Modulation of Transcriptional and Inflammatory Responses in Murine Macrophages by the Mycobacterium tuberculosis Mammalian Cell Entry (Mce) 1 Complex

The outcome of many infections depends on the initial interactions between agent and host. Aiming at elucidating the effect of the M. tuberculosis Mce1 protein complex on host transcriptional and immunological responses to infection with M. tuberculosis, RNA from murine macrophages at 15, 30, 60 min, 4 and 10 hrs post-infection with M. tuberculosis H37Rv or Δ-mce1 H37Rv was analyzed by whole-genome microarrays and RT-QPCR. Immunological responses were measured using a 23-plex cytokine assay. Compared to uninfected controls, 524 versus 64 genes were up-regulated by 15 min post H37Rv- and Δ-mce1 H37Rv-infection, respectively. By 15 min post-H37Rv infection, a decline of 17 cytokines combined with up-regulation of Ccl24 (26.5-fold), Clec4a2 (23.2-fold) and Pparγ (10.5-fold) indicated an anti-inflammatory response initiated by IL-13. Down-regulation of Il13ra1 combined with up-regulation of Il12b (30.2-fold), suggested switch to a pro-inflammatory response by 4 hrs post H37Rv-infection. Whereas no significant change in cytokine concentration or transcription was observed during the first hour post Δ-mce1 H37Rv-infection, a significant decline of IL-1b, IL-9, IL-13, Eotaxin and GM-CSF combined with increased transcription of Il12b (25.1-fold) and Inb1 (17.9-fold) by 4 hrs, indicated a pro-inflammatory response. The balance between pro-and anti-inflammatory responses during the early stages of infection may have significant bearing on outcome

In-Depth Molecular Characterization of Mycobacterium tuberculosis from New Delhi – Predominance of Drug Resistant Isolates of the ‘Modern’ (TbD1−) Type

BACKGROUND: India has the highest estimated burden of tuberculosis in the world, accounting for 21% of all tuberculosis cases world-wide. However, due to lack of systematic analysis using multiple markers the available information on the genomic diversity of Mycobacterium tuberculosis in India is limited. METHODOLOGY/PRINCIPAL FINDINGS: Thus, 65 M. tuberculosis isolates from New Delhi, India were analyzed by spoligotyping, MIRU-VNTR, large deletion PCR typing and single nucleotide polymorphism analysis (SNP). The Central Asian (CAS) 1 _DELHI sub-lineage was the most prevalent sub-lineage comprising 46.2% (n = 30) of all isolates, with shared-type (ST) 26 being the most dominant genotype comprising 24.6% (n = 16) of all isolates. Other sub-lineages observed were: East-African Indian (EAI)-5 (9.2%, n = 6), EAI6_BGD1 (6.2%, n = 4), EAI3_IND, CAS and T1 with 6.2% each (n = 4 each), Beijing (4.6%, n = 3), CAS2 (3.1%, n = 2), and X1 and X2 with 1 isolate each. Genotyping results from five isolates (7.7%) did not match any existing spoligopatterns, and one isolate, ST124, belonged to an undefined lineage. Twenty-six percent of the isolates belonged to the TbD1+ PGG1 genogroup. SNP analysis of the pncA gene revealed a CAS-lineage specific silent mutation, S65S, which was observed for all CAS-lineage isolates (except two ST26 isolates) and in 1 orphan. Mutations in the pncA gene, conferring resistance to pyrazinamide, were observed in 15.4% of all isolates. Collectively, mutations in the rpoB gene, the katG gene and in both rpoB and katG genes, conferring resistance to rifampicin and isoniazid, respectively, were more frequent in CAS1_DELHI isolates compared to non-CAS_DELHI isolates (OR: 3.1, CI95% [1.11, 8.70], P = 0.045). The increased frequency of drug-resistance could not be linked to the patients' history of previous anti-tuberculosis treatment (OR: 1.156, CI95% [0.40, 3.36], P = 0.79). Fifty-six percent of all new tuberculosis patients had mutations in either the katG gene or the rpoB gene, or in both katG and rpoB genes. CONCLUSION: CAS1_DELHI isolates circulating in New Delhi, India have a high frequency of mutations in the rpoB and katG genes. A silent mutation (S65S) in the pncA gene can be used as a putative genetic marker for CAS-lineage isolates

Functional analysis of tilS homologues in Bacteria and Archaea

Out of all of the microorganisms sequenced so far about one third of the genes have unknown function. Several studies have shown that information on evolutionary relationships between unknown genes can aid in the prediction of the function of these genes. As a result of all this new information, new methods of identification have been established which sort genes based on sequence similarity between both paralogues and orthologues genes. The Clusters of Orthologous Groups (COG) is one such method, which group genes on the basis of sequence similarity where all groups containing at least three proteins from distant genomes are assumed to belong to the same orthologous group. At the start of this project all, but one of the COGs containing universal genes had been assigned a function. In the last group, COG0037, the genes had been suggested to be ATPases based on conserved motifs. In September, 2003 an article was published where the function of one of the Escherichia coli members in COG0037, TilS (previously known as YaeN), had been determined and TilS was shown to be an RNA modification enzyme. Based on this knowledge it was decided to analyse TilS orthologues for similar function. In this work four TilS homologues; AF1595 and AF1321 from Archaeoglobus fulgidus, Sso0586 from Sulfolobus solfataricus and YdaO from E. coli were expressed in Escherichia coli, and tested for solubility. Out of the four proteins only one, YdaO proved to be soluble. This protein was purified by affinity chromatography and analysed further and shown to exhibit ATPase activity and ability to autophosphorylate. An attempt to verify whether this protein is expressed under normal conditions was unsuccessful. An attempt to determine whether the gene coding for YdaO could be used as a signature gene for E. coli was also unsuccessful

Predominance of Mycobacterium tuberculosis EAI and Beijing Lineages in Yangon, Myanmar ▿

Isolates of the Mycobacterium tuberculosis Beijing lineage are associated with high rates of transmission, hypervirulence and drug resistance. The Beijing lineage has been shown to dominate the tuberculosis (TB) epidemic in East Asia; however, the diversity and frequency of M. tuberculosis genotypes from Myanmar are unknown. We present the first comprehensive study describing the M. tuberculosis isolates circulating in Yangon, Myanmar. Thus, 310 isolates from pulmonary TB patients from Yangon, Myanmar, were genotyped by spoligotyping and IS6110-based restriction fragment length polymorphism analysis (IS6110 RFLP). The most frequent lineages observed were the East African-Indian (EAI; 48.4%; n = 150) and Beijing (31.9%; n = 99) lineages. Isolates belonging to the most frequent shared types (STs), ST1 (n = 98; Beijing), ST292 (n = 28; EAI), and ST89 (n = 11; EAI), had ≥75% similarity in their IS6110 patterns. Five of 11 Beijing isolates comprising five clusters with identical IS6110 RFLP patterns could be discriminated by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis. Of the 150 EAI isolates, 40 isolates (26.7%) had only one IS6110 copy, and 17 of these isolates could be discriminated by MIRU-VNTR analysis. The findings from this study suggest that although there is a predominance of the ancient EAI lineage in Yangon, the TB epidemic in Yangon is driven by clonal expansion of the ST1 genotype. The Beijing lineage isolates (21.4%) were more likely (P = 0.009) than EAI lineage isolates to be multidrug resistant (MDR) (1.3%; odds ratio, 3.2, adjusted for the patients' history of exposure to anti-TB drugs), suggesting that the spread of MDR Beijing isolates is a major problem in Yangon

Increased level of acute phase reactants in patients infected with modern Mycobacterium tuberculosis genotypes in Mwanza, Tanzania

Background: There is increasing evidence to suggest that different Mycobacterium tuberculosis lineages cause variations in the clinical presentation of tuberculosis (TB). Certain M. tuberculosis genotypes/lineages have been shown to be more likely to cause active TB in human populations from a distinct genetic ancestry. This study describes the genetic biodiversity of M. tuberculosis genotypes in Mwanza city, Tanzania and the clinical presentation of the disease caused by isolates of different lineages. Methods: Two-hundred-fifty-two isolates from pulmonary TB patients in Mwanza, Tanzania were characterized by spoligotyping, and 45 isolates were further characterized by mycobacterium interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). The patients’ level of the acute phase reactants AGP, CRP and neutrophil counts, in addition to BMI, were measured and compared to the M. tuberculosis lineage of the infectious agent for each patient. Results: The most frequent genotype was ST59 (48 out of 248 [19.4%]), belonging to the Euro-American lineage LAM11_ZWE, followed by ST21 (CAS_KILI lineage [44 out of 248 [17.7%]). A low degree of diversity (15.7% [39 different ST’s out of 248 isolates]) of genotypes, in addition to a high level of mixed M. tuberculosis sub-populations among isolates with an unreported spoligotype pattern (10 out of 20 isolates [50.0%]) and isolates belonging to the ST53 lineage (13 out of 25 [52%]) was observed. Isolates of the ‘modern’ (TbD1-) Euro-American lineage induced higher levels of α1-acid glycoprotein (β = 0.4, P = 0.02; 95% CI [0.06-0.66]) and neutrophil counts (β = 0.9, P = 0.02; 95% CI [0.12-1.64]) and had lower BMI score (β = −1.0, P = 0.04; 95% CI[−1.89 – (−0.03)]). LAM11_ZWE (‘modern’) isolates induced higher levels of CRP (β = 24.4, P = 0.05; 95% CI[0.24-48.63]) and neutrophil counts (β = 0.9, P = 0.03; 95% CI[0.09-1.70]). Conclusion: The low diversity of genotypes may be explained by an evolutionary advantage of the most common lineages over other lineages combined with optimal conditions for transmission, such as overcrowding and inadequate ventilation. The induction of higher levels of acute phase reactants in patients infected by ‘modern’ lineage isolates compared to ‘ancient’ lineages may suggest increased virulence among ‘modern’ lineage isolates

High Diversity of Mycobacterium tuberculosis Genotypes in South Africa and Preponderance of Mixed Infections among ST53 Isolates ▿

The reemergence of tuberculosis (TB) has become a major health problem worldwide, especially in Asia and Africa. Failure to combat this disease due to nonadherence or inappropriate drug regimens has selected for the emergence of multiple-drug-resistant (MDR) TB. The development of new molecular genotyping techniques has revealed the presence of mixed Mycobacterium tuberculosis infections, which may accelerate the emergence of drug-resistant strains. There are some studies describing the local distribution of circulating strains in South Africa, but to date, reports describing the frequency and distribution of M. tuberculosis genotypes, and specifically MDR genotypes, across the different provinces are limited. Thus, 252 isolates (of which 109 were MDR) from eight of the nine provinces of South Africa were analyzed by spoligotyping. Spoligotyping showed 10 different lineages, and ST53 (11.1%) and ST1 (10.3%) were the most frequent genotypes. Of the 75 different spoligopatterns observed, 20 (7.9%) were previously unreported. Analysis of the mycobacterial interspersed repetitive units of variable-number tandem repeats of the ST53 and ST1 isolates revealed that ∼54% of the ST53 isolates were of mixed M. tuberculosis subpopulations. Drug resistance (defined as resistance to at least isoniazid and/or rifampin) could only be linked to a history of previous anti-TB treatment (adjusted odds ratio, 4.0; 95% confidence interval, 2.27 to 7.10; P = <0.0001). This study describes a high diversity of circulating genotypes in South Africa in addition to a high frequency of mixed M. tuberculosis subpopulations among the ST53 isolates. MDR TB in South Africa could not be attributed to the spread of any single lineage

Primers used for the analysis of single nucleotide polymorphisms in 65 <i>M. tuberculosis</i> isolates from New Delhi, India.

<p>Primers used for the analysis of single nucleotide polymorphisms in 65 <i>M. tuberculosis</i> isolates from New Delhi, India.</p