Tuberculosis

Editorial Board

2010-01-01

Tuberculosis research in the European Union: Past achievements and future challenges

Hannu Lång, GianLuca Quaglio, Ole F. Olesen — 2009-11-02

Summary: The European Commission (EC) supports a large number of research activities in tuberculosis through the EU Framework Programmes for Research and Development (FP). By utilizing a variety of funding instruments, the EC has established a mixed portfolio of research projects, ranging from small discovery projects to large multidisciplinary consortia with sufficient critical mass to undertake translational and clinical research. The European investments in TB research have generated promising results with new vaccine candidates, drug leads, diagnostic markers and basic research results starting to emerge. In the light of a rapidly changing global research environment it has therefore become timely to review and update the priorities for TB research. To facilitate this process, a high-level conference on “Challenges for the future: research on HIV/AIDS, Malaria and Tuberculosis” was convened in Brussels on November 2008. This review gives an overview of the present portfolio of EC funded TB research, and summarises the conclusions from the conference on future perspectives for TB research in Europe and beyond.

Tuberculosis research: Going forward with a powerful “Translational Systems Biology” approach

Judy Day, Larry S. Schlesinger, Avner Friedman — 2010-01-04

Summary: Due to the complexity of the immune response to a Mycobacterium tuberculosis infection, identifying new, effective therapies and vaccines to combat it has been a problematic issue. Although many advances have been made in understanding particular mechanisms involved, they have, to date, proved insufficient to provide real breakthroughs in this area of tuberculosis research. The term “Translational Systems Biology” has been formally proposed to describe the use of experimental findings combined with mathematical modeling and/or engineering principles to understand complex biological processes in an integrative fashion for the purpose of enhancing clinical practice. This opinion piece discusses the importance of using a Translational Systems Biology approach for tuberculosis research as a means by which to go forward with the potential for significant breakthroughs to occur.

Variable-number tandem repeats typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 in Thailand

2009-11-13

Summary: Spoligotyping and variable-number tandem repeats (VNTR) typing have been increasingly used for differentiating Mycobacterium tuberculosis strains with low copy numbers of IS6110. However, there are few studies comparing their potential to type the strains originating from South and Southeast Asia where many of the isolates have only a few copies, or even single copy, of IS6110. Here, we evaluated the genotyping of 187M. tuberculosis isolates harboring 1–6 copies of IS6110, available from a population-based study in Chiangrai, northern Thailand during 1998–2000, using spoligotyping and VNTR typing. The low-copy-number isolates constituted about 34% of all M. tuberculosis isolated in the province. Discriminating capacities and cluster identification by the two methods were compared with each other and to those obtained by the standard IS6110-restriction fragment length polymorphism (RFLP) method. We found that VNTR typing based on the studied 10-loci set generated more distinct patterns (151 patterns) than spoligotyping (54 patterns) and IS6110-RFLP (65 patterns). Most of the RFLP- or spoligotyping-defined clusters were subdivided by VNTR typing. Combining IS6110-RFLP with VNTR typing produced 164 distinct patterns and 21.9% of clustered isolates whereas the combination of IS6110-RFLP and spoligotyping gave 103 different patterns and 59.4% of clustered isolates. Our results confirm the utility of VNTR typing as the secondary method of choice for investigating the epidemiology of M. tuberculosis with low copy numbers of IS6110.

ATP-dependent MurE ligase in Mycobacterium tuberculosis: Biochemical and structural characterisation

2009-11-30

Summary: New therapies are required against Mycobacterium tuberculosis and its cell wall peptidoglycan biosynthesis is a potential therapeutic target. UDP-MurNAc-tripeptide ligase (MurE) is a member of the ATP-dependent ligase family, which incorporate amino acids including meso-diaminopimelic acid (m-DAP) into peptidoglycan during synthesis in a species-specific manner. In the present study, we have cloned, over-expressed, and characterised MurE from M. tuberculosis (Mtb-MurE). The crystal structure has been determined at 3.0Å resolution in the presence of the substrate UDP-MurNAc-l-Ala-d-Glu (UAG). The activity of the enzyme was measured through estimating inorganic phosphate released in a non-radioactive high-throughput colourimetric assay. UDP-MurNAc-l-Ala-d-Glu-m-DAP (UMT) formation coupled to inorganic phosphate release was confirmed by HPLC and mass spectrometric analyses. Kinetic constants were determined for a range of natural substrates using optimised conditions. From our findings, it is evident that Mtb-MurE is highly specific in adding m-DAP to UDP-MurNAc-dipeptide and ATP-hydrolysis is an absolute requirement for its activity.

Identification of promoter-binding proteins of the fbp A and C genes in Mycobacterium tuberculosis

2009-12-04

Summary: The antigen 85 (Ag85) complex of Mycobacterium tuberculosis represents a promising candidate as a novel drug target and pathogenesis factor. Ag85 comprises three proteins Ag85A, B and C, (encoded by the genes fbpA, B, and C), which participate in cell wall biosynthesis, and interact with the host macrophage as fibronectin-binding proteins (fbps). Ag85 is also involved in the response to isoniazid (INH) treatment. The objective of this study was to identify potential fbp gene activators involved in the over-expression of fbp genes in response to INH.The biotinylated upstream promoter regions of fbpA and fbpC were used together with streptavidin-coated magnetic beads in DNA-binding assays, to isolate proteins with high-binding affinities from cytosolic extracts of INH-treated M. tuberculosis. Resolution of the DNA-binding proteins by 1D SDS-PAGE revealed 6 proteins with high-affinity for the fbpA promoter, and 7 with specificity the fbpC promoter. Mass spectrometric analyses [LC-ES(MS/MS)] identified proteins associated with drug resistance and stress/treatment responses, intermediary metabolism and cellular division, hypothetical proteins including a member of the MarR family of bacterial transcriptional regulators. The DNA-binding MarR protein shows potential as an authentic activator of fbp genes and functional validation of this factor is warranted.

Evaluation of 24 locus MIRU-VNTR genotyping of Mycobacterium tuberculosis isolates in Canada

2010-01-08

summary: The current gold standard for Mycobacterium tuberculosis complex (MTBC) genotyping is insertion sequence (IS) 6110 restriction fragment length polymorphism (RFLP) as it provides the highest discriminatory power of all available MTBC genotyping methods. However, RFLP is labour intensive and the interpretation of data from this method can be susceptible to errors. In 2001 a rapid, reproducible variable number of tandem repeat (VNTR) based typing method using 12 mycobacterial interspersed repetitive units (MIRU) was developed. Despite this advancement, this method lacked the discriminatory power of IS6110-RFLP. More recently a set of 24 MIRU-VNTR loci was reported to have greater discriminatory power than the original 12 locus system and may exceed that of RFLP when combined with spoligotyping. We compared the 24 locus method to the 12 locus method in order to improve surveillance of tuberculosis in Canada.A random sample of 650 MTBC isolates from British Columbia, Saskatchewan, Manitoba and Quebec Canada was genotyped using the 24 MIRU loci. Comparison of the data for the 12 and 24 MIRU loci showed an increase of the Hunter–Gaston discriminatory index (HGDI) from 0.895 (12 loci) to 0.920 (24 loci). The implementation of the 24 locus MIRU-VNTR methods offers improvement in discriminatory power over the traditional 12 locus method. For long-term surveillance of MTBC within Canada, the use of 24 MIRU-VNTR loci will provide rapid, highly discriminatory molecular epidemiology information.

GSTT1 and GSTM1 null mutations and adverse reactions induced by antituberculosis drugs in Koreans

2009-12-28

Summary: Adverse reactions induced by antituberculosis drugs (ATD) often result in serious morbidities, impeding scheduled treatment and cure. In the development of ATD-induced adverse reactions, glutathione S-transferase has been suggested to play a protective role as an intracellular scavenger by conjugating toxic reactive metabolites of ATD. This study examined the association of null mutations in GST enzyme genes (GSTT1 and GSTM1) with the development of ATD-induced hepatitis and cutaneous reactions. We compared the frequencies of GSTT1 and GSTM1 null mutations in 57 patients with hepatitis, 94 patients with cutaneous adverse reactions, and 190 ATD-tolerant controls. The frequency of null mutations in GSTT1 and GSTM1 in patients with ATD-induced hepatitis was not significantly different from that of controls (59.6% vs. 54.2% and 45.6% vs. 54.7%, respectively). Additionally, no significant difference was observed in the frequency of either null mutation in patients with ATD-induced cutaneous reactions, including maculopapular eruption, compared with controls (58.5% vs. 54.1% for GSTT1 and 59.6% vs. 54.6% for GSTM1). These findings indicate that GSTT1 and GSTM1 null mutations are not associated with the development of ATD-induced hepatitis or cutaneous reactions in this Korean population, and suggest that glutathione S-transferase enzymes do not play important roles in the pathogenesis of these conditions.

Cytokine genes are associated with tuberculin skin test response in a native Brazilian population

2009-12-14

Summary: Tuberculosis was a major cause of population decline among Brazilian indigenous peoples and remains a leading cause of morbidity and mortality among them. Despite high BCG coverage, results of Tuberculin Skin Test (TST) reactivity have shown high rates of anergy in Amazonian Indians. Given the high prevalence of anergy in these populations and the fact that genetic host factors play an important role in susceptibility to Mycobacterium tuberculosis (MTB), the aim of this study was to evaluate the association of nineteen polymorphisms in fifteen genes related to immune response and anergy in the Xavante, an indigenous group from Brazil. A total of 481 individuals were investigated. TST anergy was observed in 69% of them. Polymorphisms in four genes showed absence or very low variability: SP110, PTPN22, IL12RB1 and IL6. IFNG +874 A/T heterozygotes and IL4-590 C/C homozygotes were more frequent in those individuals who presented a positive TST (prevalence ratios of 1.9 and 2.0 respectively). The risk of anergy was 1.5 in IL10-1082 G/G homozygotes when compared to carriers for the A allele. In indigenous groups such as the Xavante exposure to a variety of infections, associated with specific genetic factors, may disturb the T-helper 1 and T-helper 2 balance leading to increased immunological susceptibility.

Mce2 operon mutant strain of Mycobacterium tuberculosis is attenuated in C57BL/6 mice

2009-12-07

Summary: Mycobacterium tuberculosis genome contains four related sets of an operon called mce (mce1-4). The disruption of one of these operons, mce1, causes M. tuberculosis to become hypervirulent, whereas the mce3 and mce4 operon mutants are attenuated in mice. This study examined the phenotype of the mce2 operon mutant. The deletion of mce2 operon in M. tuberculosis H37Rv had no effect on bacterial growth in 7H9 liquid broth or survival within macrophages. However, RAW macrophage-like cells infected with the mutant strain were reduced in their ability to produce TNF-α, IL-6 and MCP-1. In C57BL/6 mouse lungs, the mce2 operon mutant and wild type H37Rv replicated similarly up to 20 weeks of infection. However, by 56 weeks of infection, all mice infected with the wild type H37Rv had died, while 80% of those infected with the mutant remained alive (P<0.0001). The proportion of affected lung parenchyma in mice infected with the mutant was substantially less than that of mice infected with the wild type for the same time periods of infection. These observations suggest that the mce2 operon mutant is attenuated, and that this attenuation is related not to the bacterial burden but to the mutant's decreased ability to elicit a type of immune response and lung pathology detrimental to the survival of the animal.

“The heat is on”: Rapid microcalorimetric detection of mycobacteria in culture

O. Braissant, D. Wirz, B. Göpfert, A.U. Daniels — 2009-12-07

Summary: Detection of mycobacterial infection can be achieved by different means; however, culture-based methods remain the gold standard. In this paper, we present a new culture-based method using real-time microcalorimetric detection of growth of Mycobacterium species including Mycobacterium tuberculosis. Microcalorimetric detection of heat production by 6 different growing species of Mycobacterium was achieved between 20 and 310h depending on their type (fast vs. slow-growing mycobacteria) and initial concentration. This study demonstrates that microcalorimetric detection of mycobacterial growth is a potential advantageous alternative to methods using fluorescent or radiolabeled growth indicators.

Immune response to Mycobacterium tuberculosis specific antigen ESAT-6 among south Indians

2009-11-30

Summary: The 6-kDa early secreted antigenic target (ESAT-6) is a T-cell antigen recognized by individuals infected with Mycobacterium tuberculosis. The aim of the study was to identify “protective epitopes” of ESAT-6 protein in the south Indian population. Proliferative and Interferon gamma (IFN-γ) responses to ESAT-6 peptides were studied by flow cytometry and Enzyme linked immunosorbent assay (ELISA). Healthy household contacts (HHC) recognized Esp1 (10/17) and Esp6 (9/17) peptides. Among pulmonary tuberculosis patients (PTB), Esp1 (3/11) and Esp6 (5/11) were recognized. Maximal response (7/10) was found for Esp1 and Esp8 in treated patients (TR). Median values for the responding subjects gave the following results: Esp1 (76pg/ml), Esp6 (64pg/ml), induced IFN-γ production in HHC; PTB gave low IFN-γ responses for the peptides. TR responded to the peptides Esp1 (141pg/ml), Esp8 (102pg/ml). The proliferation of CD4 cells was similar in both PTB and TR for all peptides; but HHC showed an increase for Esp1 (p<0.05) and Esp6 (p<0.01). Esp1 (amino acids aa 1–20) and Esp6 (aa 51–70) were the immunogenic peptides recognized by the alleles HLA DRB1*04 and HLA DRB1*10 among HHC. But the association of the alleles with ESAT-6 peptide presentation needs to be confirmed in a large cohort of subjects. We speculate that ESAT-6 can be used along with other immune-eliciting proteins for vaccine design strategies in south Indian population.