Toxin-Antitoxin Systems Alter Adaptation of Mycobacterium smegmatis to Environmental Stress.

Toxin-antitoxin (TA) systems are ubiquitous genetic elements in prokaryotes, but their biological importance is poorly understood. Mycobacterium smegmatis contains eight putative TA systems. Previously, seven TAs have been studied, with five of them being verified as functional. Here, we show that Ms0251-0252 is a novel TA system in that expression of the toxin Ms0251 leads to growth inhibition that can be rescued by the antitoxin Ms0252. To investigate the functional roles of TA systems in M. smegmatis, we deleted the eight putative TA loci and assayed the mutants for resistance to various stresses. Deletion of all eight TA loci resulted in decreased survival under starvation conditions and altered fitness when exposed to environmental stresses. Furthermore, we showed that deletion of the eight TA loci decreased resistance to phage infection in Sauton medium compared with the results using 7H10 medium, suggesting that TA systems might have different contributions depending on the nutrient environment. Furthermore, we found that MazEF specifically played a dominant role in resistance to phage infection. Finally, transcriptome analysis revealed that MazEF overexpression led to differential expression of multiple genes, including those related to iron acquisition. Altogether, we demonstrate that TA systems coordinately function to allow M. smegmatis to adapt to changing environmental conditions. IMPORTANCE Toxin-antitoxin (TA) systems are mechanisms for rapid adaptation of bacteria to environmental changes. Mycobacterium smegmatis, a model bacterium for studying Mycobacterium tuberculosis, encodes eight putative TA systems. Here, we constructed an M. smegmatis mutant with deletions of all eight TA-encoding genes and evaluated the resistance of these mutants to environmental stresses. Our results showed that different TA systems have overlapping and, in some cases, opposing functions in adaptation to various stresses. We suggest that complementary TA modules may function together to regulate the bacterial stress response, enabling adaptation to changing environments. Together, this study provides key insights into the roles of TA systems in resistance to various environmental stresses, drug tolerance, and defense against phage infection.

Effects of Bedaquiline on Antimicrobial Activity and Cytokine Secretion of Macrophages Infected with Multidrug-Resistant Mycobacterium tuberculosis Strains.

Background: Bedaquiline (Bdq) exerts bactericidal effects against drug-susceptible and drug-resistant Mycobacterium tuberculosis strains, including multidrug-resistant M. tuberculosis strains (MDR-MTBs). However, few reported investigations exist regarding Bdq effects on MDR-MTBs-infected macrophages activities and cytokine secretion. Here, Bdq bactericidal activities against MDR-MTBs and related cellular immune mechanisms were explored. Methods: Macrophages infected with MDR-MTBs or H37Rv received Bdq treatments (4 h/8 h/24 h/48 h) at 1 × the minimum inhibitory concentration (1 × MIC), 10 × MIC and 20 × MIC. Intracellular colony-forming units (CFUs) and culture supernatant IL-12/23 p40, TNF-α, IL-6, and IL-10 were determined using the Luminex® 200TM system. Normally distributed continuous data (mean ± standard deviation) were analyzed using t-test or F-test (SPSS 25.0, P < 0.05 deemed statistically significant). Results: (1) 100% of Bdq-treated macrophages (all doses applied over 4-48 h) survived with 0% inhibition of proliferation observed. (2) Intracellular CFUs of Bdq-treated MDR-MTBs-infected macrophages decreased over 4-48 h of treatment, were lower than preadministration and control CFUs, decreased with increasing Bdq dose, and resembled H37Rv-infected group CFUs (48 h). (3) For MDR-MTBs-infected macrophages (various Bdq doses), IL-12/23 p40 levels resembled preadministration group levels and exceeded controls (4 h); TNF-α levels exceeded preadministration group levels (24 h/48 h) and controls (24 h); IL-12/23 p40 and TNF-α levels resembled H37Rv-infected group levels (4 h/8 h/24 h/48 h); IL-6 levels exceeded preadministration and H37Rv-infected group levels (24 h/48 h) and controls (24 h); IL-10 levels resembled preadministration and H37Rv-infected group levels (4 h/8 h/24 h/48 h) and were lower than controls (24 h/48 h); IL-12/23 p40 and IL-10 levels remained unchanged as intracellular CFUs changed, with IL-12/23 p40 levels exceeding controls (4 h) and IL-10 levels remaining lower than controls (24 h/48 h); TNF-α and IL-6 levels increased as intracellular CFUs decreased (24 h/48 h) and exceed controls (24 h). Conclusion: Bdq was strongly bactericidal against intracellular MDR-MTBs and H37Rv in a time-dependent, concentration-dependent manner. Bdq potentially exerted immunomodulatory effects by inducing high-level Th1 cytokine expression (IL-12/23 p40, TNF-α) and low-level Th2 cytokine expression (IL-10).

The Activities and Secretion of Cytokines Caused by Delamanid on Macrophages Infected by Multidrug-Resistant Mycobacterium tuberculosis Strains.

Background: Delamanid (Dlm) is an effective drug against drug-susceptible and drug-resistant Mycobacterium tuberculosis strains, including Multidrug-resistant Mycobacterium tuberculosis (MDR-MTB). There are few reports on the activity and secretion of cytokines caused by Dlm on macrophages infected by MDR-MTB strains. Therefore, this article aims to observe the bactericidal activity and secretion of cytokines of the macrophages infected by MDR-MTB strains after Dlm was administered, so as to provide a basis for further perfecting the mechanism of Dlm. Methods: Samples were respectively collected to count the intracellular colony-forming unit (CFU) of macrophages infected by MDR-MTB or H37Rv strains at 4, 8, 24, and 48 h after Dlm at MIC, 10MIC, and 20MIC were administered. Samples were respectively collected to detect the level of IL-12/23 p40, TNF-α, IL-6, and IL-10 in the culture supernatant of macrophages infected by MDR-MTB or H37Rv strains at 4, 24, and 48 h after Dlm at MIC were administered. The levels of four cytokines in the culture supernatant were measured using the Luminex® 200™ (Luminex, USA) according to the manufacturer's instructions. Data were analyzed by SPSS 25.0 software. The continuous data in normal distribution were expressed as mean ± standard deviation ( x¯ ± s) and analyzed by t or F test. P<0.05 was considered statistically significant. Results: (1) After Dlm was applied to macrophages infected by MDR-MTB strains:(A) The intracellular CFU gradually decreased, reached the lowest value at 48 h, and was lower than that of Dlm before administration and infection group (P<0.05). (B) The intracellular CFU was further reduced after increasing Dlm dose to 10MIC and 20MIC, and the latter was lower than that of the former (P<0.05). (C) The intracellular CFU of MDR-MTB group was higher than that of H37Rv group at 4~48 h after administration (P<0.05). (2) After Dlm at MIC dose was applied to macrophages infected by MDR-MTB strains: (A) The level of IL-12/23 p40 at any time didn't change compared with that of Dlm before administration (P>0.05), while the level of IL-12/23 p40 at 4 h was higher than that of the infection group (P<0.05). The levels of TNF-α at 24 and 48 h were higher than that of Dlm before administration (P<0.05), but were similar to that of the infection group (P>0.05). In addition, the levels of IL-12/23 p40 and TNF-α at any time were similar to that of the H37Rv group after administration (P>0.05). (B) The levels of IL-6 at 24 and 48 h were higher than that of Dlm before administration (P<0.05), but were similar to that of H37Rv group (P>0.05) and were lower than that of infection group (P<0.05). The level of IL-10 at any time didn't change compared with that of Dlm before administration (P>0.05), but was lower than that of the infection group at 4~48 h and was lower than that of the H37Rv group at 24 h (P<0.05). (C) The level of IL-12/23 p40 and IL-10 didn't change with the change of intracellular CFU (P<0.05), while the level of TNF-α and IL-6 increased with the intracellular CFU decreasing, and the increase level of TNF-α was lower than that of the infection group (P<0.05). Conclusions: Dlm had strong bactericidal activity against intracellular MDR-MTB, which was time-dependent and concentration-dependent. Its bactericidal activity against intracellular MDR-MTB strains was weaker than that against drug-susceptible tuberculosis strains. Dlm might have immunomodulatory effect, inducing low expression of Th2 cytokines IL-6 and IL-10 at different times after administration.

Anti-Mycobacterium tuberculosis Terpenoids from Resina Commiphora.

Four new compounds including two new sesquiterpenoid dimers, commiphoroids E (1) and F (2), a new triterpenoid (3), and a new sesquiterpenoid (4), along with three known terpenoids (5-7) were isolated from Resina Commiphora, whose structures were identified by NMR spectra, HRESIMS, and X-ray diffraction analysis. Compounds 1 and 2 both bear an O-bridge ring and feature a plausible [4 + 2] Diels-Alder cycloaddition reaction. Antimycobacterial activities show that all the tested compounds (200 µM) could inhibit the growth of both sensitive and clinically multi-drug resistant (MDR) isolated strains. In addition, cellular toxicity of the isolates against human cancer cells and THP-1 monocyte cells was examined.

Inflammation responses in patients with pulmonary tuberculosis in an intensive care unit.

Pulmonary tuberculosis caused by Mycobacterium tuberculosis remains a global problem. Inflammatory responses are the primary characteristics of patients with pulmonary tuberculosis in intensive care units (ICU). The aim of the present study was to investigate the clinical importance of inflammatory cells and factors for patients with pulmonary tuberculosis in ICU. A total of 124 patients with pulmonary tuberculosis in ICU were recruited for the present study. The inflammatory responses in patients with pulmonary tuberculosis in ICU were examined by changes in inflammatory cells and factors in the serum. The results indicated that serum levels of lymphocytes, plasma cells, granulocytes and monocytes were increased in patients with pulmonary tuberculosis in ICU compared with healthy controls. The serum levels of inflammatory factors interleukin (IL)-1, IL-6, IL-10, IL-12, and IL-4 were upregulated in patients with pulmonary tuberculosis in ICU. Lower plasma concentrations of IL-2, IL-15 and interferon-γ were detected in patients with pulmonary tuberculosis compared with healthy controls. It was demonstrated that high mobility group box-1 protein expression levels were higher in the serum of patients with pulmonary tuberculosis compared with healthy controls. Notably, an imbalance of T-helper cell (Th)1/Th2 cytokines was observed in patients with pulmonary tuberculosis. Pulmonary tuberculosis caused by M. tuberculosis also upregulated expression of matrix metalloproteinase (MMP)-1 and MMP-9 in hPMCs. In conclusion, these outcomes demonstrated that inflammatory responses and inflammatory factors are associated with the progression of pulmonary tuberculosis, suggesting that inhibition of inflammatory responses and inflammatory factors may be beneficial for the treatment of patients with pulmonary tuberculosis in ICU.

Identification of latent tuberculosis infection-related microRNAs in human U937 macrophages expressing Mycobacterium tuberculosis Hsp16.3.

BACKGROUND: Latent tuberculosis infection (LTBI) relies on a homeostasis of macrophages and Mycobacterium tuberculosis (Mtb). The small heat shock protein, Mtb Hsp16.3 (also known as latency-associated antigen), plays an important role in Mtb persistence within macrophages. However, the mechanism of LTBI remains elusive. The aim of this study was to delineate LTBI-related miRNA expression in U937 macrophages expressing Mtb Hsp16.3 protein. U937 macrophages were infected with an integrase-deficient Lentivirus vector to transiently express Mtb Hsp16.3, and green fluorescent protein (GFP) as a control. We used a microRNA (miRNA) microarray chip containing more than 1000 probes to identify the significant differentially expressed miRNAs in the infected U937 cells, and employed real-time quantitative polymerase chain reaction (qRT-PCR) for validation. Furthermore, we confirmed these candidate LTBI-related miRNAs in peripheral blood mononuclear cells from subjects with LTBI and in healthy control individuals. Functional annotation prediction of miRNA target genes and pathway enrichment analyses were used to explore the putative links between these miRNAs and LTBI. RESULTS: Analysis of the miRNA expression profile identified 149 miRNAs that were differentially expressed in U937 macrophages expressing Mtb Hsp16.3 compared with the control expressing GFP. The expression level of seven miRNAs (miR-424-5p, miR-493-5p, miR-296-5p, miR-27b-3p, miR-377-5p, miR-3680-5p, miR-191-5p) were validated by qRT-PCR. The expression level of four miRNAs (miR-424-5p, miR-27b-3p, miR-377-5p, miR-3680-5p) in the peripheral blood mononuclear cells samples from LTBI and healthy participants reflected the altered patterns observed in the microarray profile. The bioinformatic analyses suggest that the miRNAs may regulate Mtb latent infection by affecting the development of macrophage cells. CONCLUSIONS: The results suggest that miRNA expression may play a considerable role in the pathogenesis of LTBI, and this would increase our understanding of the molecular basis of Hsp16.3-facilitated Mtb survival in macrophages.

[Early secretory antigenic target protein-6/culture filtrate protein-10 fusion protein-specific Th1 and Th2 response and its diagnostic value in tuberculous pleural effusion].

OBJECTIVE: To detect the Th1 and Th2 cell percentage in pleural effusion mononuclear cells (PEMCs) stimulated by early secretory antigenic target protein-6 (ESAT-6)/culture filtrate protein-10 (CFP-10) fusion protein (E/C) with flow cytometry (FCM), and therefore to explore the local antigen specific Th1 and Th2 response and its diagnostic value in tuberculous pleuritis. METHODS: Forty patients with tuberculous pleural effusion and 30 patients with malignant pleural effusion were included in this study from Sep.2008 to Mar.2009. PEMCs were isolated and cryopreserved. After resuscitation, the cells were cultured with E/C (simultaneously with positive control and negative control), and antigen-specific Th1 and Th2 cells were detected with intracellular cytokine staining of FCM. Normal distribution data using t test, abnormal distribution data using Wilcoxon test. RESULTS: In the TB group,the medians (quartile range) of Th1 cells and Th1/Th2 ratio among PEMCs stimulated by ESAT-6/CFP-10 fusion protein were 3.06% (1.59%-6.92%) and 17 (7.38-35.53), significantly higher than those of the negative control [0.38% (0.02%-1.80%) and 3.59 (0.49-25.09)], the differences being statistically significant (Z = -5.345 and 3.314, P < 0.01). The percentage of Th2 cells [(0.22 ± 0.19)%] was also increased compared with that of the negative control [(0.10 ± 0.08)%], the difference being statistically significant (t = 4.108, P < 0.01). In the malignant effusion group, the medians (quartile range) of Th1 percentage and Th1/Th2 ratio were 0.12% (0.05%-0.39%) and 1.05 (0.25-2.52), which were significantly different as compared with those of the TB group (Z = -6.624 and -5.536, P < 0.01). The Th2 percentage in the 2 groups were (0.22 ± 0.19)% and (0.15 ± 0.02)%, respectively (t = 1.954, P > 0.05). The receiver operating characteristic curve indicated that the area under the curve (AUC), sensitivity, and specificity were 0.937, 85.4%, and 90.6% respectively for Th1 to diagnose tuberculous pleurisy. For Th1/Th2, the AUC, sensitivity, and specificity were 0.883, 81.5%, and 90.6% respectively. CONCLUSIONS: The feature of ESAT-6/CFP-10 fusion protein-specific Th1 and Th2 response in tuberculous pleurisy was a mixed reaction of Th1 and Th2 with Th1 predominance. Th1 percentage and Th1/Th2 ratio could be diagnostic indexes for identifying tuberculous from malignant pleural effusions.

Application of hyperbranched rolling circle amplification for direct detection of mycobacterium tuberculosis in clinical sputum specimens.

BACKGROUND: Global tuberculosis (TB) control is encumbered by the lack of a rapid and simple detection method for diagnosis, especially in low-resource areas. An isothermal amplification method, hyperbranched rolling circle amplification (HRCA), was optimized to detect Mycobacterium tuberculosis (Mtb) in clinical sputum specimens. METHODS: A clinical validation study was performed to assess the diagnostic accuracy of HRCA. In order to analyze the detection limit of HRCA under optimal conditions, the method was initially used to detect purified H37Rv strain DNA and culture suspensions. Next, three strains of Mycobacterium tuberculosis complex (MTC) and eight strains of non-tuberculosis mycobacterium (NTM) were analyzed in order to evaluate specificity. Sputum specimens from 136 patients with diagnosed pulmonary TB, 38 lung cancer patients, and 34 healthy donors were tested by HRCA to validate the clinical application of HRCA for the rapid detection of Mtb. RESULTS: The detection limit of HRCA for purified H37Rv DNA and culture suspensions was 740 aM and 200cfu/ml, respectively. The results of all MTC strains were positive in contrast to the NTM specimens which were all negative. The detection sensitivity for the 136 sputum specimens from TB patients was 77.2% (105/136), which was slightly lower than that of quantitative real-time PCR(79.4%, 108/136) and culture (80.9%,110/136). The sensitivity of all three methods was statistically higher than smear microscopy (44.9%, 61/136). The overall specificity of HRCA was 98.6% (71/72) which was similar to that of quantitative real-time PCR (qRT-PCR) and smear/culture methods (100%, 72/72). CONCLUSIONS: Use of the HRCA assay for detection of Mtb within clinical sputum specimens was demonstrated to be highly sensitive and specific. Moreover, the performance of HRCA is simple and cost-effective compared with qRT-PCR and is less time consuming than culture. Therefore, HRCA is a promising TB diagnostic tool that can be used routinely in low-resource clinical settings.

[Comparison of two interferon-gamma release assays in the diagnosis of tuberculosis].

OBJECTIVE: To compare the diagnostic performance of A.TB, an ELISA-based interferon-gamma release assay (IGRA), and T-SPOT.TB, an ELISPOT-based IGRA, and therefore to evaluate the value of A.TB assay in the routine clinical practice. METHODS: From March to May of the year of 2011, 112 hospitalized patients were enrolled from 2 chest hospitals in Beijing and Harbin, including 75 cases in the TB group (43 male and 32 female) with the average age of (44 ± 18) years, spanning from 28 to 57 years, and 37 cases in the non-TB group (21 male and 16 female) with the average age of (54 ± 10) years, spanning from 24 to 82 years. During the same period, 34 healthy volunteers (4 male and 30 female), with the average age of (20 ± 0.6) years, spanning from 19 to 22 years, were recruited in Beijing Chest Hospital. A head-to-head comparison of the 2 IGRAs was performed on the 146 subjects to evaluate their overall diagnostic performance. Chi-square test or Fisher's exact test was used for statistical analysis of enumeration data. RESULTS: The sensitivity and specificity of A.TB were 81.3% (61/75, 95%CI = 72.5 - 90.2) and 83.1% (59/71, 95%CI = 74.4 - 91.8) respectively, compared to 90.7% (68/75, 95%CI = 84.1 - 97.3) and 78.9% (56/71, 95%CI = 69.4 - 88.4) for T-SPOT.TB. There was no significant difference in sensitivity or specificity (chi square values were 2.77 and 0.17 respectively, both P > 0.05). The area under the ROC curve was 0.90 (95%CI = 0.84 - 0.95) for A.TB and 0.91 (95%CI = 0.86 - 0.96) for T-SPOT.TB. The observation agreement between the 2 methods was 87.2% (123/141), with a kappa value of 0.74. T-SPOT.TB produced indeterminate results at a rate of 3.4% (5/146). CONCLUSIONS: There was comparable diagnostic performance between the 2 assays. However, when compared to T-SPOT.TB, the A.TB testing procedure, with less technical demand and without requirement of well-equipped lab, is simpler and the interpretation of results is less subjective.

Detection of MET and SOX2 amplification by quantitative real-time PCR in non-small cell lung carcinoma.

Non-small cell lung carcinoma is a leading cause of cancer-related death. Amplification of the two oncogenes MET and SOX2 is frequently encountered in non-small-cell lung carcinoma. This study aimed to use real-time quantitative PCR to assess the correlation of MET and SOX2 amplification with clinicopathological factors. This study was conducted using 115 tissue samples including 57 squamous cell carcinomas (SCCs), 50 adenocarcinomas (ADCs) and 8 adenosquamous carcinomas (ADSCs). A total of 67 patients (58.3%) had a history of smoking. Our results showed that the frequency of MET amplification in SCCs was significantly higher compared to ADCs (χ(2)=8.0, P=0.005). SOX2 showed a markedly preferential amplification in SCCs compared to ADCs in the smoking group cases (P=0.014). Lymph node invasion correlated with MET amplification in SCCs marginally more significantly compared to ADCs (P=0.02). The amplified MET occurred more frequently in SCCs compared to ADCs correlated to tumor dimension at a small scale (<5 cm) (P=0.01). No significant difference in SOX2 amplification was found with regards to lymph node metastasis or tumor dimension. SOX2 and MET amplifications were not associated with gender or age. However, MET amplification in SCCs among patients younger than 64 years of age was higher compared to ADCs and ADSCs (P=0.03). Among ADSCs, MET was not amplified among patients who had never been smokers or were younger than 64 years of age. Neither MET nor SOX2 were amplified in tumors with dimensions <5 cm and without lymph node invasion. Findings of this study showed that MET and SOX2 amplifications are more common in the SCCs of smokers. Moreover, MET amplification is intrinsic in SCCs particularly among smokers, with regards to tumor growth, lymph node invasion and negative correlation to SOX2 amplification. The incidence of discrepancy in the amplifications of MET and SOX2 in SCCs and ADCs suggests that the MET and SOX2 genes play different roles in SCC and ADC tumorigenesis, respectively, particularly among smokers.

[The relationship between cyclic adenosine monophosphate response element-binding protein and interferon-γ gene promoter in tuberculosis patients].

OBJECTIVE: To study the relationship between cAMP response element binding protein (CREB) and the interferon-γ (IFN-γ) proximal promoter in patients with tuberculosis. METHODS: CD3(+) T cells were isolated from 25 pulmonary tuberculosis patients, who had been treated in Beijing Chest Hospital from January to December 2007, and 18 PPD-positive healthy donors. After extraction of nuclear proteins, electrophoretic mobility shift assay (EMSA) was performed to determine nuclear protein binding to the IFN-γ proximal promoter in vitro, and the specificity of binding complex was tested by competitive EMSA. Chromatin immunoprecipitation (ChIP) with anti-CREB Ab was used to determine whether CREB binded to the IFN-γ proximal promoter in vivo in live T cells exposed to microbial Ags. Western blotting with anti-CREB Ab was performed to compare the expression level of CREB in tuberculosis patients and PPD-positive healthy donors. Western blotting with Abs specific for serine 133-phosphorylated CREB was performed to determine whether M.tuberculosis Ags elicited phosphorylation of CREB. RESULTS: The results of EMSA showed a low-mobility complex binding to the IFN-γ promoter, and the binding pattern observed was similar for T cells from all 18 PPD-positive healthy donors. However, for T cells from 18 of 25 tuberculosis patients, the low-mobility complex was absent. The results of competitive EMSA showed that these nuclear proteins specifically bound to the IFN-γ promoter region and contained CREB. The results of ChIP showed a 204 bp band yielded in CD3(+) T cells from 10 PPD-positive healthy donors, but 12 tuberculosis patients didn't yield the band. CREB expression markedly decreased in tuberculosis patients compared with healthy donors detected by Western blotting. Furthermore, M. tuberculosis Ags also elicited phosphorylation of CREB in CD3(+) T cells from PPD-positive healthy donors, but not in CD3(+) T cells from tuberculosis patients. CONCLUSIONS: CREB protein binding to IFN-γ proximal promoter was reduced in tuberculosis patients compared with healthy donors. Tuberculosis patients had diminished CREB protein levels, and reduced ability of binding to the IFN-γ promoter.

CREB is a positive transcriptional regulator of gamma interferon in latent but not active tuberculosis infections.

Gamma interferon (IFN-gamma) is a crucial cytokine for protection against Mycobacterium tuberculosis, but the mechanism of IFN-gamma transcription is still unclear. The cyclic AMP (cAMP) responsive element binding (CREB) proteins belong to the bZip (basic leucine zipper) family of transcription factors and are essential for T-cell function and cytokine production. This study focused on the capacity of CREB proteins to regulate IFN-gamma transcription in CD3(+) T cells obtained from tuberculosis (TB) patients and persons with latent tuberculosis infection (LTBI) in China. The electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and Western blotting were used to demonstrate the regulatory role of CREB. EMSA (in vitro) and ChIP (in vivo) experiments suggested CREB could bind to the IFN-gamma proximal promoter in persons with LTBI, whereas no binding was detected in TB patients. Western blotting confirmed the expression of CREB proteins, especially serine-133-phosphorylated CREB, was markedly reduced in TB patients compared with persons with LTBI. These results suggested that CREB could promote the transcription and production of IFN-gamma through binding with the IFN-gamma proximal promoter, but the regulatory role of CREB was decreased in tuberculosis patients owing to diminished expression of CREB proteins, which in turn reduced the IFN-gamma production.

[A study on the haplotype of the solute carrier family 11 member 1 gene in Tibetan patients with pulmonary tuberculosis in China].

OBJECTIVE: To investigate the association of the haplotype of the solute carrier family 11 member 1 (SLC11A1) gene with susceptibility to pulmonary tuberculosis in Tibetans. METHODS: Four polymorphisms of the SLC11A1 gene [5' (GT)n, INT4, D53N, and 3' UTR] were investigated by denaturalization high performance liquid chromatography and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 140 patients (the patient group) and 139 PPD-positive healthy controls (the control group) of the Tibetan nationality from June 2004 until January 2005. The relationship between the haplotype and susceptibility to pulmonary tuberculosis in these patients was studied by chi2 test, and the linkage disequilibrium as well as the haplotype were analyzed by the SHESIS software. RESULTS: The haplotype frequencies of 5'(GT)9/INT4 G, 3'UTR TGTG/D543N G, 3'UTR TGTG del/D543N A were 64.8% (181/280), 76.6% (215/280), 12.0% (34/280) among the patients and 78.1% (217/276), 84.4% (235/276), 6.4% (18/276) among the controls. 5' (GT)9/INT4 G, 3'UTR TGTG/D543N G haplotypes rendered a lower risk (chi2 = 11.026, P<0.01, chi2 = 6.547, P<0.05, respectively), but 3'UTR TGTG del/D543N A haplotype a higher risk (chi2 = 6.547, P<0.05) for tuberculosis. CONCLUSIONS: 5' (GT)9/INT4 G, 3'UTR TGTG/D543N G and 3'UTR TGTG del/D543N A haplotypes of the SLC11A1 gene may be associated with the susceptibility of the Tibetan population to pulmonary tuberculosis.

[Prokaryotic expression, purification, and immunogenicity analysis of Mycobacterium tuberculosis specific excretive proteins].

OBJECTIVE: To obtain the recombinant rv1837c and rv3803c of Mycobacterium tuberculosis using gene engineering technology and explore their prokaryotic expression, purification, and immunogenicity. METHODS: The Mycobacterium tuberculosis rv1837c and rv3803c genes were amplified by polymerase chain reaction, and then cloned into the vector pTA2, followed by the subclone into the expression vector pET30a (+). The resulting plasmids, named pET30a (+): rv1837c and pET30a (+): rv3803c, encode recombinant protein containing a hexa-histidine tag on its N-terminus. pET30a (+): rv1837c and pET30a (+): rv3803c were introduced into E. coli BL21 (DE3) by transformation respectively, and the recombinant gene was induced with 0.4 mmol/L isopropyl-D-thiogalactopyranoside. The expressed products were identified by Western blot with hexa-histidine tag antibody and serum from tuberculotic patients. The histidine tagged protein was purified by nickel nitrilotriacetic acid His-Bind resin. Rabbits were immunized with purified recombinant Rv1837c and Rv3803c proteins. Then the purified recombinant Rv1837c and Rv3803c proteins were used to detect antibody in rabbit serum, which had been immunized by Western blot. RESULTS: After transformation of the E. coli and induction with 0.4 mmol/L of isopropyl-D-thiogalactopyranoside, recombinant target proteins Rv1837c (relative molecular mass: 92000) and Rv3803c (relative molecular mass: 38 000) were expressed in pET30a (+): rv1837c and pET30a (+): rv3803c system. The expressed protein existed in cytoplasm in an unsoluble form and amounted to 30% and 50% of the total proteins of E. coli. The purity of the purified protein reached 90%. The immunogenicity of the recombinant proteins Rv1837c and Rv3803c was strong, as identified by Western blot. CONCLUSION: The prokaryotic expression recombinant plasmids pET30a (+): rv1837c and pET30a (+): rv3803c was successfully constructed and the recombinant proteins Rv1837c and Rv3803c were obtained, which laid a basis for the optimized diagnosis of active tuberculosis.

[Establishment and evaluation of nitrate reductase combined with mycobacteriophage assay].

OBJECTIVE: To establish a rapid, inexpensive, and simple drug susceptibility test (DST) for Mycobacterium tuberculosis (M. tb) and evaluate its feasibility. METHOD: We used nitrate reductase combined with mycobacteriophage assay (PhaB-NRA) to test 49 clinical M. tb isolates of, and the results were compared with those of PhaB-NRA and traditional absolute concentration method. RESULTS: The sensitivity, specificity, and accuracy of PhaB-NRA for rifampicin were 89.1%, 91.67%, and 89.8%; on the contrary, those of isonicotinyl hydrazide were 86.21%, 90.0%, and 87.8%, respectively. The coincidence between PhaB-NRA and traditional assay were 0.746 for rifampicin and 0.750 for isonicotinyl hydrazide. CONCLUSIONS: PhaB-NRA is an inexpensive, rapid, and simple DST method. It is a promising rapid screening technique for DST of M. tb.

[Relationship between the resuscitation promoting role of resuscitation promoting factor and the initial bacteria amount of dormant Mycobacterium tuberculosis].

OBJECTIVE: To investigate the relationship between the resuscitation promoting role of resuscitation promoting factor and the initial bacteria amount of dormant Mycobacterium tuberculosis. METHODS: Mycobacterium tuberculosis (dormant bacteria) was cultured for 100 days, then diluted into 1 mg/ml concentration with 7H9, and further diluted into 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg/ml. Twelve new tubes added with 5 ml 7H9 and divided into two groups: the first group was added with the resuscitation-promoting factor protein, and the second group as control was added with 7H9. In each group the above diluted solutions were added. The tubes were located at 37 degrees C for culture. Optical density (OD) was detected on day 15, 25, 30, and 35. From each tube 1 microl culture solution was plated on 7H11 medium for colony counting. RESULTS: OD detection showed that bacteria proliferation in each group had positive linear correlation (P < 0.05, P < 0.01), indicating that the resuscitation-promoting factor played a similiar role in solutions with different dilution concentrations. 7H11 results and the OD results show that these two detection methods in each group had linear correlation (P < 0.05, P < 0.01), indicating that these two methods showed consistent test results. CONCLUSION: The resuscitation-promoting factor has no effect on the resuscitation of dormant Mycobacterium tuberculosis and its initial bacteria amount.

[Role of enzyme-linked immunospot assay and tuberculin skin test in the auxiliary diagnosis of initial pulmonary tuberculosis].

OBJECTIVE: To compare enzyme-linked immunospot assay (ELISPOT) and tuberculin skin test (TST) and explore their roles in the auxiliary diagnosis of initial pulmonary tuberculosis. METHODS: Totally 123 patients with initial pulmonary tuberculosis (tuberculosis group) and 102 patients with non-tuberculosis pulmonary disease (control group) were enrolled. The peripheral blood mononuclear cells of all participants were co-cultured with early secretiny antigen target-6/culture filtrate protein-10 fusion protein (ESAT-6/CFP-10), and spot forming cells (SFCs) were enumerated by ELISPOT (ESAT-6/CFP-10-ELISPOT). TST was also performed simultaneously. RESULTS: ESAT-6/CFP-10-ELISPOT showed significantly higher numbers of SFCs after stimulation in tuberculosis group than in control group (P = 0.000). The sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, positive predictive value, and negative predictive value of ESAT-6/CFP-10-ELISPOT were 91.1% (111/123), 81.4% (82/102), 4.60, 0.12, 0.85, and 0.87 respectively, while the above values of TST were 65.6% (59/90), 45.1% (46/102), 1.31, 0.76, 0.51, and 0.60, respectively. The sensitivity and specificity of ESAT-6/CFP-10-ELISPOT were significantly higher than those of TST (all P = 0.000). The number of SFCs were not significantly different between smear-positive tuberculosis subgroup and smear-negative tuberculosis subgroup (P = 0.166). The sensitivities were 91.8% (67/73) and 88.0% (44/50) in these two subgroups, respectively, (P = 0.448). CONCLUSIONS: ESAT-6/CFP-10-ELISPOT may be a more accurate approach for the auxiliary diagnosis of initial pulmonary tuberculosis; meanwhile, it offers certain diagnostic evidences for smear-negative tuberculosis. However, its specificity may be affected by latent tuberculosis infection. On the contrary, TST has poor value in the auxiliary diagnosis of initial pulmonary tuberculosis.

[Potential value of Mycobacterium tuberculosis RD1 region-encoded proteins in the diagnosis of tuberculosis].

Nine proteins encoded by Mycobacterium tuberculosis RD1 region are important protective antigens that become absent in long passaging of Mycobacterium tuberculosis. They only exist in pathogenic Mycobacteria and are absent in Bacille Calmette-Guerin and environmental Mycobacteria. With good immunogenicities, they may play an important role in the diagnosis and prevention of Mycobacterium tuberculosis. This article reviews recent studies on using RD1-encoded proteins as antigens in the diagnosis of active tuberculosis and tuberculous pleurisy.