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

Mycobacterium (M.) vaccae is a fast-growing species of saprophytic bacteria that is widely distributed. To understand the host immune responses induced by M. vaccae isolated from bovine submaxillary lymph nodes, C57BL/6 mice were infected with reference strain M. vaccae Bacillus Calmette-Guérin (BCG) and isolated M. vaccae using intraperitoneal injections. Comparison of the bacterial replication and organ pathology between M. vaccae and M. vaccae BCG revealed that M. vaccae was more malignant than M. vaccae in mice. We also demonstrated that serum from the M. vaccae-infected mice contained a higher expression level of gamma-interferon (IFN-γ), tumor necrosis factor alpha, monocyte chemoattractant protein-1, interleukin (IL)-4, IL-12, IL-10 and transforming growth factor beta than did the other groups, especially after week 4. Furthermore, when the numbers of CD3⁺CD4⁺IFN-γ⁺ and CD3⁺CD4⁺IL4⁺ cells in the infected mice were observed by flow cytometry, we found that a powerful T helper 1 (Th1) response was induced by M. vaccae infection, which was associated with the emergence of CD3⁺CD4⁺IFN-γ⁺ cells. However, the Th1 response declined over time, which was associated with appearance of the CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25⁺CD152⁺Treg cell reaction. In addition, a strong Th2 response was found. Finally, we found that M. vaccae infection increased the production of type I IFNs, which was associated with a reduced Th1 response.
Animals ; Bacillus ; Bacteria ; Chemokine CCL2 ; Flow Cytometry ; Injections, Intraperitoneal ; Interferon-gamma ; Interleukin-10 ; Interleukin-12 ; Interleukins ; Lymph Nodes ; Mice* ; Mycobacterium bovis ; Mycobacterium* ; Pathology ; Transforming Growth Factor beta ; Tumor Necrosis Factor-alpha

Amino Acid Sequence ; Genes, Bacterial ; Hydrogen Peroxide ; pharmacology ; Molecular Sequence Data ; Mutation ; genetics ; Mycobacterium ; drug effects ; genetics ; growth & development ; Peptides ; chemistry ; metabolism ; Stress, Physiological ; drug effects

BACKGROUND: The purpose of this study was to compare the turnaround time for liquid culturing and primary anti-tuberculous drug susceptibility testing (DST) performed using the mycobacteria growth indicator tube (MGIT) 960 system (Becton Dickinson, USA) with that for conventional culturing and DST (by the absolute concentration method) performed using solid culture medium and to determine the concordance rates of DST results obtained using these 2 methods. METHODS: In this retrospective study, we compared the turnaround times from receiving the request for mycobacterial culture to reporting the DST results before and after the introduction of the MGIT 960 system. Further, we determined the concordance between DST results for isoniazid and rifampin for Mycobacterium tuberculosis isolates obtained using the MGIT 960 system and the absolute concentration method, which was conducted at the Korean Institute of Tuberculosis. RESULTS: The overall turnaround time for mycobacterial culturing and DST was 27 days for liquid culturing and DST using the MGIT 960 system versus approximately 70 days for culturing on solid medium and DST with the absolute concentration method (P<0.001). There was a good concordance between findings of DST obtained with the 2 methods (97.2%, kappa coefficient=0.855 for rifampin; and 95.6%, kappa coefficient=0.864 for isoniazid), for 1,083 clinical isolates. CONCLUSIONS: The automated MGIT 960 system for culturing and DST of M. tuberculosis was successfully introduced in a hospital laboratory setting in Korea with significant shortening of the turnaround time.
Antitubercular Agents/*pharmacology ; Automation ; *Drug Resistance, Multiple, Bacterial/drug effects ; Humans ; Isoniazid/pharmacology ; *Microbial Sensitivity Tests/instrumentation/methods ; Mycobacterium tuberculosis/*drug effects/growth & development/isolation & purification ; Retrospective Studies ; Rifampin/pharmacology ; Time Factors ; Tuberculosis/*diagnosis

The presence of galectin-3 was immunohistochemically quantified in bovine intestines infected with paratuberculosis (Johne's disease) to determine whether galectin-3 was involved in the formation of granulation tissue associated with the disease. Mycobacterium avium subsp. paratuberculosis infection was histochemically confirmed using Ziehl-Neelsen staining and molecularly diagnosed through rpoB DNA sequencing. Galectin-3 was detected in the majority of inflammatory cells, possibly macrophages, in the granulomatous lesions within affected tissues, including the ileum. These findings suggest that galectin-3 is associated with the formation of chronic granulation tissues in bovine paratuberculosis, probably through cell adhesion and anti-apoptosis mechanisms.
Animals ; Cattle ; Cattle Diseases/*pathology ; Chronic Disease ; Galectin 3/*metabolism ; Immunohistochemistry ; Intestine, Small/microbiology/*pathology ; Mycobacterium avium subsp. paratuberculosis/growth & development/isolation & purification ; Paratuberculosis/*pathology ; RNA Polymerase II/genetics

BACKGROUND: The combined use of liquid media and solid media is recommended for mycobacterial culture. We evaluated diagnostic performance of combination of BACTEC Mycobacteria Growth Indicator Tube (MGIT; Becton Dickinson, USA) and 2% Ogawa media (Korean Institute of Tuberculosis, Korea) for recovery of mycobacteria. METHODS: In September 2007, 1,764 specimens from 1,059 patients were cultured with MGIT and Ogawa. Acid fast bacilli (AFB) smear was fluorochrome-stained. The isolates were identified into Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) with PCR using Seeplex TB Detection Kit (Seegene, Korea). Recovery rate, time to detection (TTD), contamination rate, mixed growth rate and species distribution were analyzed. RESULTS: Two hundred thirty-five specimens (13.3%) from 165 patients (15.6%) were positive for mycobacterial culture. Recovery rates of mycobacteria from the group using both media, MGIT only, and Ogawa only were 13.3%, 12.1%, and 7.8%, respectively. While MGIT recovered 98.9% of MTB and 79.7% of NTM, Ogawa recovered 65.9% of MTB and 54.1% of NTM. TTDs of total mycobacteria/MTB/NTM in MGIT and Ogawa were 10.6/11.4/9.7 days and 31/29/33 days, respectively. MGIT TTDs of total mycobacteria/MTB/NTM from AFB-positive specimens were significantly shorter than those of AFB-negative specimens; 8.2/9.5/4.4 days vs 11.6/12.7/10.7 days. Contamination and mixed growth rate of MGIT were 9.6% and 3.7%. Primary culture of Ogawa recovered 1 MTB and 1 NTM among the 170 MGIT-contaminated specimens and 38 mycobacteria among 66 specimens that showed mixed cultures of MGIT. CONCLUSIONS: MGIT warrants sensitive and rapid isolation of mycobacteria. However, the combination of MGIT and Ogawa is more desirable to recover mycobacteria in the case of contaminations or mixed cultures.
*Culture Media ; False Positive Reactions ; Humans ; Mycobacterium/*growth & development/isolation & purification ; Mycobacterium Infections/*diagnosis/microbiology ; Mycobacterium tuberculosis/*growth & development/isolation & purification ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; Sputum/microbiology ; Time Factors

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

BACKGROUND: PCR is a widely used method for rapid and accurate diagnosis of mycobacteriosis. The sensitivity and specificity of a real time PCR kit newly developed in Korea were evaluated for detecting mycobacteria in respiratory specimens. METHODS: One hundred twenty nine Mycobacterium tuberculosis (TB) culture positive respiratory specimens (82 AFB stain positive and 47 stain negative specimens) were used for evaluation of the sensitivity. Nine non-tuberculous mycobacteria (NTM) culture positive specimens were also included. For evaluation of the specificity, 48 AFB stain and culture negative respiratory specimens from patients who were initially not fully excluded from mycobacterial diseases (specificity group 1) were used. Other 51 respiratory specimens from patients who were not suspected of mycobacterial diseases were also included (specificity group 2). Real time PCR was performed by using AdvanSure TB/NTM real time PCR Kit (LG Lifescience, Korea) and SLAN real time PCR detection system (LG Lifescience). The target genes of TB and NTM were IS6110 and rpoB, respectively. RESULTS: Among 129 TB culture positive specimens, 82 of 82 AFB stain positive specimens (100%) and 35 of 47 (74.5%) stain negative specimens revealed real time PCR positivity for TB, resulting in sensitivity of 90.7%. Five of nine NTM culture positive specimens resulted in real time PCR positivity for NTM (55.6%). Forty seven of 48 specimens (97.9%) and all 51 specimens (100%) of the specificity group 1 and 2, respectively, were real time PCR negative for TB and NTM. CONCLUSIONS: AdvanSure TB/NTM real time PCR Kit should be useful for detecting TB in respiratory specimens with high sensitivity and specificity.
DNA, Bacterial/analysis ; Humans ; Mycobacterium tuberculosis/genetics/growth & development/*isolation & purification ; Polymerase Chain Reaction/*methods ; Reagent Kits, Diagnostic ; Respiratory System/microbiology ; Sensitivity and Specificity ; Specimen Handling ; Tuberculosis/*diagnosis

PURPOSE: Many medical centers routinely culture bronchoscopy samples for Mycobacterium tuberculosis, even when tuberculosis is not strongly suspected. The value of this practice, however, is controversial. We evaluated the role of that procedure in the diagnosis of pulmonary tuberculosis in an intermediate tuberculosis-burden country. PATIENTS AND METHODS: A prospective, observational study was conducted in a tertiary referral center and included 733 consecutive patients who underwent bronchoscopy examination. RESULTS: M. tuberculosis was isolated in 47 patients (6.4%). According to radiographic features, the rate of positive culture for M. tuberculosis was relatively high in patients with atelectasis (5/33, 15.2%) and those with pulmonary infiltrations of suspicious infections (26/183, 14.2%). M. tuberculosis was isolated even in patients with pulmonary masses (9/266, 3.4%) and those with pulmonary nodules (5/175, 2.9%). In 16/47 (34.0%) patients with positive cultures for M. tuberculosis, active pulmonary tuberculosis was not suspected at the time of bronchoscopy. CONCLUSION: These results suggest that routinely culturing for M. tuberculosis during bronchoscopy is still useful in the diagnosis of pulmonary tuberculosis in an intermediate tuberculosis-burden country.
Adult ; Aged ; Bacteriological Techniques/methods ; Bronchoscopy ; Female ; Humans ; Lung/microbiology/pathology ; Lung Neoplasms/microbiology ; Male ; Middle Aged ; Mycobacterium tuberculosis/growth & development/*isolation & purification ; Prospective Studies ; Pulmonary Atelectasis/microbiology ; Reproducibility of Results ; Sensitivity and Specificity ; Tuberculosis, Pulmonary/*diagnosis/microbiology

Antigens, Bacterial ; genetics ; Bacterial Proteins ; Cloning, Molecular ; Interleukin-2 ; genetics ; Mycobacterium bovis ; genetics ; growth & development ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis