Tubercle and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease最新文献

Novel bioinformatics routines have been used to provide a more detailed definition of the proteome ofMycobacterium tuberculosis H37Rv. Over half of the current proteins result from gene duplication or domain shuffling events while one-sixth show no similarity to polypeptides described in other organisms. Prominent among the genes that appear to have been duplicated on numerous occasions are those involved in fatty acid metabolism, regulation of gene expression, and the unusually glycine-rich PE and PPE proteins. Protein similarity analysis, coupled with inspection of the genetic neighbourhood, was used to explore possible functional relatedness. This uncovered four large mce operons whose proteins may mediate initial interactions between the tubercle bacillus and host cells, together with a cluster of genes that might encode components of a structure required for secretion of ESAT-6 like proteins. Close linkage of the mmpL genes, encoding large membrane proteins, with those required for fatty acid metabolism suggests involvement in lipid transport. Compared to free-living bacteria,M. tuberculosis has a significantly smaller transport protein repertoire and this may reflect its intracellular lifestyle.

Setting T helper cells can be divided into 2 subsets on the basis of their cytokine generation. T helper 1 cells secreting gamma interferon and interleukin 2 appear to be more prominent in patients with limited tuberculous disease.

Objective The purpose of this study was to evaluate human T helper cell immune responses to mycobacterial antigens in vitro and correlate these with the clinical features of patients with tuberculous infection or disease.

Design We studied 51 subjects and 11 controls who were grouped according to disease involvement as follows: 1) Mantoux negative, BCG negative, no disease; 2) Mantoux positive, no disease; 3) localized extrapulmonary; 4) healed pulmonary; 5) active pulmonary; and 6) miliary/disseminated. Peripheral blood mononuclear cells were cultured with PHA, PPD or Tetanus Toxoid, proliferation assessed and the supernatant analysed using an ELISA for IFNγ. ELISA was also used to measure M. tuberculosis specific antibodies in the serum.

Results Mantoux size correlated with PPD proliferationr = 0.5, P= 0.005 and γIFN production r= 0.36, P<0.01. All groups produced abundant γIFN although there was a trend toward higher production in groups 3 and 4. M. tuberculosis specific IgA (P= 0.003) and IgG₁(P= 0.002) was higher in groups 5 and 6. Those patients with limited disease (groups 2–4) had significantly lower levels of IgG₄than patients with severe disease (groups 5 & 6) (P<0.02).

Conclusion In conclusion patients with healed or extrapulmonary disease have immune responses in vitro suggestive of a TH₁(cell mediated immune) response, whereas patients with miliary/disseminated disease have antibody production suggestive of a TH₂response, together with high γIFN production. Both TH₁and TH₂responses may be necessary for host protection if there is a high bacillary load.

Setting The insertion sequence IS 6110 is widely used as a DNA fingerprinting probe for the classification of Mycobacterium tuberculosis strains. This study has focused on the characterization of regions disrupted by insertion of the IS 6110 element.

Objective To characterize IS 6110 insertion loci in clinical isolates of M. tuberculosis, in terms of their genomic location and genetic identity, to ascertain whether IS6110 transposition could be a mechanism driving phenotypic change.

Design Thirty-three IS 6110 insertion loci were cloned from 8 clinical isolates of M. tuberculosis. Clones representing DR locus insertions were identified by hybridization (n=4), and all other clones were characterized by DNA sequencing (n=29). The sequence data was analyzed in conjunction with that of 43 other insertion loci identified in published literature and DNA sequence databases.

Results The 76 sequences analyzed represented 66 unique insertion loci (including 9 unique insertions into the ipl locus). When mapped to the H37Rv genome, the majority of unique insertion loci demonstrated disruption of coding regions by IS 6110 (n=42; including the ipl insertions), while the remainder either occurred within intergenic regions (n=17), or could not be mapped to the H37Rv genome sequence (n=7). Mapping of the insertion loci reveals distribution throughout the chromosome, with isolated preferential insertion loci.

Conclusions This study has demonstrated the occurrence of 66 unique IS 6110 insertion loci dispersed throughout the M. tuberculosis genome, with an unexpectedly high incidence of IS 6110 insertions occurring within coding regions. However, the IS 6110 -mediated coding region disruptions identified here may only have limited impact on phenotype, as most of the coding regions disrupted are members of multiple gene families. Disruption of individual members of a family of genes may have no effect on phenotype or could have a minor or major impact, depending on the specificity and activity of the encoded protein.

Setting Mycobacterium tuberculosis isolates from patients in communities endemic for tuberculosis in South Africa.

Objective To develop a reliable PCR-based dot-blot hybridization strategy to detect mutations conferring drug resistance.

Design Different loci in six genes associated with drug resistance to isoniazid, rifampacin, streptomycin and ethambutol were selected to develop the PCR-based dot-blot hybridization strategy.

Results Primers and probes to detect mutations at codons 315, 463 (kat G) 269 (kas A), 531, 526 (rpo B) 43 (rps L), 513 (rrs) and 306 (emb B) were designed and used to develop a PCR-based dot-blot hybridization strategy. The dot-blot hybridization strategy with wild-type probes can efficiently be used to detect drug resistant mutations since these do not hybridize to mutant loci. Stripped blots and mutant probes can be used to identify the precise mutation. The emb B gene (ethambutol resistance) was used to show how the dot-blot strategy can assist with the prediction of drug resistance more accurately. The method is rapid, reproducible, not technically demanding and samples can be done in batches. Additional loci can easily be incorporated.

Conclusions A PCR-based dot-blot hybridization strategy is described which can accurately identify drug resistant strains and the method is useful for patients at risk and in areas endemic for tuberculosis.

Mycobacterium tuberculosis strain CDC 1551 exhibited unusually high levels of infectivity and virulence during a recent outbreak of tuberculosis in a rural community. Mice infected intravenously or aerogenically with M. tuberculosis CDC 1551 developed infections in the lungs and spleen which were almost identical with those for M. tuberculosis strains Erdman, H37Rv and Indian. There was also no significant difference in the survival rates of mice infected intravenously with CDC 1551, Erdman or H37Rv over a 3-month period. Studies designed to detect differences in the growth rates of CDC 1551 and Erdman in 3 inbred strains of mice failed to explain the high level of infectiousness and virulence expressed by CDC 1551 in human populations exposed to this organism.

Setting: Acquired immune deficiency syndrome (AIDS) patients have increased iron deposition in different tissues which may favour the growth of Mycobacterium avium, a common bacterial opportunist in these patients.

Objective: To test whether reducing the iron loads in macrophages in vitro and in vivo reduces M. avium proliferation.

Design: Mycobacterial proliferation was evaluated in vitro either in axenic media or cultured macrophages and in vivo in mice after manipulation of the iron status.

Results: Three different compounds – desferrioxamine (DFO), N,N′bis(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED) and a 1-amino-3-(2-bipyridyl)isoquinoline derivative (VUF8514) – were found to inhibit the growth of M. avium in axenic medium. DFO and HBED were also active in inhibiting the intramacrophagic growth of M. avium, while the use of VUF8514 was prevented by its toxicity towards the host cell. Both DFO and HBED enhanced the mycobacteriostatic effect induced in bone marrow derived macrophages by interferon gamma. In vivo, an iron poor diet led to reduced M. avium proliferation whereas the intraperitoneal administration of either DFO or HBED had small effects as they impacted little on the iron status of mice.

Conclusion: these results confirm that iron withholding is a means of inhibiting the growth of M. avium. In vitro data suggest that iron chelating compounds may be useful as adjunct therapy against M. avium, once their in vivo activity is optimized.

Setting: Low iron availability in the host induces the expression of iron acquisition systems and virulence genes in many pathogens. IdeR is a mycobacterial iron dependent regulator that controls the iron starvation and oxidative stress responses in Mycobacterium smegmatis. It is important to determine the role of IdeR and its regulon inM. tuberculosis , as identification of iron regulated genes can aid in the design of new drugs and generation of attenuated strains.

Objective: A potential IdeR binding site was found in the M. tuberculosis genome flanked by two divergently oriented open reading frames, irg1 and irg2. The aim of this study was to determine whetherirg1 and irg2 were iron and IdeR regulated genes.

Design: Interaction of IdeR with the putative binding sequence was examined by gel shift and footprinting assays. Transcriptional fusions of irg1 and irg2 tolacZ were used to study the effect of iron levels on the expression of these genes.

Results: IdeR binds to the predicted binding site, which overlaps with the irg1 promoter. irg1 and irg2 expression was decreased by iron in M. tuberculosis and in wild type M. smegmatis, but not in a M. smegmatis ideR mutant.

Conclusion: Two M. tuberculosis iron/IdeR regulated genes were identified. irg1 is predicted to be the M. tuberculosis hisE gene, which is involved in histidine biosynthesis. It is directly upstream of theM. tuberculosis hisG irg2 encodes a putative membrane protein that is a member of the PPE family.

Setting: Mycobacterium tuberculosis strain CDC1551 outbreak area in Tennessee and Kentucky and selected locations in the USA.

Objective: Develop a PCR assay to distinguish the highly transmissible CDC1551 from strains which have similar 4-band IS6110 fingerprints.

Design: Compare the IS6110 insertion sites in CDC1551 with those in 10 isolates which have similar 4-band IS6110 fingerprints. Utilize unique characteristics of insertion sites in CDC1551 to design a multiplex PCR to identify this strain.

Results: A multiplex PCR was developed which targets an IS6110 insertion conserved in most IS6110 low copy number strains and a deletion within the direct repeat region adjacent to an IS6110 insertion. Of 139 isolates with similar 4-band fingerprints, the CDC1551 PCR pattern was generated by only the 14 outbreak associated isolates. Of 154 isolates with different fingerprints, only four generated the CDC1551 pattern and these could be distinguished from CDC1551 by their IS6110 fingerprint.

Conclusions: The multiplex PCR used in conjunction with the IS6110 fingerprint should be a useful tool to aid in the continued surveillance of the outbreak area and follow the spread of this highly transmissible strain of M. tuberculosis.

Setting: Tuberculosis is endemic in south India: sputum positive pulmonary tuberculosis is predisposed by HLA-DR2 in south India and few other populations of the world.

Objective: To study HLA-DRB1, DQB1, DQA1 and DPB1 allelic polymorphism in pulmonary tuberculosis patients and endemic controls from south India.

Design: One hundred and twenty-six, sputum positive pulmonary tuberculosis patients and 87, endemic controls, from Madurai were studied for MHC class II allelic polymorphism by PCR-SSOP method. XI IHWC primers and probes and non-radioactive probing methods were employed.

Results: HLA DRB1*1501 and DQB1*0601 predisposed for pulmonary tuberculosis (DRB1*1501: odds ratio (OR)=2.68, 95% confidence interval (CI)=1.30–5.89, P value (P)=0.013, aetiological fraction (EF)=0.17; DQB1*0601: OR=2.32, CI=1.29–4.27, P=0.008, EF=0.26). Haplotype DRB1*1501–DQB1*0601 was higher in patients (1324 per 10000, X²=27.07) than controls (F=404÷10000, X²= 8.84). In a subset of 63 caste matched samples, DPB1*04 was preventive (OR=0.45, CI=0.21-0.95, P=0.036, PF=0.26): the distributions of DRB1*1501-DQB1*0601-DPB1*04 phenotypes were different between patients and controls (P=0.0092). These alleles were predominant in patients and controls of T5SU caste.

Conclusion: HLA-DRB1*1501 and DQB1*0601 predisposed to sputum positive pulmonary tuberculosis, and DPB1*04 was preventive and epistatic to this risk. Caste T5SU is an ideal model to study immunology of tuberculosis.