Mycobacterium smegmatis (MC2 155) and M. tuberculosis H37Ra were grown in Middlebrook 7H9 broth or Middlebrook 7H10 agar (DIFCO, United States) supplemented with 10% ADC (BD, United States) and kept in a biosafety P1 laboratory. The media for recombinant M. smegmatis and M. tuberculosis H37Ra that over-expressed Rv2629 were supplemented with 50 μg/mL kanamycin, while for recombinant M. smegmatis that expressed low levels of MSMEG_1130, the medium was supplemented with 50 μg/mL kanamycin and 0.1% acetamide (w/v). Transformed Escherichia coli DH5α was cultured in Luria-Bertani broth and/or agar with the appropriate antibiotic (50 μg/mL kanamycin).

Strains isolated from clinical drug-resistant patients (strains 1570, 934, 945, 1573, 1176, 522, 1219, and 1221, all of the Beijing type) were obtained and cultured in a P2 laboratory at the Shanghai Pulmonary Hospital. All clinical strains included were rifampicin-resistant. Strain 934 was rifampicin (RIF) resistant with a minimum inhibitory concentrations (MIC) of 32 μg/mL. Strains 1570, 945, 1573, 1176, 1219, 522, and 1221 were MDR. All strains were identified and drug-susceptibility was evaluated as previously described (Ramaswamy et al., 2003; Wang et al., 2007). Clinical isolates and M. tuberculosis H37Rv were cultured under the same conditions as M. tuberculosis H37Ra (Kwon et al., 2017).

THP-1 cells were grown in house and were cultured in RPMI-1640 (Gibco, Foster City, CA, United States) supplemented with 10% (w/v) fetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin (Gibco). THP-1 cells were maintained at 37°C in a humidified atmosphere with 5% CO₂.

Recombinant plasmids encoding wild type Rv2629 (Rv2629^W) and the 191C mutant (Rv2629^M) were constructed. The full length Rv2629 genes were amplified from genomic DNA extracted from H37Rv (for Rv2629^W) and from clinical strains (for Rv2629^Mgenotype). Recombinant plasmids encoding the antisense strand of MSMEG_1130, an ortholog of Rv2629 in M. smegmatis, was constructed. The amplification was conducted by PCR using the appropriate primers (listed in Supplementary Table S1). The PCR products were digested with the corresponding enzymes (NEB, United States) (Supplementary Table S1) for 3 h at 37°C. Plasmid pMV261 (for over-expression) and/or pACT (an acetamide-inducible expression vector, for reduced expression) (Invitrogen, Waltham, MA, United States) were digested with the same restriction enzymes. The two fragments were ligated, transformed into E. coli DH5α and plated on LB agar containing kanamycin (50 μg/mL). Following overnight incubation at 37°C, single colonies were randomly selected and grown in LB broth. The plasmids were isolated from E. coli DH5α culture using an Axygen Miniprep Kit (Axygen, Union City, CA, United States) and confirmed by DNA sequencing.

The recombinant pMV261 and/or pACT plasmids were transformed into the M. smegmatis strain MC2 155 (MS) and/or M. tuberculosis H37Ra, respectively by electroporation (Reed et al., 2007). The study of M. tuberculosis genes in recombinant M. smegmatis is a recognized approach (Falcone et al., 1995; Daugelat et al., 2003; Andreu et al., 2004; Zimhony et al., 2004; Altaf et al., 2010; Sweeney et al., 2011; Bae et al., 2017; Sun et al., 2017; Yu et al., 2017; Wang et al., 2017). Recombinant M. smegmatis over-expressing Rv2629 was identified as MSW (Rv2629 wild type) and MSM (Rv2629 mutant). Recombinant M. tuberculosis H37Ra over-expressing Rv2629 was identified as RaW (Rv2629 wild type) and RaM (Rv2629 mutant). Recombinant M. smegmatis expressing low levels of MSMEG_1130 was identified as MSL. Recombinant strains with the plasmid pMV261and/or pACT were constructed (MSP, RaP, or MS pACT) and used as controls. The types of strains with the corresponding genes and plasmids are presented in Supplementary Table S2.

Total RNA was isolated from M. smegmatis and M. tuberculosis using an RNA extraction kit (Omega, United States), according to the manufacturer’s instructions. Contaminating DNA was digested with RNase-free DNase I (Omega, United States) for 15 min at 37°C. Prior to further analysis the quality check of the RNA was determined by the estimation of the A260/A280 ratio. For RT-qPCR, cDNA was synthesized according to the manufacturer’s instructions (ReverTraAce qPCR RT kit, Toyobo, Japan).

qPCR analysis was conducted using Rv2629-specific primers (see Supplementary Table S1) with SYBR Green Real-Time PCR Master Mix (QPK-201) (Toyobo, Japan). 16S rRNA and Rv2703 (SigA) were used as reference sequences. The reaction conditions were: 94°C for 10 min, followed by 40 cycles of 94°C for 30 s, 56 to 65°C for 45 s, and 72°C for 30 s. A standard curve of copy number (range: 10¹–10⁸) was constructed. Data were collected using a BIO-RAD iCycler (BIO-RAD, United States). RT-negative (without reverse transcriptase) reactions were used to account for residual DNA, and the copy numbers were normalized to those of 16S rRNA. The normalized values were used to determine the relative fold change in expression. The estimation of the relative expression of each gene was carried out using the ddCT method. The experiments were carried out in triplicate, and the results are expressed as mean ± SD.

Whole cells were harvested from 20 mL cultures of M. smegmatis strains and M. tuberculosis isolates. The bacterial pellets were washed twice with 0.02 M PBS, resuspended, and sonicated in 200 μL of 0.02M PBS (supplemented with 1 mM PMSF and 10 mM EDTA) and centrifuged. The supernatant was treated with lysis buffer (8 M urea, 2 M thiourea, 140 mM DTT, 0.5% Biolyte pH 4–7, and 4% CHAPs). The protein concentration was estimated using the Bradford assay. Equal amounts of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 12% gel), transferred to a PVDF membrane and detected with anti-Rv2629 rabbit antiserum (in house preparation) and/or the FtsZ monoclonal antibody (4°C, overnight) followed by a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase (Sigma, United States) (37°C, 1 h). The blots were visualized with BCIP/NBT solution (room temperature, 20 min). The reaction was terminated by addition of deionized water. FtsZ was used as a loading control.

Minimum inhibitory concentrations assay of recombinant M. smegmatis was carried out to detect the relationship between over-expressing Rv2629 proteins and the susceptibility to anti-TB drugs (streptomycin, isoniazid, rifampicin, ethambutol, ofloxacin, levofloxacin, moxifloxacin, amikacin, kanamycin, and capreomycin). Antibiotic dilutions and 96-well plate preparations (Falcon 3072; Becton Dickinson, Lincoln Park, NJ, United States) were carried out as described by Caviedes et al. (2002). Briefly, Rv2629 overexpressing strains were grown in 7H9/ADC at 37°C until an OD₆₀₀ value of 0.5 was obtained. The bacterial suspensions were diluted 1000 times in 7H9 broth and the final diluted broth was dispensed into each well of a 96-well cell culture plate (100 μL/well). The drug storage solutions were diluted to prepare two-fold serial diluted solutions in 7H9 broth and subsequently added to the wells containing the bacterial suspensions. The plate was incubated for 48 h at 37°C. The wells containing bacterial pellets were denoted as the positive wells, whereas the wells containing clear broth were denoted as the negative wells. The minimum drug concentrations of the negative wells were the MICs of the drugs. All the experiments were carried out in triplicate.

Total RNA from each sample was quantified using the NanoDrop 1000 (Thermo Fisher, United States). RNA integrity was assessed using standard denaturing agarose gel electrophoresis. For microarray analysis, the Agilent Array platform was used according to the manufacturer’s standard protocols for sample preparation and microarray hybridization. Briefly, total RNA (1 μg) was amplified and transcribed into fluorescent cRNA, following the manufacturer’s Quick Amp Labeling protocol (Version 5.7, Agilent Technologies). The labeled samples were hybridized toward the M. tuberculosis H37Ra custom Oligo Array (8x15K, Agilent array) and were scanned using the Agilent Scanner G2505B (Agilent Technologies, United States). The Agilent Feature Extraction Software (version 10.5.1.1) (Agilent Technologies, United States) was used to analyze acquired array images. The median normalization and subsequent data processing was carried out using the GeneSpring GX v11.0 software package (Agilent Technologies, United States). Following Median normalization of the raw data, genes of at least one out of nine samples that exhibited flags in Present and/or Marginal (“All Targets Value”) were selected for further data analysis. Differentially expressed genes were identified using Volcano Plot filtering. Pathway and GO analyses were conducted in order to reveal the biological functions of this subset of differentially expressed genes. Finally, hierarchical clustering was carried out in order to show distinguishable gene expression profiling among samples.

For ultrathin cryo sections, log-phase M. Smegmatis cultures were harvested (5 mL). The cells were washed twice with 0.1 M phosphate buffer (PB) and fixed with 2.5% glutaraldehyde in 0.1 M PB (2 h). The log phase was estimated as determined previously (Belanger and Hatfull, 1999). Notably, a culture of M. smegmatis mc²155 was grown at 37°C, until an OD₆₀₀ reading of approximately 0.8 was obtained. The cells were rinsed three times (15 min per wash) with 0.1 M PBS. After fixation in 1% osmium tetroxide (3 h), the cells were rinsed as previously described prior to dehydration with solutions containing increasing concentrations of ethanol and/or acetone at 4°C. The samples were subjected to gradual infiltration with Epon resin (3 days). Subsequently, the samples were solidified by incubation at increasing temperature for a total period of 2 days. Thin sections (70–80 nm) were prepared and stained with 3% uranyl acetate and lead citrate prior to examination under a Philips CM-120 transmission electron microscope (TEM) (Philips, United States).

To determine whether Rv2629 expression levels among strains would lead to observable phenotypic growth and survival differences, recombinant M. tuberculosis H37Ra and M. smegmatis strains were subcultured thrice in Middlebrook 7H9 broth supplemented with 10% ADC and 0.05% Tween-80 (OD₆₀₀ = 0.4). The M. smegmatis cultures were diluted when an OD₆₀₀ value of 0.05 was obtained. Aliquots of 50 mL were dispensed in conical flasks and grown under continuous shaking at 220 rpm and aerobic conditions. The growth and survival were assayed by optical density readings (OD₆₀₀) every 3 h following inoculation. The experiments were performed in triplicate.

The growth curve of recombinant M. tuberculosis H37Ra was detected using the BACTEC MGIT 960 Mycobacterial Detection System (Becton, Dickinson and Company, United States), as determined by the manufacturer’s instructions.

THP-1 cells (5 × 10⁵) were stimulated with 5 ng/mL PMA (Sigma Chemical Co, St. Louis, MO, United States) for 24 h prior to infection. The infection was conducted using H37Rv, clinical M. tuberculosis strain 934, and recombinant M. smegmatis in the log-phase at a multiplicity of infection (MOI) value of 1:10 (37°C, 5% CO₂, 4 h). Extracellular bacilli were removed by washing cells three times with PBS and the cells were cultured for 1 h in complete medium in the presence of 100 μg/mL gentamycin. The medium was replaced every 12 h during incubation, and the cells were lysed using 1% Triton X-100 solution at specific time points (0, 24, 48, 72, and 96 h). The precipitates were collected by centrifugation (2000 g, 20 min) for total RNA extraction, as described above. The number of CFU of intracellular mycobacteria and the viable bacteria present in the supernatant were determined on Middlebrook 7H10 agar with 10 % ADC.

The culture of MSP and MSW was conducted in 5 mL of Middlebrook7H9 medium supplemented with 10% ADC and 500 mg sterile 2-mm glass beads at 37°C under continuous shaking. The cultures isolated during the log phase were centrifuged at 500 g in order to remove clumps, and the cells were washed twice with sterile saline. The cell suspension was adjusted to an OD₆₀₀ of 0.8, corresponding to a concentration of 5 × 10⁷ CFU/mL. Female Balb/c mice (6–8-week-old) were infected intravenously with 0.1 mL of the bacterial suspension. The size of inoculum was confirmed by CFU counts.

The ability of survival and dissemination of each strain in vivo was assessed in six mice per group that were sacrificed by cervical dislocation on 0, 3, 6, 9, 12, 15, 20, and 35 days after infection. Lung, spleen, and kidney tissue specimens were weighed and homogenized in saline. The specimens were cultured in Middlebrook 7H10 medium (Becton Dickinson, United States) supplemented with 10% OADC following appropriate dilutions. CFUs were enumerated following 3–4 days of incubation at 37°C. All of the animal procedures were approved by the animal care committee of the Fudan University.

Whole cell extracts were prepared from 100 mL of cultures of each M. smegmatis strain in the log-phase. Bacterial pellets were washed twice and centrifuged (15°C, 8000 rpm, 15 min) in 0.02 M PBS (pH 7.4). The cells were sonicated in 500 μL of 0.02 PBS (0.02 M supplemented with 1 mM PMSF and 10 mM EDTA) and centrifuged at 12,000 rpm for 20 min at 4°C. The cells were treated with lysis buffer (8 M urea, 2 M thiourea, 140 mM DTT, 0.5% Biolyte pH 4-7, and 4% CHAPs) for 1 h and centrifuged (4°C, 12,000 rpm, 15 min). The supernatant was harvested and the protein concentration was estimated using the Bradford assay. Total protein (80 μg) were loaded on linear pH 4-7 IPG strips (Bio-Rad, United States) that were allowed to rehydrate at 50 V for 13 h. Isoelectric focusing was carried out using a Protean IEF (Bio-Rad, United States). The proteins were separated using 2-dimensional SDS-PAGE (12% gels) and stained with silver nitrate, as reported previously (Scheler et al., 1998; Gharahdaghi et al., 1999). The gels were scanned using Molecular Image FX (Bio-Rad, United States) and analyzed with the PD Quest 6.0 software (Bio-Rad, United States). The proteins of interest were excised from the silver-stained 2D gels and identified by MALDI-TOF-MS (Fu and Fu-Liu, 2007).

Statistical analysis was conducted using SPSS 11.5 (SPSS Inc., Chicago, IL, United States). Statistical differences of the continuous variables were analyzed by one-way analysis of variance (ANOVA) and the Student–Newman–Keuls test and/or Student t-test. The statistical level of significance was set at P < 0.05.

Previous studies have demonstrated an increase in the expression of Rv2629 at the transcriptional and/or translational levels induced by hypoxia and nitric oxide in clinical isolates of M. tuberculosis (Voskuil et al., 2003; Kassa et al., 2012; Zhang et al., 2014). Thus, we investigated the expression of the Rv2629 protein and mRNA levels among clinical isolates of M. tuberculosis. Quantitative RT-PCR was conducted using cDNA synthesized from total RNA isolated from bacilli from seven clinical strains (1573, 1570, 945, 1176, 1219, 522, and 1221) and the M. tuberculosis H37Rv control strain in the log and/or stationary-phases respectively (Figure 1A). The stationary phase was observed notably following 10 days of growth and the bacterial cultures were harvested at 3 h intervals for further analysis. It was observed that the average transcriptional level of Rv2629 in clinical isolates during the logarithmic phase was approximately two-fold lower than that noted in M. tuberculosis H37Rv, while during the stationary phase, this level was higher in the clinical isolates than in the control strain. The expression of Rv2629 at the protein level was further measured by Western blot analysis (Figures 1B,C). The cytoplasmic fraction of the cell extracts obtained from the clinical isolates indicated a two-fold increase in the stationary phase compared to that noted in M. tuberculosis H37Rv. The over-expression of Rv2629 at both the protein and the mRNA levels in clinical M. tuberculosis isolates was related to the stationary phases of the strains. Rv2629 exhibited reduced expression during the log-phase, when bacilli were actively duplicating.

In order to investigate the association of Rv2629 with dormancy, the THP-1 human leukemic monocyte cell line was infected with H37Rv and/or a clinical RIF-resistant isolate 934 in the log-phase at MOI = 10, following stimulation with 5 ng/mL PMA for 24 h. The mRNA levels of Rv2629 expressed in bacteria were measured at 0, 3, 24, and 72 h. Strain 934 was cultured in the absence of THP-1 cells and served as control. Rv2629 gene intracellular expression was further determined during several time periods (Figure 2). The results indicated that the ratios of the Rv2629 mRNA levels of strain 934 to H37Rv at 3 and 24 h were higher in the extracellular compared with the intracellular compartment (P < 0.01), whereas the opposite was noted for the 72 h period (P < 0.001) (Figure 2). Moreover, a high upregulation (45-fold) of intracellular Rv2629 expression in the clinical strain was observed at 72 h post-infection compared with the extracellular control (Figure 2), supporting the hypothesis that intracellular bacilli can remain in a dormant phenotype up to day 3 by over-expressing Rv2629.

M. smegmatis over-expressing Rv2629^W (MSW) and Rv2629^M (MSM) were produced in order to simulate Rv2629 expression levels in clinical M. tuberculosis strains and investigate the function of the Rv2629 protein. Western blot and RT-qPCR analysis of four M. smegmatis strains during the log-phase confirmed the increased expression of Rv2629 in the MSW and MSM compared with the control MS and MSP strains (Figures 3A,B).

The ortholog of Rv2629 in H37Ra and M. smegmatis strains are MRA_2657 and MSMEG_1130, respectively. The DNA and amino acid sequences of MRA_2657 are completely identical with Rv2629, while MSMEG_1130 and Rv2629 are highly homologous, in which the overlap value between MSMEG_1130 and Rv2629 was 366 based on the data of KEGG database. M. smegmatis MSL corresponded to a bacterial strain expressing low levels of MSMEG_1130. The strain was constructed to investigate the function of Rv2629, whereas the plasmid pACT, an acetamide-inducible expression vector, served as control. For generating the MSL strain, the plasmid pACT was subjected to recombination with the DNA fragment, whose mRNA production could complement with the MSMEG_1130 mRNA sequence reversely and thus could inhibit the expression of MSMEG_1130 gene. Western blot analysis in the two M. smegmatis strains during log-phase confirmed low expression of MSMEG_1130 in MSL compared with control MSpACT (Figure 3C). The use of M. smegmatis as a parental strain for the overexpression of Rv2629 has been reported in a previous study (Wang et al., 2007).

In order to investigate the association of Rv2629 with drug-resistance, 10 anti-TB drugs were selected including four first-line drugs (streptomycin, isoniazid, rifampicin, and ethambutol) and six second-line drugs (ofloxacin, levofloxacin, moxifloxacin, amikacin, kanamycin, and capreomycin). The MIC of recombinant M. Smegmatis was determined. The results are shown in Table 1. The Rv2629 over-expressing strains enhanced drug-resistance of recombinant M. smegmatis to ethambutol, moxifloxacin, and levofloxacin at least two-fold compared with the MSP strain.

The present study aimed to investigate the differences in gene expression caused by Rv2629 overexpression in M. tuberculosis strains and the potential interplay in the biological function of the Rv2629 gene. Microarray analysis was conducted on recombinant M. tuberculosis H37Ra overexpressing Rv2629 in the log phase. The results indicated that recombinant M. tuberculosis H37Ra overexpressing Rv2629 (RaW) and H37Ra overexpressing 191 A/C mutated Rv2629 (RaM) remained in the log phase of growth, respectively. In addition, 122 genes correlated with wild type Rv2629 (Supplementary Table S3), whereas 92 genes correlated with 191A/C mutated Rv2629. The genes from the two strains were shown in different sizes (Figure 4). As shown in Figure 4, two genes are connected if the correlation between them is greater than a selected threshold (0.9). The correlation is based on a correlation coefficient between each particular gene and the two modules. A total of 30 genes correlated with wild type Rv2629 and to a lesser extent with 191 A/C mutated Rv2629 (Table 2). The correlation coefficient was lower than the selected threshold in RaM. The remaining 92 genes correlated with Rv2629 in the two bacterial strains.

The GO and pathway analysis indicated that 122 genes correlated with wild type Rv2629; they were involved in ribosome construction, toluene degradation, arginine and proline metabolism, oxidative phosphorylation, and homologous recombination. The genes were notably related to the ribosome construction and the Fisher P-value was 4.28 × 10^-10 (Supplementary Table S4). The genes that solely correlated with wild type Rv2629 were notably related to ribosome production, oxidative phosphorylation, and virulence (Table 2). The results indicated that the 191A/C mutation in Rv2629 could influence the function of wild type Rv2629 partially. The mutation may lead to the decreased effect of Rv2629 on ribosome production, oxidative phosphorylation, and virulence.

The effects of the Rv2629 gene on the morphological features of MSW and MSM at the log-phase were investigated by TEM, as demonstrated by previous studies (Tumminia et al., 1991). No difference was noted in cell wall thickness and morphology, although recombinant M. smegmatis MSW revealed apparent differences in their cytoplasmic component compared to the MSM, MSP, and MS strains (Figure 5). The cytoplasm of MSW cells exhibited lower electron density and the cytoplasmic ribosomal content was reduced significantly compared to the MS and MSP strains, with the exception of the MSM strain (Figure 5).

The microarray results indicated that Rv2629 might be related to ribosome production. Consequently, the growth curves of the MSW and MSM strains were investigated by harvesting cultures at 3-h intervals and by measuring the OD₆₀₀. MS and MSP served as controls. The results of the two independent experiments demonstrated comparatively slow growth of MSW (Figure 6A) and indicated that Rv2629 overexpression inhibits bacterial growth and delays entry in the log phase. The growth of MSM was more rapid compared with MSW and slower than MSP and MS, which suggested that the 191A/C mutation of the Rv2629 gene weakens the inhibitory function of Rv2629. A similar effect was noted in the RaW and RaM strains (Supplementary Figure S1).

MSL strains that expressed low levels of MSMEG_1130 were investigated, as described above. MSpACT was used as the control. The results of two independent experiments indicated comparatively rapid growth of MSL (Figure 6B), while the low expression of MSMEG_1130 promoted bacterial growth and entry to the log phase.

The enhanced and/or attenuated in vitro growth phenotypes of recombinant M. smegmatis prompted the examination of the correlation between Rv2629 expression and dormant phenotype. THP-1 cells were infected with the bacteria. No major differences in the intracellular number of viable bacteria were observed at 24 h, suggesting that recombinant M. smegmatis strains that expressed high and low levels of Rv2629 exhibited similar rates of growth to that of control strains within the first day after the infection (Figure 7A). Nevertheless, the bacterial growth of the over-expressing Rv2629 strains was impaired at day 2 post-infection, whereas the viable bacilli number at the turn point of growth from lag to the log-phase was decreased. This result is consistent to that found in the in vitro experiments. Furthermore, over-expression of Rv2629 decreased the growth rate of the bacilli and further prevented them from achieving higher cell density in the stationary phase. This conclusion was further confirmed by comparison of the growth capability observed in THP1 cells of low-expressing the MSMEG_1130 M. smegmatis strains. The bacilli showed increased growth rate compared with the control samples from 48 to 96 h post-infection (Figure 7B). The growth of MSL in THP-1 cells was evaluated at MOI values of 1:5 and 1:20. The results were consistent with the results observed at MOI of 1:10 (Supplementary Figure S2).

The attenuated reproductive capacity of MSW in macrophage infection prompted us to investigate whether MSW could further decrease the virulence and pathogenicity of host tissues. Balb/c mice were infected with MSP, MSW, and MSM in order to identify the different pathogenicity and survival ability among these strains in vivo. The CFU values of bacteria in the lung, spleen, and kidney tissues were counted. The results indicated that the MSW strain exhibited a longer survival time, notably in the lung tissues compared with MSP, while with regard to the MSW strain the detection of the bacterial growth was evident in the lungs of the mice following 20 days of infection (Figure 8A). This observation occurred despite the absence of CFU counts between MSP and MSW at each time point. The acid-fast staining of lung tissue indicated that overexpression of wild type Rv2629 could promote the survival of M. smegmatis in granulomas of the lung. No significant differences among the three strains were noted at the 15th day after infection. The number of MSW strains that overexpressed the wild type Rv2629 increased significantly compared to the other two strains in the granuloma of the lung at the 20th day after infection. No visible MSP and/or MSM could be found in the diseased location at the 35th day post-infection, although a high number of MSW strains formed granulomas in the lung (Figure 8B). Additional pathological tests indicated that the pathogenicity of MSW increased and additional damage was caused in the lung tissues compared to the MSP and/or MSM strains. The pulmonary alveolus of mice infected with MSP was damaged slightly and it was the sole cause of bleeding in the bronchial lumen in the absence of granuloma formation during the 15th day following infection. Sporadic granuloma could be found in the small part of the lungs at the 20th day after infection, whereas the inflammatory exudate was absorbed and the main part of the lung recovered to the normal structure 35 days post-infection (Figure 9A). Apparent changes could be observed in the lung of MSW-infected mice. The normal structure of the major part of the pulmonary alveolus disappeared, which was permeated with inflammatory exudate and associated with the infiltration of a great number of inflammatory cells. The alveolar wall indicated congestion and incrassation, whereas apparent granuloma could be found in the lungs 15 days post-infection. The area and degree of damage increased further 20 days post-infection, and the normal structure of the complete part of the pulmonary alveolus disappeared. The structure of the majority of the pulmonary alveolus recovered, although a certain part of granuloma could be found in the lung (Figure 9B). The pathogenicity that was caused by the Rv2629 expressing strains decreased when a missense mutation (Asp64 to Ala64, A/C) was present at position 191 of the genetic sequence of Rv2629. The pulmonary alveolus of mice infected with MSM was damaged partly and sporadic granuloma could be found in certain parts of the lungs 15 days post-infection. The damaged structure of pulmonary alveolus recovered gradually and the majority of the lung tissue recovered to the normal structure at the 20th and the 35th day week post-infection, respectively, as noted for the MSP strain (Figure 9C). Consequently, the overexpression of the wild type Rv2629 could increase the survival and pathogenicity of M. smegmatis, whereas the 191 A/C mutation in Rv2629 could weaken the pathogenicity of the Rv2629-expressing strains.

Protein expression maps of M. smegmatis overexpressing Rv2629 were obtained by 2-DE (Figure 10). A total of eight differential proteins were found by comparing the protein differential expressing map on 2-DE gels. A total of five proteins, namely, CHP, UK, TF, AA, and FFP, were identified by MALDI-TOF-MS (Table 3). Nevertheless, the expression pattern of these proteins differed among the strains. TF and FFP were present only on the MSW 2-DE gel, as opposed to the MSP gel. AA was not detected on the MSW 2-DE gel. The expression of UK decreased, while the expression of CHP increased in the MSW compared with the MSP strains.