Th2-type immune response observed in healthy individuals to sonicate antigen prepared from the most prevalent Mycobacterium tuberculosis strain with single copy of IS6110

Different Mycobacterium tuberculosis strains operate different immune evasion strategies for their survival in the host. This mainly depends on the virulence of the strain and the host immune responses. The most virulent strains are actively involved in the transmission, widely spread in the community and induce differential immune responses. We evaluated the immune response of a sonicate antigen prepared from one predominant strain (S7) from M. tuberculosis harbouring a single copy of IS6110. Significant lymphoproliferative response against purified protein derivative from tubercle bacillus (PPD) and H37Rv antigens was observed in PPD positive normal individuals and tuberculosis patients. Interferon-gamma (IFN-gamma) levels against these antigens were significantly increased in normal individuals but not in tuberculosis patients. The antigen S7 showed marginal T-cell proliferation but did not induce IFN-gamma secretion in both groups. Conversely, it induced significantly high levels of cytokine interleukin 4 (IL-4) in normal individuals. The macrophage cytokines, IL-12 and tumour necrosis factor alpha (TNF-alpha), did not show S7 antigen specific stimulation. The intracellular cytokine further confirmed an increase in IL-4(+)/CD4+ T-cells and a decrease in IFN-gamma(+)/CD4+ T-cells upon stimulation. The antibody response showed an increase in IgG and IgA levels against this antigen in normal individuals. These observations suggest that antigen S7 modulates the immune response towards T helper cell type 2 by suppressing T helper cell type 1 protective immune response in PPD positive normal individuals. We speculate that some components of this sonicate antigen are associated with immunosuppressive response.     

Use of the insertion element IS6110 for DNA fingerprinting of Mycobacterium tuberculosis isolates presenting various profiles of drug susceptibility

Abstract IS 6100 is an insertion sequence of the IS3 family and it is present in multiple copies in the chromosome of Mycobacterium tuberculosis . Four to 15 copies are present in various strains of M. tuberculosis . In this study, the value of IS 6110 as an epidemiological marker of tuberculosis was examined. Unrelated clinical strains from Greek patients presented, in restriction fragment length polymorphism analysis, a high degree of polymorphism, whereas patterns of related clinical strains from familial outbreaks were identical. Since RFLP analysis with acetylaminofluorene labeled IS 6110 as the probe gave satisfactory results, it is suggested that this non-radioactive probe can be used in hospitals and health centres for the epidemiological survey of M. tuberculosis infections.     

Molecular features of Mycobacterium avium human isolates carrying a single copy of IS1245 and IS1311 per genome

Human clinical isolates of the Mycobacterium avium complex, from hospitals in Bogotá, were studied using a wide range of molecular tests including PCR restriction-enzyme analysis (PRA) of the hsp65 gene. Up to 21 of the isolates were identified as M. avium PRA variant III (Mav III), a variant obtained only from isolates on the American continent. In contrast to previous reports, restriction fragment length polymorphism analysis using IS1245 and IS1311 showed a single copy for each insertion sequence (IS) in the majority (19/21) of the Colombian Mav III isolates under study. In order to analyse whether the ISs were inserted in a relevant genomic region, experimental conditions were established to determine the insertion loci of each single copy of both ISs in the genome. Analysis of genomic insertion loci indicated that both IS1245 and IS1311 were present in areas containing putatively truncated integrases and/or transposases, which may have an influence on the mobility of the inserted IS. In addition, a conserved genomic region was identified for the insertion of IS1311; this region could be part of the IS1311 itself.     

Estimating change rates of genetic markers using serial samples: applications to the transposon IS6110 in Mycobacterium tuberculosis

In infectious disease epidemiology, it is useful to know how quickly genetic markers of pathogenic agents evolve while inside hosts. We propose a modular framework with which these genotype change rates can be estimated. The estimation scheme requires a model of the underlying process of genetic change, a detection scheme that filters this process into observable quantities, and a monitoring scheme that describes the timing of observations. We study a linear "birth-shift-death" model for change in transposable element genotypes, obtaining maximum-likelihood estimators for various detection and monitoring schemes. The method is applied to serial genotypes of the transposon IS6110 in Mycobacterium tuberculosis. The estimated birth rate of 0.0161 (events per copy of the transposon per year) and death rate of 0.0108 are both significantly larger than the estimated shift rate of 0.0018. The sum of these estimates, which corresponds to a "half-life" of 2.4 years for a typical strain that has 10 copies of the element, substantially exceeds a previous estimate of 0.0135 total changes per copy per year. We consider experimental design issues that enable the precision of estimates to be improved. We also discuss extensions to other markers and implications for molecular epidemiology.     

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.     

结核分枝杆菌诊断技术研究进展

近年来,结核病的诊断方法逐渐得到完善,将传统细菌学检验方法、免疫学方法和分子生物学的方法相结合,加速了对结核病的诊断研究。特别是分子生物学技术的发展,对于阐明分枝杆菌和结核分枝杆菌的遗传变异规律及分子进化等都有重要的意义。我们结合近年来结核分枝杆菌诊断在免疫学检测、PCR技术、基因技术等方面的进步,从细菌学检测、免疫学检测、分子生物学检测等3个方面做简要综述。     

Mycobacterium tuberculosis Phylogeny Reconstruction Based on Combined Numerical Analysis with IS1081, IS6110, VNTR, and DR-Based Spoligotyping Suggests the Existence of Two New Phylogeographical Clades

This paper deals with phylogenetic relationships among a set of 90 clinical strains representative of the worldwide diversity of the Mycobacterium tuberculosis complex (Kremer et al. 1999) using eight independent genetic markers: IS6110, IS1081, the direct repeat (DR) locus, and five variable number of tandem DNA repeat loci (VNTR). In a preliminary experiment, phylogenetic trees based on single markers were constructed that led to the detection of some similarities between the VNTR-based and the spoligotyping-based phylogenetic trees. In the second step, a more global phenetic approach based on pairwise comparison of strains within each typing system was used, followed by calculations of mean genetic distances based on all the eight loci and the use of the neighbor-joining algorithm for tree reconstruction. This analysis confirmed our preliminary observations and suggested the existence of at least two new phylogeographical clades of M. tuberculosis, one defined as the ``East African–Indian family' (EA-I), which may find its origin on the African or Asian continents, and the other as the ``Latin American and Mediterranean' (LA-M) family. The existence of these two families was also validated by an independent phylogenetic analysis of spoligotyping on a larger set of shared types (n= 252) and further corroborated by VNTR and katG–gyrA results. The potential origin of these families of bacilli is discussed based on cattle domestication and human migration history. In conclusion, the information contained in insertion sequence and repetitive DNAs may serve as a model for the phylogenetic reconstruction of the M. tuberculosis complex.     

Extensive sequence homology between the mycobacterium leprae LSR (12 kDa) antigen and its Mycobacterium tuberculosis counterpart

The Mycobacterium leprae LSR (12 kDa) protein antigen has been reported to mimic whole cell M. leprae in T cell responses across the leprosy spectrum. In addition, B cell responses to specific sequences within the LSR antigen have been shown to be associated with immunopathological responses in leprosy patients with erythema nodosum leprosum. We have in the present study applied the M. leprae LSR DNA sequence as query to search for the presence of homologous genes within the recently completed Mycobacterium tuberculosis genome database (Sanger Centre, UK). By using the BLASTN search tool, a homologous M. tuberculosis open reading frame (336 bp), encoding a protein antigen of 12.1 kDa, was identified within the cosmid MTCY07H7B.25. The gene is designated Rv3597c within the M. tuberculosis H37Rv genome. Sequence alignment revealed 93% identity between the M. leprae and M. tuberculosis antigens at the amino acid sequence level. The finding that some B and T cell epitopes were localized to regions with amino acid substitutions may account for the putative differential responsiveness to this antigen in tuberculosis and leprosy.     

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.     

Polymorphisms of 20 regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans and animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and other members of the MTC evolved. The genome of M. bovis is over >99.95% identical to that of M. tuberculosis but with seven deletions ranging in size from 1 to 12.7 kb. In addition, 1200 single nucleotide mutations in coding regions distinguish M. bovis from M. tuberculosis. In the present study, we assessed 75 M. tuberculosis genomes and 23 M. bovis genomes to identify non‐synonymous mutations in 202 coding sequences of regulatory genes between both species. We identified species‐specific variants in 20 regulatory proteins and confirmed differential expression of hypoxia‐related genes between M. bovis and M. tuberculosis.     

Drug resistance and IS6110‐RFLP patterns of Mycobacterium tuberculosis in patients with recurrent tuberculosis in northern Thailand

The emergence of drug resistant Mycobacterium tuberculosis has become a global threat to tuberculosis (TB) prevention and control efforts. This study aimed to determine the drug resistance profiles and DNA fingerprints of M. tuberculosis strains isolated from patients with relapsed or retreatment pulmonary TB in Chiang Rai province in northern Thailand. Significant differences in multidrug resistance (MDR) (P = 0.025) and resistance to isoniazid (P = 0.025) and rifampin (P = 0.046) between first and second registrations of patients with retreatment TB were found. However, there were no significant differences in resistance to any drugs in patients with relapsed TB. The rate of MDR‐TB strains was 12.2% among new patients at first registration, 22.5% among patients with recurrence who had previously undergone treatment at second registration and 12.5% at third registration. Two retreatment patients whose initial treatment had failed had developed MDR‐TB with resistance to all TB drugs tested, including rifampin, isoniazid, streptomycin and ethambutol. IS6110‐RFLP analysis revealed that 66.7% (10/15 isolates) of MDR‐TB belonged to the Beijing family. In most cases, IS6110‐RFLP patterns of isolates from the same patients were identical in relapse and retreatment groups. However, some pairs of isolates from retreatment patients after treatment failure had non‐identical IS6110‐RFLP patterns. These results suggest that, after failure and default treatment, patients with retreatment tuberculosis have a significantly greater risk of MDR‐TB, isoniazid and rifampin resistance than do other patients.     

结核分枝杆菌的致病机理

结核病仍是全球健康的主要威胁,其致病菌结核分枝杆菌的致闰机理与众不同。脂类代谢在致病中具有重要的作用。巨噬细胞被入侵的细菌修的机理也是研究细菌持续感染致病的重要突破点,研究细菌的致病机理可以为开发新的疫苗和治疗药物奠定坚实的基础。     

结核分枝杆菌对异烟肼耐药的分子机制

抗结核一线药物异烟肼是应用最广泛的抗结核药物之一,自1952年应用于临床以来,异烟肼就成了治疗结核和潜在感染的基础药物.有报道,我国异烟肼耐药已排在首位.结核分枝杆菌对异烟肼耐药的分子机制十分复杂,涉及katG、inhA、kasA、ndh、axyR等多种基因,本研究仅就此方面的研究作一综述.     

结核分枝杆菌重要诊断用抗原研究进展

血清学试验是结核诊断的重要依据。随着科学技术的进步,新的结核诊断用抗原不断被发现。我们简要综述了结核菌素蛋白衍生物、抗原85复合体、38kDa磷酸盐转运蛋白、6kDa早期分泌性蛋白、10kDa培养滤液蛋白、免疫性蛋白MPT64、主要分泌性免疫蛋白MPB70、表面脂蛋白MPB83等8种结核分枝杆菌重要抗原作为结核诊断用抗原的研究进展。     

结核分枝杆菌免疫优势抗原研究进展

结核病是当今影响人类健康、流行性最广、病死率最高的感染性疾病之一。结核病的诊断和疫苗的构建成为当前的研究热点,筛选出结核分枝杆菌免疫优势抗原是快速准确的诊断结核病及研制安全有效的疫苗的关键。拟对近年来国内外学者发现的结核分枝杆菌免疫优势抗原的分子生物学特性研究进展进行综述。     

结核分枝杆菌RD1区研究进展

RD1区是结核分枝杆菌(MTB)在长期传代过程中丢失的重要保护性抗原,RD1区仅存在于致病性分枝杆菌中,而在卡介苗(BCG)及环境分枝杆菌中缺失。RD1区基因全长9.5kb,共有9个开放读码框,分别编码9个蛋白。RD1区是MTB毒力的关键因素之一,同时RD1区存在一种新分泌表达体系,能保证ESAT6和CFP10蛋白分泌表达。RD1区蛋白有较强的免疫原性,在MTB的预防和诊断中将可能发挥巨大作用,并有可能成为筛选抗MTB药物的理想靶抗原。     

Mycobacterium tuberculosis mammalian cell entry operon (mce) homologs in Mycobacterium other than tuberculosis (MOTT)

The cloned mammalian cell entry gene mce1a from Mycobacterium tuberculosis confers to non-pathogenic Escherichia coli the ability to invade and survive inside macrophages and HeLa cells. The aim of this work was to search for and characterize homologs of the four M. tuberculosis mammalian cell entry operons (mce1, mce2, mce3 and mce4) in mycobacteria other than tuberculosis (MOTT). The dot-blot and polymerase chain reaction (PCR) experiments performed on 24 clinical isolates representing 20 different mycobacterial species indicated that the mce operons were widely distributed throughout the genus Mycobacterium. BLAST search results showed the presence of mce1, mce2 and mce4 homologs in Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. A homologous region for the mce3 operon was also found in M. avium and M. smegmatis. DNA and protein alignments were done to compare the M. tuberculosis mce operons and the deduced M. bovis, M. avium, and M. smegmatis homologs. The deduced proteins of M. bovis mce1, mce2 and mce4 operons had 99.6-100% homology with the respective M. tuberculosis mce proteins (MTmce). The similarity between M. avium mce proteins and the individual M. tuberculosis homologs ranged from 56.2 to 85.5%. The alignment results between M. smegmatis mce proteins and the respective MTmce proteins ranged from 58.5% to 68.5%. Primer sets were designed from the M. tuberculosis mce4a gene for amplification of 379-bp fragments. Amplification was successful in 14 strains representing 11 different mycobacterial species. The PCR fragments were sequenced from 10 strains representing eight species. Alignment of the sequenced PCR products showed that mce4a homologs are highly conserved in the genus Mycobacterium. In conclusions, the four mce operons in different mycobacterial species are generally organized in the same manner. The phylogenetic tree comparing the different mce operons showed that the mce1 operon was closely related to the mce2 operon and mce3 diverged from the other operons. The wide distribution of the mce operons in pathogenic and non-pathogenic mycobacteria implicates that the presence of these putative virulence genes is not an indicator for the pathogenicity of the bacilli. Instead, the pathogenicity of these factors might be determined by their expression.     

Dynamics of fluoroquinolones induced resistance in DNA gyrase of Mycobacterium tuberculosis

DNA gyrase is a validated target of fluoroquinolones which are key components of multidrug resistance tuberculosis (TB) treatment. Most frequent occurring mutations associated with high level of resistance to fluoroquinolone in clinical isolates of TB patients are A90V, D94G, and A90V–D94G (double mutant [DM]), present in the larger subunit of DNA Gyrase. In order to explicate the molecular mechanism of drug resistance corresponding to these mutations, molecular dynamics (MD) and mechanics approach was applied. Structure-based molecular docking of complex comprised of DNA bound with Gyrase A (large subunit) and Gyrase C (small subunit) with moxifloxacin (MFX) revealed high binding affinity to wild type with considerably high Glide XP docking score of ?7.88 kcal/mol. MFX affinity decreases toward single mutants and was minimum toward the DM with a docking score of ?3.82 kcal/mol. Docking studies were also performed against 8-Methyl-moxifloxacin which exhibited higher binding affinity against wild and mutants DNA gyrase when compared to MFX. Molecular Mechanics/Generalized Born Surface Area method predicted the binding free energy of the wild, A90V, D94G, and DM complexes to be ?55.81, ?25.87, ?20.45, and ?12.29 kcal/mol, respectively. These complexes were further subjected to 30 ns long MD simulations to examine significant interactions and conformational flexibilities in terms of root mean square deviation, root mean square fluctuation, and strength of hydrogen bond formed. This comparative drug interaction analysis provides systematic insights into the mechanism behind drug resistance and also paves way toward identifying potent lead compounds that could combat drug resistance of DNA gyrase due to mutations.