-
zymes. The cleavage sites for each enzyme are indicated by arrows, and
the N-terminal sequence obtained for each fragment is indicated by
boldface type. The individual glycosylation sites of native protein are
enclosed in brackets, and glycosylated recombinant peptides are un-
derlined.
FIG. 5. MALDI-TOF peptide map of the tryptically digested re-
combinant 47-kDa protein. The signal comprising amino acids 150 to
165 showed a molecular ion 76 Da lower than expected. MS-MS
analysis unequivocally demonstrated that there was a change from Y to
S at position 161. Both the N-terminal peptide T1-73 and the C-
terminal peptide T239-282 were found to be posttranslationally mod-
ified by hexose residues.
682 LARA ET AL. APPL.ENVIRON.MICROBIOL.

shown). This change could have been due to an error during
PCR amplification of the gene. Both the N-terminal peptide
T1-73 and the C-terminal peptide T239-282 were found to be
posttranslationally modified by hexose residues, which could
be identified as mannose due to the binding characteristics of
the protein to ConA. Zero to nine mannose residues were
detected in the T1-73 peptide, as indicated by a ladder of
molecular ions differing by 162 Da, which is characteristic of
hexose residues, whereas zero to four mannose residues were
found to be linked to the T239-282 peptide (Fig. 5). Glycosy-
lation on the 45-kDa protein was also found at the same po-
sition of the 47-kDa protein but with a different mannosylation
pattern, the unglycosylated N-terminal peptide had the highest
signal intensity, and peptides with one to five mannose residues
were detectable with less than one-third the intensity of the
unglycosylated peptide. In the C-terminal peptide, the intensity
of the glycopeptides with one to three mannose residues was
less than 20% of the intensity of the unglycosylated peptide
(data not shown).
ELISA. Comparison of reactivities of human tuberculosis
and control sera by the ELISA showed that only the ConA-
binding 47-kDa protein was recognized by sera of individuals
infected with M. tuberculosis; in contrast, MAb 6A3 recognized
both the 45- and 47-kDa proteins (Fig. 6). The interaction of
antibodies with the 47-kDa protein was lost after treatment
with jack bean ␣-D-mannosidase.
DISCUSSION
There have been several reports of mycobacterial gene ex-
pression in Streptomyces. In particular, expression of M. bovis
BCG and M. leprae genes in S. lividans has been reported
elsewhere (16, 20). More recently, two major antigens of M.
tuberculosis, the 38- and 19-kDa proteins (Rv0934 and
Rv3763), were overproduced by S. lividans as secreted extra-
cellular proteins when their genes were cloned in an engi-
neered expression-secretion vector (39). In this work, the 45/47
kDa glycoprotein of M. tuberculosis was expressed in S. livi-
dans. The gene that encodes this protein (modD) is the fourth
gene of a putative molybdenum transport operon. Even though
the fragment cloned in the vector pIJ486 does not carry the
entire operon, expression was observed. Transcriptional read-
through from the vector can be ruled out as the cause of ex-
pression, since the gene was cloned downstream of a strong
transcriptional terminator (4). Therefore, it is very likely that
the fragment cloned in pIJ486, which carried 1 kb of DNA
upstream of the gene, carried an internal promoter capable of
driving expression of the M. tuberculosis 45/47 kDa protein
gene in S. lividans. This shows that Streptomyces is able to rec-
ognize M. tuberculosis promoters, which is not surprising given
the relatedness of these organisms in the actinomycetes group,
which has been shown to extend to the genome level (1). The
presence of promoters which are internal to operons in myco-
bacteria has been described recently for the phosphate-specific
transport operon, which encodes the PstS-1 protein, and for
genes of the putative mpt70-mpt83 operon (14, 37).
Studies on glycosylation of mycobacterial proteins by S. livi-
dans are scarce. However, a few reports have shown that this
microorganism has protein glycosylation ability (18, 21, 28).
In the present work, the well-known M. tuberculosis 45/47
kDa glycoprotein was expressed in S. lividans, and we showed
that the protein is glycosylated. Like the native protein, the
recombinant protein was secreted into the medium and mi-
grated as a double band in SDS-PAGE gels; however, in con-
trast to the native mycobacterial protein, only the upper 47-
kDa band of the recombinant protein from S. lividans reacted
with ConA. This suggests either that the protein is glycosylated
in S. lividans by sugars other than mannose or that it is glyco-
sylated in a configuration not recognized by ConA. In addition,
only the 45-kDa lower band of the recombinant protein could
be recognized by the anti-C45 antibody. This difference in
reactivity between the native and recombinant proteins can be
explained by differences in glycosylation. The same explanation
can be extrapolated to the native 45/47 kDa protein, in which
the existence of the two bands can be attributed to changes in
glycosylation rather than to degradation or C-terminal modi-
fications. Our observations support the hypothesis that differ-
ences in glycosylation are the cause of the two forms of the
recombinant protein, since no differences in N-terminal se-
quences or isoelectric points could be found between the 45-
and 47-kDa proteins. In addition, evidence supporting the hy-
pothesis that the recombinant protein is glycosylated at the
same position as the native protein was obtained from the
ConA reactivity of all peptides generated by Glu-C and Lys-C
digestion of the S.lividans r45/47 kDa protein (data not shown).
This finding was further supported by the MALDI-TOF MS
analysis that showed that, as in the native protein, the glyco-
sylation sites of the r45/47 kDa protein are located in both the
N and C termini of the molecule. Interestingly, the glycosyla-
tion pattern of the recombinant N terminus from zero to nine
hexose residues was very similar to that of the native proteins
from three M. tuberculosis reference strains (12). In contrast,
differences in the degree of glycosylation were found between
recombinant 47- and 45-kDa forms of the protein, as reported
elsewhere for the native protein (12); these differences could
explain the existence of two forms of the molecule, as well as
the differences in ConA reactivity and biotin labeling. There-
fore, it is important to determine if the individual glycosylation
sites of the r45/47 kDa protein are the same as those defined
for the native protein (5). Finally, an interesting observation
FIG. 6. ELISA of purified 45- and 47-kDa proteins: reactivities of
antibodies with the ConA-binding 47-kDa protein (open bars), with
the ␣-D-mannosidase-treated 47-kDa protein (gray bars), and with the
non-ConA-binding 45-kDa protein (solid bars). Reactivity was tested
with MAb 6A3 (6A3), with sera from human tuberculosis patients
(TB) (2), and with sera from healthy individuals (Healthy) (3). The
results are expressed as mean optical. densities and are representative
of three separate experiments.
VOL. 70, 2004 S. LIVIDANS, GLYCOSYLATION, AND M. TUBERCULOSIS 683

was the different reactivities of human tuberculosis-infected
sera with the ConA-binding 47-kDa protein and the nonbind-
ing 45-kDa protein, as analyzed by the ELISA. Antibodies
from tuberculosis patients recognized only the upper ConA-
binding fraction, suggesting that antibodies in the sera had
been generated against the carbohydrate residues. This obser-
vation was confirmed by the loss of antibody binding to degly-
cosylated 47-kDa protein.
Carbohydrates that decorate the surfaces of infectious
agents are considered pathogen-associated molecular patterns,
and they are recognized by pattern recognition receptors, such
as the mannose receptor and the dendritic cell-specific intra-
cellular adhesion molecule 3-grabbing nonintegrin receptor,
which play a key role in innate and adaptive immunity (9, 35).
ConA-binding carbohydrates have been found in lipoarabino-
mannan and other important antigens of M. tuberculosis, like
the 19- and 38-kDa antigens (7, 11, 13). The interaction of
LAM with the mannose receptor has been widely documented
(3, 15), and more recently the dendritic cell-specific intracel-
lular adhesion molecule 3-grabbing nonintegrin receptor has
been defined as the major M. tuberculosis receptor on human
dendritic cells with the capacity to discriminate between My-
cobacterium species through selective recognition of the man-
nose caps on LAM (22, 36). These observations suggest that
the presence of mannose residues in mycobacterial molecules
could be an important signal for host recognition through
mannose receptors or other carbohydrate recognition recep-
tors, and therefore, carbohydrate motifs in glycoproteins of
mycobacteria could play an important role in both the cellular
(12, 32) and humoral immune responses.
ACKNOWLEDGMENTS
This work was supported by grant IN221599 from DGAPA-Univer-
sidad Nacional Auto´noma de Me´xico and by grants 33580-M and
CONACYT-DLR from CONACYT.
The Tropist 3 cosmid library of M. tuberculosis DNA was kindly
provided by K. De Smet (Tuberculosis and Related Infections Unit,
Medical Research Council, Clinical Sciences Centre, London, United
Kingdom). We thank Rafael Cervantes and Gabriela Gonza´lez-Cero´n
for technical assistance, Rita Getzlaff for protein sequencing, and
Isabel Pe´rez Montfort for reviewing the English version of the manu-
script.
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