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Infection and Immunity, April 2000, p. 2323-2327, Vol. 68, No. 4
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification of Secreted Proteins of
Mycobacterium tuberculosis by a Bioinformatic
Approach
Manuel
Gomez,
Sadie
Johnson, and
Maria Laura
Gennaro*
Public Health Research Institute, New York,
New York
Received 3 September 1999/Returned for modification 18 October
1999/Accepted 13 December 1999
 |
ABSTRACT |
Proteins secreted by Mycobacterium tuberculosis are
usually targets of immune responses in the infected host. Here we
describe a search for secreted proteins that combined the use of
bioinformatics and phoA' fusion technology. The 3,924 proteins deduced from the M. tuberculosis genome were
analyzed with several computer programs. We identified 52 proteins
carrying an NH2-terminal secretory signal peptide but
lacking additional membrane-anchoring moieties. Of these 52 proteins
the TM1 subgrouponly 7 had been previously reported to be
secreted proteins. Our predictions were confirmed in 9 of 10 TM1 genes
that were fused to Escherichia coli phoA', a marker of
subcellular localization. These findings demonstrate that the
systematic computer search described in this work identified secreted
proteins of M. tuberculosis with high efficiency and 90% accuracy.
| |
TEXT |
Proteins released by
Mycobacterium tuberculosis to the extracellular environment
have been the focus of much of the research directed at identifying
antigens that induce protective immunity or those that elicit immune
responses of diagnostic value (reviewed in references 2,
11, and 37). Two different experimental approaches have been used. One is analysis of the protein composition of M. tuberculosis culture filtrates. The M. tuberculosis culture filtrate, which contains as many as 200 proteins (29), has been investigated by means of protein
purification, by immunological methods, and by screening of expression
libraries of M. tuberculosis DNA with anti-culture filtrate
sera (for examples, see references 1, 4, 5, 19, 25,
33, and 34). A second, genetic approach
involves the screening of libraries of fusions of M. tuberculosis genes to reporter genes encoding enzymes that become active upon translocation across the cell membrane (7, 18). As a result of the combined efforts of several laboratories, more than
30 secreted proteins of M. tuberculosis have been
characterized (examples are provided in references 3, 6, 9,
10, 12, 15, 17, 18, 20, 22, 26, 30, 32, and
34). Nevertheless, much of the immunological
activity of the culture filtrate of M. tuberculosis remains
unaccounted for.
Analysis of the NH2-terminal sequences of proteins purified
from the culture filtrate vis-à-vis corresponding deduced amino acid sequences (34, 37) indicates that many proteins of
M. tuberculosis are secreted via the general export pathway
(GEP). The GEP mediates protein translocation across the cytoplasmic membrane by means of an NH2-terminal secretory signal
peptide (24, 28). Following translocation, cleavage of the
signal peptide by a signal peptidase releases the mature protein,
providing that there are no additional membrane-spanning segments or
membrane-anchoring moieties (Fig. 1). The
conserved features of signal peptides (length and amino acid
composition plus the signal peptidase cleavage site
[31]) make them amenable to identification by sequence analysis. Thus, we set out to identify proteins of M. tuberculosis secreted via the GEP by using a bioinformatic
approach that takes advantage of the recently released nucleotide
sequence of the M. tuberculosis genome (8).
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FIG. 1.
Fates of proteins having an NH2-terminal
signal peptide. The NH2-terminal signal peptide directs
translocation of a protein across the cell membrane (top left panel).
Processing of the preprotein by a type I signal peptidase releases the
mature protein (A), providing that there are no additional
transmembrane domains (B). Translocation of LPPs across the membrane is
followed by modification with glycerol and fatty acids of the
prolipoprotein and processing by a type II signal peptidase. The lipid
moiety anchors the LPP to the membrane (C). Mb, cell membrane; Cy,
cytoplasm; N, NH2-terminal end; C, COOH-terminal end.
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Prediction of secreted proteins of M. tuberculosis.
The
computer strategy used to identify proteins having secretory signal
peptides but lacking additional membrane attachment domains is
presented in Fig. 2. The amino acid
sequences of 3,924 proteins deduced from the nucleotide sequence of the
M. tuberculosis genome were downloaded from the Sanger
Center
database (http://www.sanger.ac.uk/Projects/M_tuberculosis). Segments
containing the NH2-terminal 70 amino acid residues of each
polypeptide were analyzed for secretory signal peptides with two
computer programs, SignalP (http://www.cbs.dtu.dk/services/SignalP) and
SPScan (13). The two programs assigned scores to potential signal peptides for all of the proteins except two (Rv1572c and Rv3599c) that were too short for SPScan analysis. Scores ranged from
0.039 to 0.888 for SignalP predictions and from 
3.8 to +14.8 for
SPScan predictions (Fig. 3). Cutoff values for the computer predictions
were chosen on the basis of scores assigned to nine known secreted
proteins of M. tuberculosis that contain a signal peptide (Ag85A, Ag85B, Ag85C, MPT32, MPT51, MPT53, MPT63, MPT64, and MPB70) (34). For these proteins, SignalP scores
ranged from 0.425 to 0.758 and SPScan scores ranged from 8 to 11.2. We
chose score cutoffs of 0.4 for SignalP and 8.0 for SPScan. Both values are more restrictive than the default cutoff values (0.34 for SignalP
and 3.5 for SPScan). Two hundred eight proteins (5% of the proteome)
scored above the cutoff with both programs (boxed inset in Fig.
3). This set of proteins is henceforth
referred to as the Top208 group.
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FIG. 2.
Schematic representation of the strategy utilized to
predict M. tuberculosis secreted proteins. TM,
transmembrane; SP, signal peptide; PE/PPE, families of M. tuberculosis proteins containing multiple copies of sequences rich
in small-side-chain amino acid residues (8).
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FIG. 3.
Correlation between SignalP and SPScan scores. Each
circle represents one of the 3,924 deduced proteins of M. tuberculosis. The position of the circle indicates the scores
assigned by SignalP and SPScan to the signal peptide. For this study,
the two computer programs were set in the mode for gram-positive
bacteria. SPScan was used in the adjusted mode, which penalizes signal
peptides longer than 45 amino acid residues (i.e., the maximal length
for gram-positive bacteria). The boxed area contains the Top208 group
of proteins that scored  0.4 with SignalP and 8 with SPScan. The
solid circles within the boxed area represent nine known secreted
proteins (Ag85A, Ag85B, Ag85C, MPT32, MPT51, MPT53, MPT63, MPT64, and
MPB70) (34) that were used to set cutoff values for SignalP
and SPScan scores. The solid circles outside of the boxed area
represent four known proteins (MPT46, GroES, GroEL2, and DnaK)
containing no signal peptide (34). Also outside of the boxed
area is located the 38-kDa antigen, a secreted protein which exhibits
an LPP type of signal peptide (14).
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Of the proteins in the Top208 group, 47 had been previously annotated
as members of the PE and PPE families (
8). Proteins
in these
families contain multiple copies of motifs rich in small-side-chain
amino acid residues, such as alanine and glycine. Since a biased
amino
acid composition and a repetitive primary sequence could
lead to
unreliable results in sequence analyses, the 47 PE and
PPE proteins in
the Top208 group were not further
analyzed.
Proteins that cross the membrane via the GEP are released to the
external environment after cleavage of the signal peptide
only if they
lack membrane-anchoring sequences (Fig.
1). To eliminate
from our study
those proteins that contain membrane-spanning segments,
the remaining
161 proteins in the Top208 group were analyzed with
the program TMpred
(
http: //www.ch.embnet.org/software/TMPRED_form.html)
(Fig.
2). The presence of an NH
2-terminal transmembrane
segment
(i.e., the putative signal peptide) was confirmed by
TMpred only
for 142 proteins. Of these, 49 were putative integral
membrane
proteins, for they contained transmembrane segments within the
mature protein that scored >1,000 (the default cutoff score
for
TMpred is 500) (Fig.
2). The remaining 93 proteins were analyzed
for membrane lipoprotein (LPP) lipid attachment sites with the
program
PrositeScan
(
http://www.isrec.isb-sib.ch/software/PSTSCAN_form.html)
(LPP
motif, PROSITE database entry PS00013). We defined three
subgroups
based on TMpred and PrositeScan predictions (Fig.
2).
The first
subgroup, Top208-TM1, comprised 52 proteins that were
classified as
"most likely secreted." These proteins contained
one transmembrane
domain corresponding to the signal peptide,
no additional
membrane-spanning segments having TMpred scores
above 500, and no
LPP motifs. The second subgroup, Top208-TM2,
consisted of 25 proteins
that were classified as "possibly secreted"
because the mature
proteins contained segments having TMpred scores
between 500 and 1,000. The remaining 16 proteins (the Top208-LPP
subgroup) were predicted to
be LPPs, for they contained a properly
positioned LPP
motif.
The 52 proteins comprising the Top208-TM1 subgroup are listed in Table
1. Thirty-two of the TM1 proteins were
previously
unrecognized as secreted proteins (set A in Table
1, column
2),
while 13 TM1 proteins had annotations in the Sanger Center database
that suggest the presence of NH
2-terminal signal peptides
(set
B in Table
1, column 2). Only seven TM1 proteins were previously
known as secreted proteins (Mtb8.4, MTB12, MTC28, MPT32, MPT53,
MPT63,
and MPT64) (
9,
12,
17,
19,
20,
32,
34,
36)
(set C in Table
1, column 2). Sixteen of the 52 TM1 proteins
were assigned a probable
function on the basis of the presence
of functional motifs or
similarities to proteins with known functions
(Table
1, column 6).
Analysis of TM1 proteins by phoA' fusion
technology.
A method to study protein topology with respect to the
cell membrane is to analyze the enzymatic activity of a hybrid
consisting of the test protein and the reporter Escherichia
coli PhoA protein devoid of its original signal peptide
(21). To test the prediction that the proteins in the TM1
subgroup contained a single transmembrane domain, the signal peptide
(Fig. 1), fusions were constructed between phoA' and
full-length M. tuberculosis TM1 coding sequences. To
introduce a minimum of bias in these experiments, we chose 10 proteins
at random from the TM1 subgroup. These 10 proteins (marked with
asterisks in Table 1, column 1) represented a broad range of SPScan
scores (8.3 to 14.8) and SignalP scores (0.469 to 0.787) and were
equally divided between those having (set B) and those not having (set
A) database annotations with reference to topology or localization.
Recombinant plasmids bearing fusions of
phoA' with
M. tuberculosis TM1 genes were constructed in
E. coli.
Clones carrying
the fusions were evaluated for alkaline phosphatase
activity by
plating on Luria-Bertani agar containing the
chromogenic substrate
5-bromo-4-chloro-3-indolylphosphate (BCIP).
On indicator plates,
alkaline phosphatase activity is seen as
blue-green colonies.
The colony color phenotype of one representative
clone for each
fusion is shown in Fig.
4.
Nine of 10 fusions exhibited alkaline
phosphatase activity. The only
phoA' fusion that yielded a weak,
albeit detectable,
blue-green colony color was that with Rv3354
(clone 12 in Fig.
4). All
of the hybrid proteins, except that
encoded by the
Rv3354::
phoA' fusion, were detected by Western
blot analysis of whole-cell lysates with anti-PhoA antibodies
(data not
shown). These results suggest that the hybrid protein
encoded by the
Rv3354::
phoA' fusion is either synthesized at low
levels
or rapidly degraded. The finding that 9 of 10 mycobacterial
protein
hybrids directed export of the PhoA moiety provides strong
evidence
that proteins in the Top208-TM1 subgroup are bona fide
secreted
proteins.
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FIG. 4.
Alkaline phosphatase activity of 10 M. tuberculosis TM1 proteins fused to PhoA. phoA' fusions
were constructed in E. coli plasmid pUCCMPHOA
(16), a pUC18 derivative that carries phoA'
downstream of codon 18 of lacZ. phoA' lacks the
nucleotide sequences coding for the NH2-terminal signal
peptide and the first 11 amino acid residues of the mature PhoA
protein. Hybrid gene expression in pUCCMPHOA utilizes the
lac promoter and the lacZ ribosome binding site
and start codon. Full-length M. tuberculosis coding
sequences (Mtcds) were synthesized from chromosomal DNA by
PCR, cloned into plasmid pCR2.1 (Invitrogen), and subsequently
transferred into pUCCMPHOA using appropriate restriction endonuclease
sites to obtain in-frame lacZ-Mtcds-phoA' fusions.
Recombinant plasmids were characterized by nucleotide sequence analysis
of the DNA junctions. E. coli clones bearing vector
pUCCMPHOA and 12 different phoA fusions were tested for
alkaline phosphatase activity on Luria-Bertani agar plates containing
BCIP at 40 µg ml 1. Plate sectors: 1, pUCCMPHOA
(negative control); 2, B. subtilis membrane protein ComP
(positive control) (27); 3, secreted protein MPT63 of
M. tuberculosis (positive controlalso one of the TM1
proteins) (20); 4, Rv0315; 5, Rv1268c; 6, Rv1269c; 7, Rv1566c; 8, Rv1906c; 9, Rv2223c; 10, Rv2290; 11, Rv2450c; 12, Rv3354;
13, Rv3668c.
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In summary, 52 proteins (the Top208-TM1 subgroup) were identified by
computer-based analyses as most likely secreted. The
computer
predictions were confirmed in 90% of the TM1 proteins
tested by
E. coli phoA' gene fusion methods. Only 7 of the 52
TM1
proteins had been previously reported to be secreted proteins.
Thus,
the bioinformatic method used in the present paper is highly
efficient
and accurate. The high accuracy is explained by the
use of stringent
selection criteria (i.e., high cutoff scores
for SignalP, SPScan, and
TMpred).
The computer-based approach that we have used to identify secreted
proteins differs in several important ways from genetic
screening
methods employing random fusions to
phoA' or
bla,
markers
of extracellular localization. First, methods that employ
random
fusions do not allow distinction between secretory signal
peptides
and transmembrane domains of membrane-bound proteins
(reference
35 and references therein). Second,
expression of alkaline phosphatase
activity by clones generated by
random insertion of
phoA-coupled
transposons critically
depends on the strength of promoters located
upstream of
the transposition site (
21). The same limitation
applies to the screening of random fusion libraries in promoterless
plasmids. Third, gene fusion library screening methods often yield
multiple hits in the same genes, thus making the process of gene
identification labor intensive (
7). A disadvantage of the
computer-based
approach is that it is limited to only those proteins
secreted
via the GEP. While many secreted antigens of
M. tuberculosis fall
into this category, the presence in
culture filtrates of proteins
lacking secretory signal peptides (e.g.,
ESAT-6, SodA, GlnA, and
KatG [
15,
29,
30,
38]) suggests the existence of other,
still undefined,
mechanisms of protein secretion that operate
in
M. tuberculosis.
The identification of novel secreted proteins of
M. tuberculosis opens the way to studies on their subcellular
localization
in
M. tuberculosis and to the immunological
characterization of
these proteins to define their potential for
immunological diagnosis
of tuberculosis or vaccine
design.
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ACKNOWLEDGMENTS |
We thank David Dubnau for providing plasmid pUCCMPHOA and Carol
Lusty and Karl Drlica for critical reading of the manuscript.
This work was supported by NIH grant AI-36989 (M.L.G.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Public Health
Research Institute, 455 First Ave., New York, NY 10016. Phone: (212) 578-0844. Fax: (212) 578-0804. E-mail:
gennaro{at}phri.nyu.edu.
Editor:
S. H. E. Kaufmann
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REFERENCES |
| 1.
|
Amara, R. R., and V. Satchidanandam.
1996.
Analysis of a genomic DNA expression library of Mycobacterium tuberculosis using tuberculosis patient sera: evidence for modulation of host immune response.
Infect. Immun.
64:3765-3771[Abstract].
|
| 2.
|
Andersen, A. B., and P. Brennan.
1994.
Proteins and antigens of Mycobacterium tuberculosis, p. 307-327.
In
B. R. Bloom (ed.), Tuberculosis: pathogenesis, protection, and control. American Society for Microbiology, Washington, D.C.
|
| 3.
|
Andersen, A. B., and E. B. Hansen.
1989.
Structure and mapping of antigenic domains of protein antigen b, a 38,000-molecular-weight protein of Mycobacterium tuberculosis.
Infect. Immun.
57:2481-2488[Abstract/Free Full Text].
|
| 4.
|
Andersen, P.,
D. Askgaard,
A. Gottschau,
J. Bennedsen,
S. Nagai, and I. Heron.
1992.
Identification of immunodominant antigens during infection with Mycobacterium tuberculosis.
Scand. J. Immunol.
36:823-831[CrossRef][Medline].
|
| 5.
|
Andersen, P.,
D. Askgaard,
L. Ljungqvist,
J. Bennedsen, and I. Heron.
1991.
Proteins released from Mycobacterium tuberculosis during growth.
Infect. Immun.
59:1905-1910[Abstract/Free Full Text].
|
| 6.
|
Ashbridge, K. R.,
R. J. Booth,
J. D. Watson, and R. B. Lathigra.
1989.
Nucleotide sequence of the 19 kDa antigen gene from Mycobacterium tuberculosis.
Nucleic Acids Res.
17:1249[Free Full Text].
|
| 7.
|
Chubb, A. J.,
Z. L. Woodman,
F. M. da Silva Tatley,
H. J. Hoffmann,
R. R. Scholle, and M. R. Ehlers.
1998.
Identification of Mycobacterium tuberculosis signal sequences that direct the export of a leaderless beta-lactamase gene product in Escherichia coli.
Microbiology
144:1619-1629[Abstract/Free Full Text].
|
| 8.
|
Cole, S. T.,
R. Brosch,
J. Parkhill,
T. Garnier,
C. Churcher,
D. Harris,
S. V. Gordon,
K. Eiglmeier,
S. Gas,
C. E. Barry, 3rd,
F. Tekaia,
K. Badcock,
D. Basham,
D. Brown,
T. Chillingworth,
R. Connor,
R. Davies,
K. Devlin,
T. Feltwell,
S. Gentles,
N. Hamlin,
S. Holroyd,
T. Hornsby,
K. Jagels,
B. G. Barrell, et al.
1998.
Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.
Nature
393:537-544[CrossRef][Medline].
|
| 9.
|
Coler, R. N.,
Y. A. Skeiky,
T. Vedvick,
T. Bement,
P. Ovendale,
A. Campos-Neto,
M. R. Alderson, and S. G. Reed.
1998.
Molecular cloning and immunologic reactivity of a novel low molecular mass antigen of Mycobacterium tuberculosis.
J. Immunol.
161:2356-2364[Abstract/Free Full Text].
|
| 10.
|
Content, J.,
A. de la Cuvellerie,
L. De Wit,
V. Vincent-Levy-Frebault,
J. Ooms, and J. De Bruyn.
1991.
The genes coding for the antigen 85 complexes of Mycobacterium tuberculosis and Mycobacterium bovis BCG are members of a gene family: cloning, sequence determination, and genomic organization of the gene coding for antigen 85-C of M. tuberculosis.
Infect. Immun.
59:3205-3212[Abstract/Free Full Text].
|
| 11.
|
Cooper, A. M., and J. L. Flynn.
1995.
The protective immune response to Mycobacterium tuberculosis.
Curr. Opin. Immunol.
7:512-516[CrossRef][Medline].
|
| 12.
|
Freer, G.,
W. Florio,
B. Dalla Casa,
D. Bottai,
G. Batoni,
G. Maisetta,
S. Senesi, and M. Campa.
1998.
Identification and molecular cloning of a novel secretion antigen from Mycobacterium tuberculosis and Mycobacterium bovis BCG.
Res. Microbiol.
149:265-275[Medline].
|
| 13.
|
Genetics Computer Group.
1997.
Wisconsin Package Version 9.1.
Genetics Computer Group, Madison, Wis.
|
| 14.
|
Harboe, M., and H. G. Wiker.
1992.
The 38-kDa protein of Mycobacterium tuberculosis: a review.
J. Infect. Dis.
166:874-884[Medline].
|
| 15.
|
Harth, G.,
D. L. Clemens, and M. A. Horwitz.
1994.
Glutamine synthetase of Mycobacterium tuberculosis: extracellular release and characterization of its enzymatic activity.
Proc. Natl. Acad. Sci. USA
91:9342-9346[Abstract/Free Full Text].
|
| 16.
|
Inamine, G. S., and D. Dubnau.
1995.
ComEA, a Bacillus subtilis integral membrane protein required for genetic transformation, is needed for both DNA binding and transport.
J. Bacteriol.
177:3045-3051[Abstract/Free Full Text].
|
| 17.
|
Laqueyrerie, A.,
P. Militzer,
F. Romain,
K. Eiglmeier,
S. Cole, and G. Marchal.
1995.
Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47-kilodalton secreted antigen complex.
Infect. Immun.
63:4003-4010[Abstract].
|
| 18.
|
Lim, E. M.,
J. Rauzier,
J. Timm,
G. Torrea,
A. Murray,
B. Gicquel, and D. Portnoi.
1995.
Identification of Mycobacterium tuberculosis DNA sequences encoding exported proteins by using phoA gene fusions.
J. Bacteriol.
177:59-65[Abstract/Free Full Text].
|
| 19.
|
Manca, C.,
K. Lyashchenko,
R. Colangeli, and M. L. Gennaro.
1997.
MTC28, a novel 28-kilodalton proline-rich secreted antigen specific for the Mycobacterium tuberculosis complex.
Infect. Immun.
65:4951-4957[Abstract].
|
| 20.
|
Manca, C.,
K. Lyashchenko,
H. G. Wiker,
D. Usai,
R. Colangeli, and M. L. Gennaro.
1997.
Molecular cloning, purification, and serological characterization of MPT63, a novel antigen secreted by Mycobacterium tuberculosis.
Infect. Immun.
65:16-23[Abstract].
|
| 21.
|
Manoil, C.
1991.
Analysis of membrane protein topology using alkaline phosphatase and beta-galactosidase gene fusions.
Methods Cell. Biol.
34:61-75[Medline].
|
| 22.
|
Matsumoto, S.,
T. Matsuo,
N. Ohara,
H. Hotokezaka,
M. Naito,
J. Minami, and T. Yamada.
1995.
Cloning and sequencing of a unique antigen MPT70 from Mycobacterium tuberculosis H37Rv and expression in BCG using E. coli-mycobacteria shuttle vector.
Scand. J. Immunol.
41:281-287[CrossRef][Medline].
|
| 23.
|
Mukamolova, G. V.,
A. S. Kaprelyants,
D. I. Young,
M. Young, and D. B. Kell.
1998.
A bacterial cytokine.
Proc. Natl. Acad. Sci. USA
95:8916-8921[Abstract/Free Full Text].
|
| 24.
|
Murphy, C. K., and J. Beckwith.
1987.
Export of proteins to the cell envelope in Escherichia coli, p. 967-978.
In
F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 1. American Society for Microbiology, Washington, D.C.
|
| 25.
|
Nagai, S.,
H. G. Wiker,
M. Harboe, and M. Kinomoto.
1991.
Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis.
Infect. Immun.
59:372-382[Abstract/Free Full Text].
|
| 26.
|
Ohara, N.,
H. Kitaura,
H. Hotokezaka,
T. Nishiyama,
N. Wada,
S. Matsumoto,
T. Matsuo,
M. Naito, and T. Yamada.
1995.
Characterization of the gene encoding the MPB51, one of the major secreted protein antigens of Mycobacterium bovis BCG, and identification of the secreted protein closely related to the fibronectin binding 85 complex.
Scand. J. Immunol.
41:433-442[CrossRef][Medline].
|
| 27.
|
Piazza, F.,
P. Tortosa, and D. Dubnau.
1999.
Mutational analysis and membrane topology of ComP, a quorum-sensing histidine kinase of Bacillus subtilis controlling competence development.
J. Bacteriol.
181:4540-4548[Abstract/Free Full Text].
|
| 28.
|
Pugsley, A. P.
1993.
The complete general secretory pathway in gram-negative bacteria.
Microbiol. Rev.
57:50-108[Abstract/Free Full Text].
|
| 29.
|
Sonnenberg, M. G., and J. T. Belisle.
1997.
Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry.
Infect. Immun.
65:4515-4524[Abstract].
|
| 30.
|
Sorensen, A. L.,
S. Nagai,
G. Houen,
P. Andersen, and A. B. Andersen.
1995.
Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis.
Infect. Immun.
63:1710-1717[Abstract].
|
| 31.
|
Watson, M. E. E.
1984.
Compilation of published signal sequences.
Nucleic Acids Res.
12:5145-5164[Free Full Text].
|
| 32.
|
Webb, J. R.,
T. S. Vedvick,
M. R. Alderson,
J. A. Guderian,
S. S. Jen,
P. J. Ovendale,
S. M. Johnson,
S. G. Reed, and Y. A. Skeiky.
1998.
Molecular cloning, expression, and immunogenicity of MTB12, a novel low-molecular-weight antigen secreted by Mycobacterium tuberculosis.
Infect. Immun.
66:4208-4214[Abstract/Free Full Text].
|
| 33.
|
Weldingh, K.,
I. Rosenkrands,
S. Jacobsen,
P. B. Rasmussen,
M. J. Elhay, and P. Andersen.
1998.
Two-dimensional electrophoresis for analysis of Mycobacterium tuberculosis culture filtrate and purification and characterization of six novel proteins.
Infect. Immun.
66:3492-3500[Abstract/Free Full Text].
|
| 34.
|
Wiker, H. G.,
S. L. Michell,
R. G. Hewinson,
E. Spierings,
S. Nagai, and M. Harboe.
1999.
Cloning, expression and significance of MPT53 for identification of secreted proteins of Mycobacterium tuberculosis.
Microb. Pathog.
26:207-219[CrossRef][Medline].
|
| 35.
|
Worley, M. J.,
I. Stojiljkovic, and F. Heffron.
1998.
The identification of exported proteins with gene fusions to invasin.
Mol. Microbiol.
29:1471-1480[CrossRef][Medline].
|
| 36.
|
Yamaguchi, R.,
K. Matsuo,
A. Yamazaki,
C. Abe,
S. Nagai,
K. Terasaka, and T. Yamada.
1989.
Cloning and characterization of the gene for immunogenic protein MPB64 of Mycobacterium bovis BCG.
Infect. Immun.
57:283-288[Abstract/Free Full Text].
|
| 37.
|
Young, D. B.,
S. H. Kaufmann,
P. W. Hermans, and J. E. Thole.
1992.
Mycobacterial protein antigens: a compilation.
Mol. Microbiol.
6:133-145[Medline].
|
| 38.
|
Zhang, Y.,
R. Lathigra,
T. Garbe,
D. Catty, and D. Young.
1991.
Genetic analysis of superoxide dismutase, the 23 kilodalton antigen of Mycobacterium tuberculosis.
Mol. Microbiol.
5:381-391[Medline].
|
Infection and Immunity, April 2000, p. 2323-2327, Vol. 68, No. 4
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