Expression, purification, and characterization of a functionally active Mycobacterium tuberculosis UDP-glucose pyrophosphorylase

Tuberculosis, which is caused by Mycobacterium tuberculosis, remains to be a global health problem. The thick and complex cell envelope has been implicated in many aspects of the pathogenicity of M. tuberculosis. M. tuberculosis UDP-glucose pyrophosphorylase (UGP, coded by galU, Rv0993) is involved in cell envelope precursor synthesis. UGP catalyzes the reversible formation of UDP-glucose and inorganic pyrophosphate from UTP and glucose 1-phosphate (Glc-l-P). Bacterial UGPs are completely unrelated to their eukaryotic counterparts. This enzyme is recognized as a virulence factor in several bacterial species and is conserved among mycobacterial species, which makes it a good target for mycobacterial pathogenicity research. The recombinant M. tuberculosis UGP (rMtUGP) was purified in Escherichia coli and found to be stable and catalytically active. The effects of pH, temperature and Mg2+ on enzyme activity were characterized. In addition, subcellular localization studies revealed that most of M. tuberculosis UGP protein was located in the cell wall. The purification and characterization of M. tuberculosis UGP may help to decipher the pathogenicity of M. tuberculosis.     

Skeletal tuberculosis,pelvic contraction,and parturition

Obliquely contracted pelvis was observed in association with tuberculosis and dystocia (difficult parturition) in four out of 762 childbearing women in a Southeast Asian population. It is hypothesized that the pelvic contracture results from tuberculosis of the sacro-iliac joint with destruction of the sacral ala. This gross morphologic deformity, as observed in living populations, may be a useful marker for skeletal tuberculosis in prehistoric and historic populations as well. The occurrence of obliquely contracted pelvis with consequent dystocia as a specific complication of systemic tuberculosis may have direct implications for differential reproductivity.     

Feasibility, Yield, and Cost of Active Tuberculosis Case Finding Linked to a Mobile HIV Service in Cape Town, South Africa: A Cross-sectional Study

Background

The World Health Organization is currently developing guidelines on screening for tuberculosis disease to inform national screening strategies. This process is complicated by significant gaps in knowledge regarding mass screening. This study aimed to assess feasibility, uptake, yield, treatment outcomes, and costs of adding an active tuberculosis case-finding program to an existing mobile HIV testing service.

Methods and Findings

The study was conducted at a mobile HIV testing service operating in deprived communities in Cape Town, South Africa. All HIV-negative individuals with symptoms suggestive of tuberculosis, and all HIV-positive individuals regardless of symptoms were eligible for participation and referred for sputum induction. Samples were examined by microscopy and culture. Active tuberculosis case finding was conducted on 181 days at 58 different sites. Of the 6,309 adults who accessed the mobile clinic, 1,385 were eligible and 1,130 (81.6%) were enrolled. The prevalence of smear-positive tuberculosis was 2.2% (95% CI 1.1–4.0), 3.3% (95% CI 1.4–6.4), and 0.4% (95% CI 1.4 015–6.4) in HIV-negative individuals, individuals newly diagnosed with HIV, and known HIV, respectively. The corresponding prevalence of culture-positive tuberculosis was 5.3% (95% CI 3.5–7.7), 7.4% (95% CI 4.5–11.5), 4.3% (95% CI 2.3–7.4), respectively. Of the 56 new tuberculosis cases detected, 42 started tuberculosis treatment and 34 (81.0%) completed treatment. The cost of the intervention was US$1,117 per tuberculosis case detected and US$2,458 per tuberculosis case cured. The generalisability of the study is limited to similar settings with comparable levels of deprivation and TB and HIV prevalence.

Conclusions

Mobile active tuberculosis case finding in deprived populations with a high burden of HIV and tuberculosis is feasible, has a high uptake, yield, and treatment success. Further work is now required to examine cost-effectiveness and affordability and whether and how the same results may be achieved at scale.     

Association of Serum Levels of Iron,Copper, and Zinc,and Inflammatory Markers with Bacteriological Sputum Conversion During Tuberculosis Treatment

Iron, copper, and zinc are key micronutrients that play an important role in the immune response to Mycobacterium tuberculosis. The present study aimed to evaluate the association between serum levels of those micronutrients, inflammatory markers, and the smear and culture conversion of M. tuberculosis during 60 days of tuberculosis treatment. Seventy-five male patients with pulmonary tuberculosis (mean age, 40.0?±?10.7 years) were evaluated at baseline and again at 30 and 60 days of tuberculosis treatment. Serum levels of iron, copper, zinc, albumin, globulin, C-reactive protein, and hemoglobin, and smear and cultures for M. tuberculosis in sputum samples were analyzed. Compared to healthy subjects, at baseline, patients with PTB had lower serum iron levels, higher copper levels and copper/zinc ratio, and similar zinc levels. During the tuberculosis treatment, no significant changes in the serum levels of iron, zinc, and copper/zinc were observed. Lower serum copper levels were associated with bacteriological conversion in tuberculosis treatment (tuberculosis-negative) at 30 days but not at 60 days (tuberculosis-positive). C-reactive protein levels and the C-reactive protein/albumin ratio were lower in tuberculosis-negative patients than in tuberculosis-positive patients at 30 and 60 days after treatment. Albumin and hemoglobin levels and the albumin/globulin ratio in patients with pulmonary tuberculosis increased during the study period, regardless of the bacteriological results. High serum globulin levels did not change among pulmonary tuberculosis patients during the study. Serum copper levels and the C-reactive protein/albumin ratio may be important parameters to evaluate the persistence of non-conversion after 60 days of tuberculosis treatment, and they may serve as predictors for relapse after successful treatment.     

Oxygen binding and NO scavenging properties of truncated hemoglobin, HbN, of Mycobacterium smegmatis

Unraveling of microbial genome data has indicated that two distantly related truncated hemoglobins (trHbs), HbN and HbO, might occur in many species of slow-growing pathogenic mycobacteria. Involvement of HbN in bacterial defense against NO toxicity and nitrosative stress has been proposed. A gene, encoding a putative HbN homolog with conserved features of typical trHbs, has been identified within the genome sequence of fast-growing mycobacterium, Mycobacterium smegmatis. Sequence analysis of M. smegmatis HbN indicated that it is relatively smaller in size and lacks N-terminal pre-A region, carrying 12-residue polar sequence motif that is present in HbN of M. tuberculosis. HbN encoding gene of M. smegmatis was expressed in E. coli as a 12.8kD homodimeric heme protein that binds oxygen reversibly with high affinity (P50 approximately 0.081 mm Hg) and autooxidizes faster than M. tuberculosis HbN. The circular dichroism spectra indicate that HbN of M. smegmatis and M. tuberculosis are structurally similar. Interestingly, an hmp mutant of E. coli, unable to metabolize nitric oxide, exhibited very low NO uptake activity in the presence of M. smegmatis HbN as compared to HbN of M. tuberculosis. On the basis of cellular heme content, specific nitric oxide dioxygenase (NOD) activity of M. smegmatis HbN was nearly one-third of that from M. tuberculosis. Additionally, the hmp mutant of E. coli, carrying M. smegmatis HbN, exhibited nearly 10-fold lower cell survival under nitrosative stress and nitrite derived reactive nitrogen species as compared to the isogenic strain harboring HbN of M. tuberculosis. Taken together, these results suggest that NO metabolizing activity and protection provided by M. smegmatis HbN against toxicity of NO and reactive nitrogen is significantly lower than HbN of M. tuberculosis. The lower efficiency of M. smegmatis HbN for NO detoxification as compared to M. tuberculosis HbN might be related to different level of NO exposure and nitrosative stress faced by these mycobacteria during their cellular metabolism.     

The metabolic activity of Mycobacterium tuberculosis, assessed by use of a novel inducible GFP expression system, correlates with its capacity to inhibit phagosomal maturation and acidification in human macrophages

Mycobacterium tuberculosis generally reside in phagosomes within human macrophages that resist maturation and acidification, but exhibit significant heterogeneity. In this study we have constructed an IPTG-inducible GFP expression system in M. tuberculosis to assess the relationship between the metabolic status of M. tuberculosis and the degree of phagosomal maturation. Using these recombinant bacteria, we have found that, in human macrophages, M. tuberculosis that respond to IPTG with expression of GFP fluorescence, and hence are metabolically active, reside in non-acidified phagosomes that have not fused with Texas red dextran pre-labelled lysosomes. In contrast, M. tuberculosis that fail to express GFP in response to IPTG, and hence are metabolically inactive, reside within acidified phagosomes that have fused with Texas red dextran labelled lysosomes. These studies demonstrate that metabolic activity of M. tuberculosis correlates strongly with phagosomal maturation and that the inducible GFP expression system is useful for assessing metabolic activity of intracellular M. tuberculosis.     

AIDS-related tuberculosis in Rio de Janeiro, Brazil

Background

We studied the incidence of tuberculosis, AIDS, AIDS deaths and AIDS-TB co-infection at the population level in Rio de Janeiro, Brazil where universal and free access to combination antiretroviral therapy has been available since 1997.

Methodology/Principal Findings

This was a retrospective surveillance database match of Rio de Janeiro databases from 1995–2004. Proportions of tuberculosis occurring within 30 days and between 30 days and 1 year after AIDS diagnosis were determined. Generalized additive models fitted with cubic splines with appropriate estimating methods were used to describe rates and proportions over time. Overall, 90,806 tuberculosis cases and 16,891 AIDS cases were reported; 3,125 tuberculosis cases within 1 year of AIDS diagnosis were detected. Tuberculosis notification rates decreased after 1997 from a fitted rate (fR per 100,000) of 166.5 to 138.8 in 2004. AIDS incidence rates increased 26% between 1995 and 1998 (30.7 to 38.7) followed by a 33.3% decrease to 25.8 in 2004. AIDS mortality rates decreased dramatically after antiretroviral therapy was introduced between 1995 (27.5) and 1999 (13.4). The fitted proportion (fP) of patients with tuberculosis diagnosed within one year of AIDS decreased from 1995 (24.4%) to1998 (15.2%), remaining stable since. Seventy-five percent of tuberculosis diagnoses after an AIDS diagnosis occurred within 30 days of AIDS diagnosis.

Conclusions/Significance

Our results suggest that while combination ART should be considered an essential component of the response to the HIV and HIV/tuberculosis epidemics, it may not be sufficient alone to prevent progression from latent TB to active disease among HIV-infected populations. When tuberculosis is diagnosed prior to or at the same time as AIDS and ART has not yet been initiated, then ART is ineffective as a tuberculosis prevention strategy for these patients. Earlier HIV/AIDS diagnosis and ART initiation may reduce TB incidence in HIV/AIDS patients. More specific interventions will be required if HIV-related tuberculosis incidence is to continue to decline.     

The use of discrete characters in discriminant analysis for diagnosis of pulmonary tuberculosis and for classification of patients differing in treatment efficiency based on polymorphisms at nine codominant loci-HP,GC, TF,PI, PGM1, GLO1, C3, ACP1 and ESD

Discriminant analysis was used to differentiate patients with pulmonary tuberculosis (N = 106) from healthy individuals (N = 328) and patients whose treatment was efficient (N = 71) from those whose treatment was inefficient (N = 35). The analysis involved the data on nine polymorphic codominant loci: HP, GC, TF, PI, PGM1, GLO1, C3, ACP1, and ESD. The loci were selected by significance of differences in genotype frequencies between tuberculosis patients and healthy controls (GC, TF, PI, C3, ACP1) or between the two groups of patients differing in treatment efficiency (HP, GC, PI, PGM1, C3, ESD). Discrimination was based on a graphic method of Bayes classification procedure with a single-variate nomograph allowing easy estimation of the a posteriori probabilities for an individual to be classified. The two groups of patients proved to be discriminated sufficiently well (probability of misclassification Perr = 0.24), whereas discrimination between tuberculosis patients and healthy individuals was less efficient (Perr = 0.33). The method was proposed as a means of predicting the efficiency of treatment in pulmonary tuberculosis. Along with clinical, roentgenological, and laboratory examination, discriminant analysis may be employed as an accessory test in diagnostics of pulmonary tuberculosis, especially when the diagnosis is questionable.     

Evolving concepts of the epidemiology, diagnosis, and therapy of Mycobacterium tuberculosis infection

Tuberculosis in the United States is evolving in nearly all respects--epidemiology, diagnosis, treatment, and prophylaxis. Today a relatively larger segment of the population has predisposing factors to infection with tuberculosis. There is a greater percentage of people who are elderly, who have immigrated from countries endemic for tuberculosis, or who are immunosuppressed due to medications necessary for other conditions, because of malignancies, or because of infection with HIV. Skin test classifications have been revised to give different meanings to different-sized areas of induration at the injection site for defined populations. More sensitive, more specific, and faster diagnostic laboratory tests for tuberculosis are being developed. Short-course chemotherapy of from six to nine months is now accepted as standard treatment, regardless of exactly which of the proven regimens of antibiotics or accepted lengths of therapy is used. Patient compliance is improved with the shorter courses both for treatment and for prophylaxis. Better compliance with therapy results in better treatment outcomes of infections with Mycobacterium tuberculosis.     

A novel mycolic acid cyclopropane synthetase is required for cording, persistence, and virulence of Mycobacterium tuberculosis

Colonial morphology of pathogenic bacteria is often associated with virulence. For M. tuberculosis, the causative agent of tuberculosis (TB), virulence is correlated with the formation of serpentine cords, a morphology that was first noted by Koch. We identified a mycobacterial gene, pcaA, that we show is required for cording and mycolic acid cyclopropane ring synthesis in the cell wall of both BCG and M. tuberculosis. Furthermore, we show that mutants of pcaA fail to persist within and kill infected mice despite normal initial replication. These results indicate that a novel member of a family of cyclopropane synthetases is necessary for lethal chronic persistent M. tuberculosis infection and define a role for cyclopropanated lipids in bacterial pathogenesis.     

结核分枝杆菌Rv3425蛋白的原核表达纯化及其血清学诊断价值

目的：用原核系统表达结核分枝杆菌Rv3425蛋白并纯化,评价该重组蛋白在结核病血清学诊断方面的价值。方法：以结核分枝杆菌H37Rv株基因组为模板,PCR扩增得到Rv3425基因序列,克隆至表达载体pET-28a中,转入大肠杆菌BL21（DE3）进行诱导、表达后纯化,用Western印迹和ELISA法进行抗原性初步评价。结果：在原核系统内经IPTG诱导表达后,Rv3425蛋白主要以包涵体形式存在,经复性和镍柱层析纯化后,纯度达95%以上;Western印迹和ELISA结果证明重组Rv3425具有较强的抗原活性;用纯化的Rv3425蛋白做抗原,临床诊断结核病人血清,阳性率达50%。结论：高纯度的Rv3425蛋白在结核病诊断方面具有很高的应用价值,可作为结核病诊断的备选抗原。     

一种新型抗结核先导化合物S28 对结核分枝杆菌蛋白质组表达的影响

目的 探讨从化合物库中高通量筛选得到的、可有效抑制结核分枝杆菌生长和繁殖的新型活性化合物S28 的作用机制及其可能的作用靶点。方法 采用双向电泳技术, 比较分析活性化合物作用于结核分枝杆菌H37Ra 前、后的全细胞蛋白表达差异。结果 13 个蛋白质斑点表达下调, 对其中6 个改变明显的蛋白质斑点进行基质辅助激光解吸/ 电离飞行时间质谱分析, 成功测定2 个蛋白质斑点。数据库检索分析确定这2 个差异蛋白点分别为延长因子Tu 和短链脱氢酶, 是参与蛋白质翻译和氧化呼吸、能量代谢等生理过程的重要蛋白。结论 为 进一步深入探索新型抗结核活性化合物的作用机制和可能的靶点提供研究基础和方向。     

CpG oligonucleotides partially inhibit growth of Mycobacterium tuberculosis, but not Salmonella or Listeria, in human monocyte-derived macrophages

Immunostimulatory DNA sequences and their synthetic oligonucleotide analogs (CpG-ODN) activate innate immunity and can stimulate antibacterial effects against numerous intracellular pathogens. While it has been shown previously that CpG-ODN inhibit growth of Mycobacterium avium in murine and human macrophages, we now report that Mycobacterium tuberculosis growth can be inhibited by CpG-ODN treatment of human monocyte-derived macrophages (hMDM). This inhibitory effect was reversed by IFN-gamma, which has been shown repeatedly to enhance the growth of virulent M. tuberculosis in cultured hMDM. The antibacterial effect of CpG-ODN in human macrophages was specific for M. tuberculosis when compared to other intracellular pathogens including Listeria monocytogenes and Salmonella enterica serovar Dublin. These data indicate that CpG-ODN can improve the ability of hMDM to contain growth of virulent M. tuberculosis.     

Molecular detection, identification and drug resistance detection in Mycobacterium tuberculosis

This minireview presents recent developments in molecular methods for the diagnosis of tuberculosis, including detection, identification and determination of drug resistance of Mycobacterium tuberculosis . Tuberculosis remains one of the major causes of global death from a single infectious agent. This situation is worsened by the HIV/AIDS pandemic because one-third of HIV/AIDS patients are coinfected with M. tuberculosis . Also of great concern is the emergence of drug-resistant tuberculosis because there are almost no treatment options available for patients affected by highly resistant strains of M. tuberculosis . Advances in molecular biology techniques and a better knowledge of the molecular mechanisms of drug resistance have provided new tools for the rapid diagnosis of tuberculosis. Several nucleic acid amplification technologies have been developed and evaluated. New molecular approaches are being introduced continuously. This minireview will also comment on the future perspectives for the molecular diagnosis of tuberculosis and the feasibility for the implementation of these newer techniques in the clinical diagnostic laboratory.     

Isolation of ribonucleotide reductase from Mycobacterium tuberculosis and cloning, expression, and purification of the large subunit.

Ribonucleotide reductase, an allosterically regulated, cell cycle-dependent enzyme catalyzing a unique step in the synthesis of DNA, the reduction of 2'-ribonucleotides to 2'-deoxyribonucleotides, was purified 500-fold from Mycobacterium tuberculosis Erdman strain through cell disruption, ammonium sulfate fractionation, and dATP-Sepharose affinity column chromatography. As in eucaryotes and certain bacteria and viruses, the M. tuberculosis enzyme consists of two nonidentical subunits, R1 and R2, both of which are required for activity. R1 has a molecular mass of 84 kDa, as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and photoaffinity labeling with dATP. The amino acid sequences of the N-terminal peptide and two internal peptides were determined, and a partial R1 gene was isolated by PCR with primers designed from these amino acid sequences. Additional coding sequences were isolated by screening size-selected libraries, and a full-length form of M. tuberculosis R1 was generated by PCR amplification of high-molecular-weight M. tuberculosis DNA and expressed in Eschericnia coli. This coding sequence is 2,169 nucleotides long and contains no introns. The predicted molecular mass of R1 from the DNA sequence is 82,244 Da. Recombinant M. tuberculosis R1, purified to homogeneity, was biochemically active when assayed with extracts of M. tuberculosis enriched for R2.     

Lipid peroxidation, vitamins C, E and reduced glutathione levels in patients with pulmonary tuberculosis

The present study examined the relationship between lipid peroxidation and vitamin C, vitamin E and reduced glutathione levels in plasma, erythrocytes and erythrocyte membranes of pulmonary tuberculosis patients and an equal number of age-and sex-matched healthy subjects. Enhanced plasma, erythrocytes and erythrocyte membrane lipid peroxidation with concomitant decline in vitamin C, vitamin E and reduced glutathione levels were found in pulmonary tuberculosis patients. The elevated lipid peroxidation and decreased vitamin C, vitamin E and reduced glutathione levels indicate the potential of oxidative damage to erythrocytes and erythrocyte membranes of pulmonary tuberculosis patients.     

Forecast Model Analysis for the Morbidity of Tuberculosis in Xinjiang,China

Tuberculosis is a major global public health problem, which also affects economic and social development. China has the second largest burden of tuberculosis in the world. The tuberculosis morbidity in Xinjiang is much higher than the national situation; therefore, there is an urgent need for monitoring and predicting tuberculosis morbidity so as to make the control of tuberculosis more effective. Recently, the Box-Jenkins approach, specifically the autoregressive integrated moving average (ARIMA) model, is typically applied to predict the morbidity of infectious diseases; it can take into account changing trends, periodic changes, and random disturbances in time series. Autoregressive conditional heteroscedasticity (ARCH) models are the prevalent tools used to deal with time series heteroscedasticity. In this study, based on the data of the tuberculosis morbidity from January 2004 to June 2014 in Xinjiang, we establish the single ARIMA (1, 1, 2) (1, 1, 1)₁₂ model and the combined ARIMA (1, 1, 2) (1, 1, 1)₁₂-ARCH (1) model, which can be used to predict the tuberculosis morbidity successfully in Xinjiang. Comparative analyses show that the combined model is more effective. To the best of our knowledge, this is the first study to establish the ARIMA model and ARIMA-ARCH model for prediction and monitoring the monthly morbidity of tuberculosis in Xinjiang. Based on the results of this study, the ARIMA (1, 1, 2) (1, 1, 1)₁₂-ARCH (1) model is suggested to give tuberculosis surveillance by providing estimates on tuberculosis morbidity trends in Xinjiang, China.     

PhoP, a key player in Mycobacterium tuberculosis virulence

The Mycobacterium tuberculosis PhoPR two-component system is essential for virulence in animal models of tuberculosis. Recent articles have shown that among the reasons for the attenuation of the M. tuberculosis H37Ra strain is a mutation in the phoP gene that prevents the secretion of proteins that are important for virulence. There is a need for new anti-tubercular therapies because of the emergence of multi-drug-resistant M. tuberculosis strains and also the variable efficacy of the currently used bacille Calmette-Guérin vaccine. Because of its major role in M. tuberculosis pathogenicity, PhoP is a potential target candidate. This review summarizes our understanding of PhoPR's role in virulence and discusses areas in which our knowledge is limited.