Rv0901 from Mycobacterium tuberculosis, a Possible Novel Virulent Gene Proved through the Recombinant Mycobacterium smegmatis

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1 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 Zhang 1,2, Qi Zhong 1, Lang Bao 1 *, Ying Zhang 1, Lei Gao 1, Bi Huang 1, and Hui-Dong Zhang 1 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, 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 SDS- PAGE. 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. *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 , P.R. China. Tel: , baolang@moe.edu.cn 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 recombinant plasmid bearing Rv0901 was constructed using the shuttle vector pmv261, then electroporated into avirulent Mycobacterium smegmatis mc 2 155, 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. MATERIALS AND METHODS Bacterial strains, cells, animals, and culture conditions: The genomes of M. tuberculosis (H37Rv) and M. smegmatis (mc 2 155) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Mc 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 THP- 1 (human monocytic cell line) cells were grown at 37 C in a 5% CO 2 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 mc 2 155: 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 TGTCCGCCGTGTGCTCTT- 3 (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 pmv261-26

2 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 mc 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 g/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% SDS- PAGE, 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 (Sino- American, 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 g of purified Rv0901 mixed with complete Freund adjuvant, followed by a booster inoculum of 50 g of protein and incomplete Freund adjuvant administered three times. At the third administration, the rabbit was boosted intravenously with 50 g 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 g/ml) of His-tagged protein Rv0901 for 48 h, and 40 l 1 mg/ml of XTT and 25 mol/l phenazine methosulfate (PMS) were added to each well. After the cells were incubated under 5% CO 2 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 dehydrogenase (LDH) and/or nitrogen monoxidum (NO) were released. The cells were plated in a 6-well plate at a concentration of 10 5 cells per well and treated with a serum-free medium 24 h before experiments. PE0901 were diluted to 1, 5, 10, 20, and 40 g/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 10 5 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 g/ml) and then incubated in RPMI1640 medium containing gentamicin (25 g/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, 10 6 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 10 5 cells per well and grown in 96-well plates. XTT was used to detect the proliferation state. Briefly, 50 l 10 g/ml of whole Mycobacterium protein was added into each well to be tested, and the plate was incubated under 5% CO 2 at 37 C for 5 days, and then XTT was added to each well with 25 mol/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 = A treated / A control. 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 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)- activity was quantified in the serum using a mouse IFN- ELISA kit (Biosource, 27

3 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. NC ) by DNA blast (data not shown). Results of SDS- PAGE 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 (Rmc 2 155) were obtained. Total proteins of Rmc and mc were gained after heat induction. SDS-PAGE confirmed that the expressed 19-kDa Rv0901 was present in the cell lysates of Rmc 2 155, while no such band appeared in those of mc 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 Fig. 2. Expression of Rv0901 protein in recombinant mc The Coomassie Brilliant Blue R250 stain (A) and Western blot analysis (B) of 12% SDS-PAGE reveal a 19-kDa protein expressed by Rmc containing pmv-rv0901 induced by heat shock. (A) Expression of Rv0901 protein in Rmc M, Marker; lane 1, Rmc containing pmv-rv0901 induced by heat shock; lane 2, mc induced by heat shock; lane 3, pmv261 induced by heat shock. (B) Western blot. Lane 1, mc containing pmv261 induced by heat shock; lane 2, 19-kDa Rmc 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 g/ml. Table 1. Cytotoxicity effect of the Rv0901 protein on ANA-1 cells Protein NO ( mol/l) LDH (U/L) XTT (A450) Apoptosis rate (%) ( g/ml) 24 h 48 h 24 h 48 h 48 h 48 h ± ± ± ± ± ± ± ± ± ± ± ± ± 2.58* ± 3.95* ± 74.82* 1, ± * 1.53 ± 0.15* ± 1.34* ± 4.42* ± 3.42* 1, ± * 1, ± * 1.23 ± 0.13* ± 1.2* ± 3.52* ± 2.2* 1, ± * 2, ± 240* 0.9 ± 0.06* ± 1.55* ± 3.17* ± 6.55* 1, ± * 3, ± * 0.54 ± 0.08* ± 1.1* * P < 0.05 versus control group (0 g/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

4 Table 2. Effect of Rmc and mc on THP-1 cells Group Survival ratio (%) NO ( mol/l) Apoptosis ratio (%) 3 h 24 h 48 h 48 h 96 h 24 h 72 h THP ± 3.6* 78.0 ± 6.2* 68.0 ± 6.0* ± 3.2* ± 4.3* 1.5 ± 3.0* 4.2 ± 2.7* mc ± 1.0* 56.0 ± 3.6* 50.0 ± 4.4* ± 5.2* ± 1.6* 7.2 ± 4.0* 18.7 ± 1.6* Rmc ± ± ± ± ± ± ± 2.9 * P < 0.05 versus Rmc 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 ± mc ± 0.038* 1.96 ± 0.10* Rmc ± ± 0.19 *P < 0.05 versus Rmc group. PBS immunized mice as control. Fig. 4. Apoptosis ratio assessed by flow cytometry of THP-1 cells infected with Rmc or mc Rmc mc can increase the ratio of apoptosis of THP-1 cells with the extension of time, but mc was lower than Rmc A, B: Apoptosis ratio of THP-1; C, D: Apoptosis ratio of THP-1 infected with mc 2 155; E, F: Apoptosis ratio of THP-1 infected with Rmc 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 ± ± ± 0.25 mc ± 1.51* ± 1.42* 2.19 ± 0.16 Rmc ± 3.09* ± 1.73* 2.44 ± 0.29 *P < 0.05 compare to control; P < 0.05 compare to mc PBS immunized mice as control. Table 5. Levels of IFN-, NO in blood serum of mice infected with mc or Rmc Group IFN- (ng/ml) NO ( mol/l) Fig. 5. Flow cytometric analysis of CD4 +, CD8 + subset cells. Percentages of CD4 +, CD8 + subset of mice that received the challenge infection via Rmc (C) were greater than that of mc (B). A, PBS control; B, mice immuzied by mc 2 155; C, mice immuzied by Rmc 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 Rmc 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 Rmc 2 155, consistent with the profiles of apoptosis by flow cytometry (Table 2, Fig. 4). Furthermore, the amount of NO increased dramatically in cells infected with Rmc 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 Control 0.11 ± ± 0.8 mc ± 0.14* 29.1 ± 1.3* Rmc ± 0.11* 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- and NO (Table 5) compared with the control group, but no significant differences in mice infected with M. smegmatis or recombinant M. smegmatis. 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

5 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 (mc 2 155). Protein structure prediction hints that the C-terminus of Rv0901 protein has some similarity (44.9%) to Mycobacterium leprae virulence protein TR 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 mc 2 155, 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 g/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 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- 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- in response to infected macrophages presenting mycobacterial antigens (22-24). IFN- 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. 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