Protein phosphorylation plays a key role in Mycobacterium tuberculosis, the pathogen of tuberculosis, holding promise as a new target of anti-tuberculosis drugs. We used M. smegmatis, a close relative of M. tuberculosis, as a model organism to study the protein phosphorylation at different growth phases. We identified 573 phosphorylated peptides and 816 phosphorylated sites of 385 proteins in the M. smegmatis samples at both logarithmic and stationary phases, and then established a comprehensive dataset of phosphorylated proteins in M. smegmatis. By comparing the expression levels of phosphorylated proteins between the logarithmic and the stationary phase with the selected ion monitoring (SIM) strategy, we verified 68 upregulated proteins involved in cell division and protein translation, and 69 downregulated proteins mainly involved in the tricarboxylic acid cycle pathway. The differentially expressed phosphorylated proteins were significantly enriched in important cellular cycle events such as cell elongation and division. The findings of this study provide proteome evidence for elucidating the phosphorylation in both M. smegmatis and M. tuberculosis.
Mycobacterium smegmatis/genetics* ; Bacterial Proteins/genetics* ; Phosphorylation ; Phosphoproteins/metabolism* ; Mycobacterium tuberculosis/growth & development* ; Proteome/metabolism* ; Proteomics

Cryoelectron Microscopy ; Mycobacterium smegmatis ; genetics ; ultrastructure ; RNA, Ribosomal ; ultrastructure ; Ribosomal Proteins ; genetics ; isolation & purification ; Ribosomes ; genetics ; ultrastructure

9α-hydroxy-4-androstene-3,17-dione (9-OH-AD) is an important intermediate in the steroidal drugs production. 3-ketosteroid-9α-hydroxylase (KSH), a two protein system of KshA and KshB, is a key-enzyme in the microbial steroid ring B-opening pathway. KSH catalyzes the transformation of 4-androstene-3,17-dione (AD) into 9-OH-AD specifically. In the present study, the putative KshA and KshB genes were cloned from Mycobacterium smegmatis mc(2)155 and Gordonia neofelifaecis NRRL B-59395 respectively, and were inserted into the expression vector pNIT, the co-expression plasmids of kshA-kshB were obtained and electroporated into Mycobacterium sp. NRRL B-3805 cells. The recombinants were used to transform steroids, the main product was characterized as 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), showing that kshA and kshB were expressed successfully. Different from the original strain Mycobacterium sp. NRRL B-3805 that accumulates 4-androstene-3,17-dione, the recombinants accumulates 9α-hydroxy-4-androstene-3,17-dione as the main product. This results indicates that the putative genes kshA, kshB encode active KshA and KshB, respectively. The process of biotransformation was investigated and the results show that phytosterol is the most suitable substrate for biotransformation, kshA and kshB from M. smegmatis mc(2)155 seemed to exhibit high activity, because the resultant recombinant of them catalyzed the biotransformation of phytosterol to 9-OH-AD in a percent conversion of 90%, which was much higher than that of G. neofelifaecis NRRL B-59395. This study on the manipulation of the ksh genes in Mycobacterium sp. NRRL B-3805 provides a new pathway for producing steroid medicines.
Androstenedione ; analogs & derivatives ; biosynthesis ; Bacterial Proteins ; genetics ; metabolism ; Biotransformation ; Ketosteroids ; Mixed Function Oxygenases ; genetics ; metabolism ; Mycobacterium ; metabolism ; Mycobacterium smegmatis ; enzymology ; Plasmids

With the emergence of drug resistant tuberculosis, it is very urgent to find novel anti-tuberculosis drugs, especially novel anti-drug-resistant tuberculosis drugs. Because of the slow growth and the need to work in a biosafty environment of Mycobacterium tuberculosis, the development of evaluation of drug effect is severely impeded. In order to solve these issues, non-pathogenic fast-growing Mycobacterium smegmatis is introduced as test organism. The inhA is one of a target of isoniazid (INH) overexpression or mutation of this gene in Mycobacterium tuberculosis conferring resistant to INH. A recombinant plasmid bearing inhA was constructed and electroporated into Mycobacterium smegmatis, using shuttle expression vector pMV261. Transformants were induced to express a protein of inhA, identified by SDS-PAGE. Results show that Mycobacterium smegmatis containing inhA plasmids exhibited 100-fold or greater increased resistance to INH, but it conferred no increased resistance to others first-line anti-tuberculosis drugs. Resazurin microtiter assay plate testing of Mycobacterium smegmatis susceptibility to drugs is a rapid, simple, and inexpensive method and could decrease color background of drugs by detecting fluorescence. It will be benefit for high-throughout screening of drugs of anti-isoniazid-resistant Mycobacteria.
Anti-Bacterial Agents ; pharmacology ; Antibiotics, Antitubercular ; pharmacology ; Antitubercular Agents ; pharmacology ; Bacterial Proteins ; genetics ; metabolism ; Drug Resistance, Bacterial ; Electroporation ; Ethambutol ; pharmacology ; Isoniazid ; pharmacology ; Microbial Sensitivity Tests ; Mycobacterium smegmatis ; drug effects ; genetics ; metabolism ; Oxidoreductases ; genetics ; metabolism ; Plasmids ; Rifampin ; pharmacology ; Streptomycin ; pharmacology

BACKGROUNDIsocitrate lyase (ICL) was previously demonstrated to play a pivotal role in the intracellular metabolism of Mycobacterium tuberculosis (MTB). Presently several lines of evidence suggest that ICL from MTB (MTB-ICL) may play some roles in the interaction between MTB and host macrophage. However, there has been no research on the interaction between MTB-ICL and host macrophage.

METHODSMTB-icl and M. smegmatis (MS)-icl genes were amplified by polymerase chain reaction (PCR) and cloned into the E. coli-mycobacterium shuttle plasmid pUV15 to obtain recombinant shuttle plasmids pMTB-icl and pMS-icl. Following transformation into MS by electroporation, the expression of pMTB-icl and pMS-icl was verified by reverse transcriptase (RT)-PCR. The expression of recombinant plasmids derived from pUV15 when rMS was phagocytized by macrophage was also verified via fluorescence microscope. Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were used to infect RAW264.7 cells and the survival of intracellular MS was monitored by bacterial culture at 0, 24 and 48 hours after infection. The culture supernatants from macrophage infected by Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were collected and the interferon (IFN)-gamma and nitric oxide (NO) concentrations were measured by ELISA or by Griess assay, respectively. The apoptosis of macrophage was assayed by the in situ TUNEL technique.

RESULTSRT-PCR showed that both pMTB-icl and pMS-icl could be expressed in MS. Fluorescence microscopic observation showed that recombinant plasmids derived from pUV15 (pUV15-IG) could also be expressed in MS when MS were phagocytized by macrophage. Bacterial culture data demonstrated that rMS-pMTB-icl exhibited significantly increased intracellular survival in the murine macrophage cell line RAW264.7 compared with Ms 1 - 2c, rMS-pUV15 and rMS-pMS-icl. This increased intracellular survival was not accompanied by the upregulation of IFN-gamma and NO in host macrophage. But a lower apoptosis rate of macrophages infected with rMS-pMTB-icl was observed when compared with macrophages infected with other strains of MS.

CONCLUSIONSMTB-ICL could promote the intracellular survival of MS. Suppressing the apoptosis of host macrophage may be one of the important mechanisms involved in this increased intracellular survival.

Animals ; Apoptosis ; genetics ; physiology ; Cell Line ; In Situ Nick-End Labeling ; Interferon-gamma ; metabolism ; Isocitrate Lyase ; genetics ; metabolism ; Macrophages ; cytology ; metabolism ; microbiology ; Microbial Viability ; Microscopy, Fluorescence ; Mycobacterium smegmatis ; enzymology ; genetics ; growth & development ; Mycobacterium tuberculosis ; enzymology ; genetics ; Nitric Oxide ; metabolism ; Plasmids ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transformation, Genetic

OBJECTIVEObjective To construct recombinant Mycobacterium smegmatis expressing ESAT-6 of the human pathogen Mycobacterium tuberculosis.

METHODSESAT-6 gene was amplified from M. tuberculosis genomic DNA and inserted into an E.coli-mycobacterium shuttle vector under the control of HSP60 promoter. The recombinant vector was transformed into M. smegmatis by electroporation. To assess the ability of recombinant M. smegmatis to activate macrophage, mouse macrophage ANA-1 was cocultured with recombinant M. smegmatis. The apoptosis of ANA-1 cells was detected by flow cytometry and iNOS mRNA expression of the cells was detected by reverse transcription-polymerase chain reaction (RT-PCR). The survival of M. smegmatis strains in ANA-1 cells was evaluated.

RESULTSThe recombinant vector was verified by restriction endonuclease digestion and DNA sequencing. ESAT-6 protein was expressed in M. smegmatis in response to heat shock and the molecular weight of the expression product was identical to the expected value. The growth curve of the new recombinant M. smegmatis was consistent with that of the wild-type strain, suggesting the absence of ESAT-6 protein toxicity against M. smegmatis. The recombinant M. smegmatis did not induce significant changes in mouse macrophage ANA-1 apoptosis. Coculture of the macrophages with recombinant M. smegmatis for 4 to 24 h could induce iNOS expression in the former, and the CFU of recombination M. smegmatis grown in ANA-1 cells was much less than that of the control bacteria.

CONCLUSIONThe recombinant M. smegmatis expressing M. tuberculosis ESAT-6 gene possess immunogenicity, which provides experimental evidence for the development of novel M. smegmatis-based vaccine against tuberculosis.

Animals ; Antigens, Bacterial ; biosynthesis ; genetics ; immunology ; Apoptosis ; immunology ; Bacterial Proteins ; biosynthesis ; genetics ; immunology ; Cell Line ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Flow Cytometry ; Genetic Vectors ; Humans ; Macrophage Activation ; immunology ; Macrophages ; cytology ; immunology ; metabolism ; Mice ; Mycobacterium smegmatis ; genetics ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; RNA, Messenger ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Reverse Transcriptase Polymerase Chain Reaction ; Transformation, Genetic

To construct the secretive prokaryotic shuttle expression plasmid pBCG-SP-HSP65, the signal peptide sequence of antigen 85B amplified from Bacillus Calmette-guérin (BCG) genome by PCR and the whole HSP65 DNA sequence of human M. tuberculosis obtained from the plasmid pCMV-MTHSP65 by PCR were cloned into the plasmid pBCG-2100 under the control of the promoter of Heat Shock Protein 70 (HSP70) from human M. tuberculosis. Recombinants were electroporated into Mycobacterial smegmatis and induced by heating. Results of the induced expression were detected by SDS-PAGE and the biological activity of the expressed protein was tested by Western-blot analysis. Results showed pBCG-SP-MTHSP65 was constructed successfully and confirmed by restriction endonuclease analysis, PCR detection and DNA sequencing analysis. After it was electroporated into Mycobacterial smegmatis and induced by heating, the percentage of expressed 65kD protein in Mycobacterial smegmatis detected by SDS-PAGE was 20% in total bacterial protein. But the percentage of expressed 65kD protein in recombibinant Mycobacterial smegmatis was up to 34.46% in total bacterial protein and 68.56% in the total protein of cell lysate supernants, Which demonstrated the recombinant HSP65 gene could express in recombinant with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive proteins could specially combine with antibody against human M. tuberculosis HSP65. Orally, pBCG-SP-HSP65 was successfully constructed; HSP65 gene could express in Mycobacterial smegmatis with high efficiency via it. And the expressed proteins possess the biological activity. So it provids experimental evidence for the application of the recombinant Mycobacterial smegmatis and the development of the vaccine against tuberculosis.
Bacterial Proteins ; biosynthesis ; genetics ; Chaperonin 60 ; Chaperonins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Humans ; Mycobacterium smegmatis ; genetics ; metabolism ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

The BALB/c mice were immunized with rMS-Sj26GST and rBCG-Sj26GST vaccine in Schistosoma japonicum by subcutaneous injection. After they were immunized for 8 weeks, the eyeballs were removed to get blood and macrophages of abdominal cavity and spleen cells were harvested. The lymphocytic stimulating index (SI) was used to measure the cellular proliferating ability and NO release was used to measure the phagocytic activity of the macrophages. By using ELISA kit, the levels of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) in serum and the splenic lymphocytic cultured supernatant were detected. The results showed that after the mice were immunized with 10(6) CFU of rMS-Sj26GST and rBCG-Sj26GST vaccine separately by subcutaneous injection, proliferating ability of splenic lymphocytes in the mice showed no difference (P > 0.05), but both were significantly increased as compared with that in the control group (P < 0.05); The contents of NO in the intraperitoneal macrophages of rMS-Sj26GST vaccine group were significantly lower than in the control group (P < 0.001) and rBCG-Sj26GST vaccine group (P < 0.01); The levels of serum IL-2 in the rMS-Sj26GST vaccine group were significantly increased as compared with that in the control group (P < 0.001), vector group (P < 0.01) and rBCG-Sj26GST vaccine group (P < 0.05); The contents of serum IFN-gamma in the rMS-Sj26GST vaccine group were significantly increased as compared with that in the control group (P < 0.01) and rBCG-Sj26GST vaccine group (P < 0.05), The contents of IFN-gamma in the cultured supernatant were significantly lower than those of rBCG-Sj26GST vaccine group (P < 0.001), but were significantly increased as compared with that in the control group (P < 0.01). It was indicated that both vaccines could enhance the immune response of the mice, but rMS-Sj26GST vaccine had stronger immunogenicity than rBCG-Sj26GST vaccine.
Animals ; Antigens, Helminth ; genetics ; immunology ; BCG Vaccine ; immunology ; Escherichia coli ; genetics ; Genetic Engineering ; methods ; Genetic Vectors ; immunology ; Glutathione Transferase ; genetics ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mycobacterium smegmatis ; genetics ; immunology ; Polymerase Chain Reaction ; Random Allocation ; Recombinant Fusion Proteins ; genetics ; immunology ; Schistosoma japonicum ; enzymology ; genetics ; immunology ; Vaccines, Synthetic ; genetics ; immunology