Jpn. J. Infect. Dis., 62, 26-31, 2009
Original Article
Rv0901 from Mycobacterium tuberculosis, a Possible Novel Virulent
Gene Proved through the Recombinant Mycobacterium smegmatis
Le Zhang1,2, Qi Zhong1, Lang Bao1*, Ying Zhang1, Lei Gao1,
Bi Huang1, and Hui-Dong Zhang1
1
Infection and Immunity Unit, West China Center of Medical Sciences, Sichuan University, Sichuan, and
2
College of Medicine, Shihezi University, Xinjang, China
(Received August 19, 2008. Accepted November 21, 2008)
SUMMARY: The function of protein-coding gene Rv0901 of Mycobacterium tuberculosis, which belongs to
the cell wall and cell processes category, is not yet clear. To explore its features, we amplified this gene from the
H37Rv genome, and His-tagged Rv0901 protein was expressed and purified. Also, a recombinant plasmid bearing Rv0901 was constructed and electroporated into a virulent Mycobacterium smegmatis, using shuttle expression vector pMV261. Transformants were induced to express a predicted protein of Rv0901, identified by SDSPAGE. Rv0901 protein and recombinant M. smegmatis were used to expose mammalian cells. In addition we
studied the effect of protein or recombinant M. smegmatis on cells and in animals with regard to survival ratio,
apoptosis ratio, quantum of nitric oxide, and gamma interferon. Together, gene function, protein function, and
animal test results suggest that Rv0901 has some relationship with the virulence and immunogenicity of M.
tuberculosis.
binant plasmid bearing Rv0901 was constructed using the
shuttle vector pMV261, then electroporated into avirulent
Mycobacterium smegmatis mc2155, which lacks the sequence.
This transformant was induced to express a predicted protein
of Rv0901. Then, mammalian cells were exposed to Rv0901
protein, and test cells and mice were infected with recombinant M. smegmatis.
INTRODUCTION
Tuberculosis (TB), caused by the respiratory pathogen
Mycobacterium tuberculosis, remains a global health problem and the leading curable infectious cause of death (1).
Researchers estimated that there were 8.9 million new cases
and 1.7 million deaths from TB in 2004 (2,3). A major proportion of the global TB burden comes from developing
countries (2,4), where the incidence of TB is advanced by the
prevalence of immunodeficiency caused by human immunodeficiency virus (HIV), the emergence of multiple-drugresistant strains of M. tuberculosis (3,5), and a steadily
increasing transmission rate of M. tuberculosis for many years
(6-8). In addition, investigations have confirmed that TB can
interact with such chronic conditions as diabetes, malnutrition, and respiratory diseases caused by tobacco or air pollution (9). To explore the pathogenesis of TB and find new
strategies for prevention and treatment, Cole et al. published
the genome sequence of M. tuberculosis H37Rv in 1998 (10).
The sequence was updated by Camus et al. in 2002 (11), providing important insights into the genomics and proteomics
of M. tuberculosis. The protein-encoding gene Rv0901,
annotated as unknown in 1998 and listed as conserving a
hypothetical protein, was transferred to the cell wall and cell
processes category in 2002 (11). Essential to understanding
the pathogenesis of TB is understanding the interaction
between pathogen, phagocytes, and various mycobacterium
cell wall components that may be involved in these processes.
In this research, we investigated whether Rv0901 is functionally correlated with the toxicity and immunogenicity of
M. tuberculosis. To address this question, we caused Histagged Rv0901 protein to be expressed and purified. A recom-
MATERIALS AND METHODS
Bacterial strains, cells, animals, and culture conditions:
The genomes of M. tuberculosis (H37Rv) and M. smegmatis
(mc2155) were obtained from the National Institute for the
Control of Pharmaceutical and Biological Products (Beijing,
China). Mc2155 was cultured in Sauton medium and frozen
at –70°C until use. BL21 and DH5a cells, grown on solid or
in liquid Luria-Bertani (LB) medium, were used for gene cloning and expression. ANA-1 (murine macrophages) and THP1 (human monocytic cell line) cells were grown at 37°C in a
5% CO2 incubator with RPMI1640 (Hyclone Laboratories,
Logan, Utah, USA) medium, supplemented with 10% fetal
bovine serum (FBS) and 100 U/ml ampicillin. Pathogen-free
BALB/c female mice were purchased from the Research
Institute of Antibiotics (Chengdu, China), maintained under
barriers conditions, and fed commercial mouse chow and
water. The mice were 6 - 8 weeks old at the time of experiments.
Gene amplification and construction of plasmids and
recombinant M. smegmatis mc2155: We amplified the fulllength Rv0901 gene by polymerase chain reaction (PCR) with
the following primers: 5´-TTAGGATCCATGGAACACGTG
CACTG-3´ (forward primer; the the BamHI site is underlined); 5´-GTTGAATTCTCA TGTCCGCCGTGTGCTCTT3´ (reverse primer; the EcoRI site is underlined). The target
fragment was inserted into pET32a(+) , a 6 × His-tag vector
(Novagen, Madison, Wis., USA), and shuttle expression vector pMV261 (a gift of Dr Heng Xu, Institute of Life Science,
Sichuan University). The resulting pET-Rv0901 and pMV261-
*Corresponding author: Mailing address: Infection and Immunity Unit, West China Center of Medical Sciences, Sichuan
University, No. 17, 3rd Section, Ren Min Nan Road, Chengdu,
Sichuan 610041, P.R. China. Tel: +86-28-85503376, E-mail:
[email protected]
26
nase (LDH) and/or nitrogen monoxidum (NO) were released.
The cells were plated in a 6-well plate at a concentration of
105 cells per well and treated with a serum-free medium 24
h before experiments. PE0901 were diluted to 1, 5, 10, 20,
and 40 μg/ml in medium. Cytotoxicity was determined by
measuring the released LDH and NO activity in the medium
using an LDH and NO kit (Nanjing Jiancheng Biotechnology
Institute, Nanjing, China) according to the manufacturer’s
instructions. Cells were harvested and washed with PBS twice
and fixed in 1 ml 75% ethanol. Cell apoptosis was analyzed
by flow cytometry. Experiments were performed five times.
Effects of recombinant M. smegmatis and M. smegmatis
on a mammalian cell line: Both recombinant M. smegmatis
and M. smegmatis were incubated and then measured by A600
to calculate cell concentration. THP-1 cells were seeded into
a 6-well plate at 105 cells per well. Bacterial cultures were
diluted and added to each well to achieve a multiplicity of
infection (MOI) of 10. After 2 h, the THP-1 cells were washed
twice with PBS containing gentamicin (25 μg/ml) and then
incubated in RPMI1640 medium containing gentamicin (25
μg/ml) for 3, 24, and 48 h. At each of these time points, live
cells were counted (15).
In the same way, THP-1 cells were harvested at 24 h and
72 h after infection, washed with PBS, and fixed in 1 ml 75%
ethanol, and then cell apoptosis was analyzed by flow
cytometry.
Supernatants of cells were collected at 48 h and 96 h after
infection, and NO activity was measured using a NO kit
(Nanjing Jiancheng Biotechnology Institute) according to the
manufacturer’s instructions.
Recombinant M. smegmatis challenge: Mice (n = 6) were
infected subcutaneously with 0.5 ml of PBS, 106 CFU/0.1 ml
of M. smegmatis, or recombinant M. smegmatis. They were
sacrificed after 5 weeks to prepare serum and splenocytes.
Lungs and livers were removed, fixed in 10% phosphatebuffered formalin, embedded in paraffin, sectioned, and stained
with hematoxylin and eosin.
Infected mice were sacrificed 1 week after the last injection, and spleens were removed aseptically. The spleens were
ground up, and lymphocytes were isolated by lymphocyte
separating medium, which pooled the lymphocytes in each
group. Cells were adjusted to a concentration of 105 cells per
well and grown in 96-well plates. XTT was used to detect
the proliferation state. Briefly, 50 μl 10 μg/ml of whole
Mycobacterium protein was added into each well to be tested,
and the plate was incubated under 5% CO2 at 37°C for 5 days,
and then XTT was added to each well with 25 μmol/L PMS.
The optical density at 450 nm was measured, and a stimulation index (SI) was used to describe the proliferation degree
of lymphocytes, based on the following formula: SI = Atreated/
Acontrol.
Lymphocytes were labeled with a fluorescein isothiocyanate
(FITC)-conjugated anti-CD4+ antibody and PE-conjugated
anti-CD8+ antibody for 30 min and washed twice with PBS
by centrifugation at 1,800 rpm at a concentration of 2 × 106
cells per well. Thirty thousand cells in the lymphocyte gate
were acquired on a cytofluorometer. The ratio of CD4+ to
CD8+ T cell activity was detected by flow cytometry.
Mice of each group were bled from the eyeball vein and
sacrificed by cervical vertebra luxation, and the collected blood
was incubated for 30 min at 37°C. The erythrocytes were
removed by centrifugation, and the serum was collected from
the supernatant. Interferon (IFN)-γ activity was quantified
in the serum using a mouse IFN-γ ELISA kit (Biosource,
Rv0901 were used to transform Escherichia coli. The correct
sequence was verified by DNA sequencing. Then, the recombinant shuttle plasmids pMV261-Rv0901 were transformed
into M. smegmatis mc2155 by electroporation as described
previously (12). Cells were recovered in 1 ml of SOC at 37°C
with vigorous shaking for 8 h, then plated on Sauton agar
containing kanamycin (25 μg/ml) and incubated at 37°C
for 3 days (13). Identification of the recombinant strains was
performed by PCR and Western blotting.
Expression and purification of His-tagged protein
Rv0901: Recombinant E. coli (pET-Rv0901) and control E.
coli (pET) were induced with IPTG (1 mM) at 37°C for 5 h.
Extracted from induced bacteria and separated by 12% SDSPAGE, separated proteins (PE0901) were electrophoretically
transferred to PVDF membrane. Following blocking with 5%
nonfat milk, the membrane was probed with mouse anti-His
monoclonal antibody (Lab Vision, Fremont, Calif., USA). After being washed, the membrane was incubated with alkaline
phosphatase (AP)-conjugated goat anti-mouse IgG (SinoAmerican, Henan, China; 1:5,000). Lastly, immunoreactive
bands were visualized with DAB for coloration. After confirmation of expression, His-tagged Rv0901 was purified using
a Ni-NTA column (GE Healthcare, Piscataway, N.J., USA),
and endotoxin in the purified protein was quantitatively determined by Tachypleus amebocyte lysate (TAL) assay (Institute
of Medical Analysis, Shanghai, China).
Production of Rv0901 antiserum: One New Zealand
white rabbit weighing about 2 kg was immunized through
subcutaneous injection on the back with 500 μg of purified
Rv0901 mixed with complete Freund adjuvant, followed by
a booster inoculum of 50 μg of protein and incomplete Freund
adjuvant administered three times. At the third administration, the rabbit was boosted intravenously with 50 μg of
protein. A week later, the rabbit was bled by the arteria carotis
communis, and the blood was incubated for 30 min at 37°C.
After that, erythrocytes were removed by centrifugation, and
the serum was collected from the supernatant.
Expression of recombinant plasmid in M. smegmatis:
Both recombinant M. smegmatis and M. smegmatis were incubated at 45°C for 30 min (14), and the cells were collected
by centrifugation at 10,000 rpm for 10 min. The cells were
then washed twice with prechilled PBS and resuspended in
prechilled PBS (1/50th of original volume). Next, 2 × SDS
loading buffer (identical volume) was added to the cell
suspension. The cell suspension was boiled for 10 min and
centrifugated at 10,000 rpm for 10 min, after which the
supernatants were harvested and proteins were analyzed by
SDS-PAGE and Western blotting.
Cytotoxicity assay of His-tagged protein Rv0901 in a
mammalian cell line: Cell growth and viability were assessed by XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)5-[(phenylamino) carbonyl] -2H-tetrazoium hydroxide) assay.
ANA-1 cells were inoculated and grown in 96-well plates
for 36 h. Cells were treated with a serum-free medium for 24
h, and then protein was added to the cell culture medium.
Cells were then exposed to various concentrations (1, 5, 10,
20, and 40 μg/ml) of His-tagged protein Rv0901 for 48 h,
and 40 μl 1 mg/ml of XTT and 25 μmol/L phenazine
methosulfate (PMS) were added to each well. After the cells
were incubated under 5% CO2 at 37°C for 4 h, absorbance at
450 nm was measured for each well with a Bio-Rad microplate
reader. Experiments were performed five times.
The cytotoxicity of His-tagged protein Rv0901 was measured based on cell apoptosis and whether lactate dehydroge27
in those of mc2155. This result was further confirmed by
Western blotting with the antibody Rv0901 prepared by our
laboratory as described previously (Fig. 2).
Cytotoxicity effect of Rv0901 on ANA-1 cells: To verify
the cytotoxicity of Rv0901 on ANA-1 cells, we measured the
XTT absorbance, release of LDH and NO into the culture
media from cells and apoptosis ratio of ANA-1 cells. The
addition of Rv0901 to cultured ANA-1 cells caused decreased
XTT absorbance and increased LDH and NO release from
all cells tested in a concentration-dependent manner as compared to untreated cells (Table 1). Significant apoptosis ratio
changes were observed when Rv0901 was administered to
Camarillo, Calif., USA) according to the manufacturer’s
protocol. NO activity was quantified in the serum using a NO
kit according to the manufacturer’s protocol.
Statistical analysis: Student’s t test was used to calculate
the statistical significances between data obtained from the
experimental groups. A value of P < 0.05 was considered to
be significant. All statistical analyses were conducted by SPSS
software (SPSS, Inc., Chicago, Ill., USA).
RESULTS
Expression and purification of His-tagged Rv0901: The
Rv0901 gene (528 bp) was amplified and confirmed as having the published sequence (GenBank accession no. NC000962) by DNA blast (data not shown). Results of SDSPAGE and Western blot analysis showed that the recombinant E. coli containing Rv0901 on a pET-32a(+) vector expressed a 40-kDa protein, in accord with the predicted
molecular weight of PE0901 (Fig. 1). The endotoxin detection assay indicated that the endotoxin content of purified
His-tagged Rv0901 was as low as 0.03 endotoxin units (EU)
per microgram of protein.
Expression of the Rv0901 of recombinant M. smegmatis:
M. smegmatis cells were transformed with recombinant shuttle
plasmids PMV261, and accordingly recombinant M. smegmatis
strains (Rmc2155) were obtained. Total proteins of Rmc2155
and mc2155 were gained after heat induction. SDS-PAGE
confirmed that the expressed 19-kDa Rv0901 was present in
the cell lysates of Rmc2155, while no such band appeared
Fig. 2. Expression of Rv0901 protein in recombinant mc2155. The
Coomassie Brilliant Blue R250 stain (A) and Western blot analysis
(B) of 12% SDS-PAGE reveal a 19-kDa protein expressed by
Rmc2155 containing pMV-Rv0901 induced by heat shock. (A) Expression of Rv0901 protein in Rmc2155. M, Marker; lane 1, Rmc2155
containing pMV-Rv0901 induced by heat shock; lane 2, mc2155
induced by heat shock; lane 3, pMV261 induced by heat shock. (B)
Western blot. Lane 1, mc2155 containing pMV261 induced by heat
shock; lane 2, 19-kDa Rmc2155 containing pMV-Rv0901 induced
by heat shock.
Fig. 1. Expression of His-tagged Rv0901 protein in recombinant E.
coli. The Coomassie Brilliant Blue R250 stain (A) and Western blot
analysis (B) of 12% SDS-PAGE reveal a 40-kDa protein expressed
by recombinant E. coli (pET-Rv0901) induced through IPTG (1 mM)
for 5 h, while not expressed by control E. coli (pET) under the same
inducing condition. (A) M, Marker; lane 1, BL21 control; lane 2, pET
plasmid in BL21 before induced; lane 3, pET plasmid in BL21 after
induced; lane 4, the bacterial culture liquid before the fusion protein
in BL21 induced; lane 5, the bacterial culture liquid after the fusion
protein in BL21 induced; lane 6, purified 40 kDa His-tagged Rv0901
protein. (B) Lane 1, 20.4-kDa pET protein; lane 2, 40-kDa PE0901.
Fig. 3. Apoptosis ratio assessed by flow cytometry of ANA-1 cells
exposed to different concentration of Rv0901 protein. Apoptosis ratio
increased when the concentration of Rv0901 increased. A, B, C, D,
E, F, Rv0901 protein of 0, 1, 5, 10, 20, 40 μg/ml.
Table 1. Cytotoxicity effect of the Rv0901 protein on ANA-1 cells
Protein
(μg/ml)
0
1
5
10
20
40
NO (μmol/L)
24 h
48 h
21.99 ± 3.19
34.22 ± 2.7
42.74 ± 2.58*
54.06 ± 4.42*
60.97 ± 3.52*
70.64 ± 3.17*
23.71 ± 3.21
44.26 ± 3.59
57.62 ± 3.95*
64.03 ± 3.42*
71.75 ± 2.2*
103.71 ± 6.55*
24 h
LDH (U/L)
48 h
XTT (A450)
48 h
Apoptosis rate (%)
48 h
560.4 ± 61.12
667.22 ± 51.83
905.88 ± 74.82*
1,077.94 ± 147.78*
1,243.96 ± 150.54*
1,455.41 ± 139.64*
775.11 ± 112.14
990.94 ± 138.78
1,509.04 ± 146.88*
1,938.32 ± 212.86*
2,431.94 ± 240*
3,301.47 ± 213.65*
1.84 ± 0.05
1.75 ± 0.1
1.53 ± 0.15*
1.23 ± 0.13*
0.9 ± 0.06*
0.54 ± 0.08*
7.04 ± 0.98
8.32 ± 0.91
12.48 ± 1.34*
14.85 ± 1.2*
17.74 ± 1.55*
19.56 ± 1.1*
* P < 0.05 versus control group (0 μg/ml).
Quantum of nitrogen monoxidium (NO), lactate dehydrogenase (LDH), apoptosis ratio increased and XTT absorbance decreased
when the concentration of protein increased. Average of 5 independent assays ± standard deviation.
28
Table 2. Effect of Rmc2155 and mc2155 on THP-1 cells
Group
THP-1
mc2155
Rmc2155
3h
Survival ratio (%)
24 h
86.0 ± 3.6*
58.0 ± 1.0*
28.0 ± 3.0
78.0 ± 6.2*
56.0 ± 3.6*
16.0 ± 2.6
NO (μmol/L)
48 h
96 h
48 h
68.0 ± 6.0*
50.0 ± 4.4*
12.0 ± 3.5
33.07 ± 3.2*
40.67 ± 5.2*
49.68 ± 2.7
36.56 ± 4.3*
60.09 ± 1.6*
72.33 ± 1.9
Apoptosis ratio (%)
24 h
72 h
1.5 ± 3.0*
7.2 ± 4.0*
38.6 ± 3.3
4.2 ± 2.7*
18.7 ± 1.6*
57.3 ± 2.9
* P < 0.05 versus Rmc2155 group.
The survival ratio of THP-1 cells decreased significantly and NO, apoptosis ratio increased with the extension of time.
Table 3. Proilferative responses of splenocytes from
immunized mice
Group
OD 450
SI
Control
mc2155
Rmc2155
0.291 ± 0.024
0.569 ± 0.038*
0.781 ± 0.015
–
1.96 ± 0.10*
2.70 ± 0.19
*P < 0.05 versus Rmc2155 group.
PBS immunized mice as control.
Fig. 4. Apoptosis ratio assessed by flow cytometry of THP-1 cells infected with Rmc2155 or mc2155. Rmc2155 mc2155 can increase the
ratio of apoptosis of THP-1 cells with the extension of time, but
mc2155 was lower than Rmc2155. A, B: Apoptosis ratio of THP-1;
C, D: Apoptosis ratio of THP-1 infected with mc2155; E, F: Apoptosis
ratio of THP-1 infected with Rmc2155. A, C, E (24 h), B, D, F (72 h).
Table 4. Alteration of the propotions of splenocyte subsets in
immunized mice
Group
CD4+ (%)
CD8+ (%)
CD4+/CD8+
Control
mc2155
Rmc2155
21.37 ± 1.37
23.87 ± 1.51*
29.63 ± 3.09* △
10.53 ± 2.39
13.37 ± 1.42*
14.00 ± 1.73* △
1.74 ± 0.25
2.19 ± 0.16
2.44 ± 0.29
*P < 0.05 compare to control; △ P < 0.05 compare to mc2155.
PBS immunized mice as control.
Table 5. Levels of IFN-γ, NO in blood serum of
mice infected with mc2155 or Rmc2155
Fig. 5. Flow cytometric analysis of CD4+, CD8+ subset cells. Percentages of CD4+, CD8+ subset of mice that received the challenge infection via Rmc2155 (C) were greater than that of mc2155 (B). A, PBS
control; B, mice immuzied by mc2155; C, mice immuzied by
Rmc2155.
Group
IFN-γ (ng/ml)
NO (μmol/L)
Control
mc2155
Rmc2155
0.11 ± 0.06
0.24 ± 0.14*
0.26 ± 0.11*
9.2 ± 0.8
29.1 ± 1.3*
30.2 ± 2.1*
*P < 0.05 compare to control.
PBS immunized mice as control.
recombinant M. smegmatis and M. smegmatis groups, with
the CD4/CD8 ratio increasing simultaneously. Infection with
recombinant M. smegmatis induced significantly greater
percentages of CD4+/CD8+ cells than did infection with M.
smegmatis (Table 4, Fig. 5).
Results from infection with M. smegmatis and recombinant M. smegmatis revealed significantly enhanced levels of
IFN-γ and NO (Table 5) compared with the control group, but
no significant differences in mice infected with M. smegmatis
or recombinant M. smegmatis.
ANA-1 cells compared with untreated cells (Table 1, Fig. 3).
The effect of recombinant M. smegmatis and M. smegmatis
on THP-1 cells: The survival ratio of THP-1 cells decreased
significantly after infection with Rmc2155 at 3 h, 24 h, and
48 h (Table 2). Accordingly, we found that the survival ratio
of THP-1 cells decreased in cells infected with Rmc2155, consistent with the profiles of apoptosis by flow cytometry (Table
2, Fig. 4). Furthermore, the amount of NO increased dramatically in cells infected with Rmc2155 at 48 h and 96 h (Table
2).
Efficacy against recombinant M. smegmatis challenge:
Infected mice were sacrificed 1 week after the last injection,
and no significant differences were found in visual inspection of the surfaces of the lungs, livers, or spleens, or from
histopathological examination between the two groups.
Lymphoproliferation in response to antigen restimulation
in vitro was measured 3 weeks after the last infection. Recombinant M. smegmatis induced higher lymphoproliferation
than did the control M. smegmatis by the elevated stimulation index (Table 3).
The proportions of splenocyte subsets were detected by
flow cytometry after the challenge experiment. The percentages of CD4+/CD8+ subsets increased substantially in the
DISCUSSION
TB is the infectious disease with the highest death rate,
and China’s TB population is the second largest in the world.
In 2002, Camus et al. used biological information tools (11)
to re-annotate and reclassify the complete genome sequence
of M. tuberculosis. In this work, genes previously classified
as unknown were reclassified as relating to the cell wall and
cell processes. Very little is known about the specific proteins in M. tuberculosis (11); the Rv0901 gene is one of the
genes related to the cell wall, but its function is not understood. The M. tuberculosis cell wall plays an indispensable
role in its pathogenic mechanism and virulence. Rv0901,
29
extracted from infected New Zealand white rabbits showed
that Rv0901 was highly immunogenic, which may allow for
its use as a vaccine against TB. To address this question, we
used mice infected with recombinant M. smegmatis to investigate the toxicity and immunogenicity of Rv0901. However,
no apparent histopathological changes were found from
organs of these infected animals. Moreover, the IFN-γ and
NO concentration of two infected groups was higher than
that in the control group, while no significant difference was
found between the two infected groups. This result probably
indicated that the change in a single gene was not enough to
lead to obvious pathologic changes, or that the infection time
or bacterial dose were insufficient. Most importantly, in this
animal test the recombinant M. smegmatis bearing the Rv0901
gene confirmed that the gene protected the host against the
challenging infection.
M. tuberculosis is an intracellular bacterium residing primarily in lung macrophages. Cell-mediated responses are
known to be involved in the control of this infection. Activation of both CD4+ and CD8+ T cells is seen in primo-infected
individuals (20) and in mice after experimental infection (21).
CD4+ and CD8+ T cells are thought to control infection at
different stages and sites of infection by their capacity to
produce IFN-γ in response to infected macrophages presenting mycobacterial antigens (22-24). IFN-γ in turn activates
macrophages to kill the resident bacteria via the induction of
reactive nitrogen and oxygen intermediates (25) and by promoting phagolysosome fusion (26). Percentages of CD4+ and
CD8+ in the subset of mice that received the challenge infection via recombinant M. smegmatis were greater than those
of M. smegmatis, which may contribute to better protection
because of the important role of Rv0901. This may be explained
by the close correlation of immunogenicity and toxicity, which
could result in proportional changes in critical cytokine and
splenocyte subsets.
The present study clearly indicates that Rv0901 correlates
with immunogenicity and toxicity; the signal mechanism of
this correlation has yet to be explored. Long-term observation and alternative animal model studies are needed. In our
laboratory, study of the function of the Rv0901 gene of M.
tuberculosis is ongoing at the present time.
located at positions 1,003,957 - 1,004,481 of the M. tuberculosis (H37Rv) genome gene, encodes 175 amino acids, with an
overall length of 528 bp. Bioinformatics indicate that the
Rv0901 gene only exists in the virulent M. tuberculosis
(H37Rv and CDC1551) and M. bovis (AF2122/97) strains
and not in M. smegmatis (mc2155). Protein structure prediction hints that the C-terminus of Rv0901 protein has some
similarity (44.9%) to Mycobacterium leprae virulence protein TR03307.
M. tuberculosis is a sophisticated intracellular pathogen
that can persist for months or years within the human host,
and different mechanisms related to its entry, survival, and
replication in macrophages are involved in the infection process (16). The bacilli are notably able to interfere with normal
macrophage functions, such as antigen presentation and
phagosome maturation, residing in a specialized, nonacidified
compartment. We studied the toxic effect of the Rv0901 gene
and Rv0901 protein on cultured mammalian cells. The
Rv0901 gene was knocked into mc2155, and the results indicated that after infection with recombinant M. smegmatis
bearing the Rv0901gene, the survival capacity of human acute
monocytic leukemia cell line THP-1 cells declined and the
release of NO increased dramatically. Similarly, when the
Rv0901 protein concentration equaled or exceeded 5 μg/ml
with the increase of protein concentration, the release of LDH
and NO from mouse bone marrow macrophage line ANA-1
cells increased and the XTT absorbance value decreased in a
dose-dependent manner. These results were probably caused
by the Rv0901 gene and Rv0901 protein. Macrophages play
a vital role in diseases caused by M. tuberculosis. To some
extent, the reduced survival capacity of THP-1 cells reflects
the fact that the Rv0901 gene is virulent. LDH, an intracellular enzyme, can be detected in the culture medium when
released in the case of cell injury or death and is a generally
the accepted index of cell injury. When used with phenazine
methosulfate, XTT can be reduced by mitochondrion dehydrogenase in living cells to form a water-soluble, light brown
formazan product that parallels the number of living cells,
and this method is used to detect the number of living cells
(17). In light of this, the above results show that Rv0901 protein can cause significant injury to ANA-1 cells and thus has
a toxic effect on cells. NO has a reported cytotoxic effect on
anti-M. tuberculosis; it can restrain the cytochondriome
signal and produce free radicals (18). This study reveals
that both recombinant M. smegmatis and Rv0901 protein
can increase the amount of NO released by macrophage
cells, further proving the cytotoxicity of Rv0901. In addition, Rv0901 protein has a significant toxic effect on cells,
causing significant apoptosis of ANA-1 cells, which is consistent with the conclusion that recombinant M. smegmatis
can increase the ratio of THP-1 cell apoptosis. Recent studies
show that the virulent M. tuberculosis strain H37Rv induces
substantially less macrophage apoptosis than does the attenuated strain H37Ra. This difference is believed to be caused
by the virulent genes of H37Rv (19). Rv0901 is likely to be a
virulent-related gene of H37Rv, as shown by the reactions of
the two cell lines. THP-1 is a kind of human acute monocytic
leukemia cell line, while ANA-1 is a normal mouse bone
marrow macrophage cell line. When the protein or gene functioned in different cells, results were similar, which can be
explained by differences in the two cell lines. Therefore, we
still reasonably hold that to a certain extent Rv0901 is a
virulent correlated factor of M. tuberculosis.
Stimulated production of high titer and specific antibody
ACKNOWLEDGMENTS
This research was supported by the National Natural Science Foundation
of China (No. 30872257) and the National key project of Infectious Diseases.
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