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

We report the identification of the first histone-like protein ofMycobacterium tuberculosis(MTB) (HLP_Mt). The T cell blot assay was used to identify antigens of MTB associated with human immune response in healthy contacts. Fraction 21 corresponding to proteins in the molecular weight range of approximately 30 kDa were found to be immunogenic in tuberculin reactors. None of the fractions were found to be immunogenic by this assay in non-reactors to tuberculin. All sera, irrespective of the source, showed reactivity with MTB antigen(s) over a wide molecular weight range (205 16 kDa). In the present study fraction 21 was processed for the generation of murine polyclonal sera and amino acid sequencing. The sequence of a 16-amino acid long peptide showed a 100% homology with an open reading frame (ORF) in the translated sequence of cosmid cY349 (Sanger Centre, Cambridge, UK). The ORF was predicted to code for a protein of 214 amino acids. Oligonucleotide primers were synthesized based on the nucleotide sequence located at the 5′ and 3′ regions of the gene. The gene encoding the predicted protein was PCR-amplified, cloned, sequenced and expressed inEscherichia colias a protein of 28 kDa. The expressed HLP_Mtprotein was shown to react with the polyclonal murine sera originally raised against fraction 21. Human immune response to the recombinant HLP_Mtprotein was demonstrated by its ability to induce lymphoproliferation in peripheral blood derived mononuclear cells, and the presence of anti-HLP_Mtantibodies in pooled patient sera by immunoblot. The recombinant HLP_Mtprotein elicited a vigorous lymphoproliferative response especially in healthy tuberculin reactors compared to non-reactors and patients of tuberculosis, (P⪡ 0.05). The protein has unique dual domains with homology to both bacterial histone-like proteins (HU) and eukaryotic histone H1. Homology to prokaryotic and eukaryotic deoxyribonucleic acid (DNA)-binding proteins suggested that HLP_Mtcould bind DNA. DNA-binding properties were confirmed by South-Western analysis strongly suggesting an interaction between HLP_Mtand the MTB chromosome.

A two-component system of Mycobacterium tuberculosis H37Rv, designated as devR-devS , was identified in our laboratory, that encodes a response regulator and a histidine sensor-kinase respectively. Southern analysis of the devR-devS locus in the genomes of M. tuberculosis H37Rv, H37Ra and Erdman indicated the presence of anEco RI restriction fragment length polymorphism (RFLP) in the Erdman strain. Studies employing polymerase chain reaction (PCR), suggested that the RFLP is associated with a deletion in M. tuberculosis Erdman. Deoxyribonucleic acid sequence analysis of this region in the Erdman strain confirmed a deletion of 330 bp. The deletion maps in an open reading frame (ORF) designated as orfX (which maps upstream of the devR-devS locus) that is present in M. tuberculosis H37Rv and H37Ra, and in all five isolates of M. tuberculosis examined in this study. OrfX is transcriptionally active in M. tuberculosis H37Rv and H37Ra but not in the Erdman strain. These observations point towards variations in genomic organization and in transcriptional activity of virulent strains of M. tuberculosis . OrfX gene deletion can be utilized in a rapid PCR-based assay to differentiate between H37Rv and Erdman strains.

Objective : To understand the mechanism by which IdeR is necessary for maintaining wild type levels of KatG and SodA enzyme activity and normal isoniazid (INH) resistance.

Design : To identify the step(s) of SodA and KatG function that were affected by the ideR mutation, quantitative western immunoassays and ribonucleic acid (RNA) hybridizations were performed. To see if the increased INH sensitivity of the ideR mutant was caused by lower SodA activity, the Mycobacterium smegmatis sod gene was inactivated.

Results : The levels of KatG and SodA mRNA and protein in the M. smegmatis IdeR mutant are decreased to approximately 20–40% of those observed in the wild type parent strain. This is quantitatively similar to the decrease in KatG and SodA enzyme activities originally observed in the ideR strain. The M. smegmatis sodA mutant was slightly more sensitive to INH, compared to the wild type strain and was more resistant than the ideR mutant.

Conclusion : IdeR is necessary for full expression of the M. smegmatis katG and sodA genes. It is not yet known whether this protein acts directly at the gene level. The lower levels of SodA contribute slightly to the increased susceptibility to INH of the ideR mutant, but cannot explain the magnitude of the INH sensitivity observed when IdeR is not present. These data suggest that IdeR is a regulator of the cellular stress response, as it has a protective role in cells facing environmental stresses, such as increased levels of reactive oxygen species and INH toxic intermediates. These conclusions do not necessarily apply to IdeR's role in M. tuberculosis physiology, since we have not inactivated its gene in this pathogen.

Setting : The relationship between alveolar macrophages (AM) and lymphocytes may be important in the early establishment of infection with Mycobacterium tuberculosis . AM in several species have been shown to suppress lymphoproliferation by producing inhibitors that include nitric oxide (NO).

Objective : To study this phenomenon in the guinea pig, the mitogen-induced proliferation of splenic lymphocytes was quantified under various conditions of co-culture with resident AM.

Results : Guinea pig AM consistently and profoundly suppressed proliferation in the co-cultures at AM:lymphocyte ratios of 1:4 or greater. The inclusion of a NO synthesis inhibitor, N-monomethyl-L arginine (NMMA), in the co-culture medium did not influence the suppression of Con A-induced lymphoproliferation by resident guinea pig AM. No nitrite could be detected in supernatant fluids of co-cultured AM and splenocytes. Attempts to stimulate guinea pig AM with LPS in combination with recombinant murine and human IFN--γ, infection with live Listeria monocytogenes , or incubation with the supernatants from ConA-activated guinea pig lymphocytes failed to generate NO metabolites. The addition of catalase or indomethacin to the Con A-induced AM-splenocyte co-cultures, to inhibit hydrogen peroxide (H₂O₂) or prostaglandin E₂(PGE₂), respectively, did not counteract the suppression mediated by AM. Cell contact was necessary for the co-cultures to generate their inhibitory effects on lymphoproliferation, however, the suppression was actually mediated, at least in part, by soluble factors produced in the co-cultures.

Conclusion : These results suggest that resident alveolar macrophages suppress lymphocyte proliferation in the guinea pig, but that the effect is not mediated by NO, PGE₂or H₂O₂. The failure to demonstrate NO synthesis under a variety of stimulatory conditions, which resulted in macrophage activation, suggests that the guinea pig is similar to the human in that regard.

Summary

Objective:

To characterize the correlation of the mutations in the pncA gene encoding pyrazinamidase (PZase) of Mycobacterium tuberculosis to a loss of PZase activity and development of pyrazinamide (PZA) resistance.

Design:

The association of PZase activity, minimum inhibitory concentrations (MICs), and mutations in the pncA gene of M. tuberculosis isolated in mostly Asian countries was investigated.

Results:

One hundred thirty-five out of 168 isolates were PZase positive, and 33 were negative. The MICs of PZA at pH 6.0 were over 400 g/ml for all 33 PZase-negative isolates, while those of PZase-positive isolates were equal to or less than 200 g/ml. Among 33 PZase-negative isolates sequenced, 32 (97%) had mutations within the pncA gene. A mutation was seen in various regions throughout the pncA gene. It was suprising that all three strains of in vitro selected PZA resistant mutants were PZase-positive and showed no change in the pncA gene. These results indicate that additional mechanisms may be involved in PZA resistance. No mutations were observed in all of 135 PZase-positive M. tuberculosis isolates tested, indicating that mutations in the pncA gene could be involved in the loss of PZase activity.

Conclusions:

Sequencing analysis of the pncA gene should provide rapid diagnosis of PZA resistant clinical isolates of M. tuberculosis.

This work describes the screening of a M. bovis BCG cosmid library in M. smegmatis with a hyperimmune rabbit anti-BCG serum.

Cross-reactive antibodies interfere with the detection of BCG specific antigens in M. smegmatis culture filtrates. We, therefore, screened parallel western blots with serum adsorbed with a M. smegmatis cell lysate and unadsorbed serum. Comparison of the western blots allowed distinction between BCG specific and cross-reactive M. smegmatis antigens. Thirty-one cosmids expressed BCG specific antigens. One of them, a hitherto undescribed 100 kDa antigen was subcloned, sequenced and expressed in E. coli. It shows a high degree of homology to ClpB, a member of the Clp family of proteases and was immunologically reactive with the rabbit hyperimmune serum against M. bovis BCG. A positive signal was also obtained with sera of patients with tuberculosis. This antigen is a previously unrecognized target of the human immune response to mycobacteria.

Setting: A study of tuberculosis cases and healthy blood donor controls from the Western Region of The Gambia, West Africa.

Objective: To investigate the potential role of candidate gene polymorphisms in host susceptibility to tuberculosis.

Design: Single base change polymorphisms in interleukin 1 beta (IL1β), interleukin 10 (IL10) and fucosyltransferase-2 (FUT-2), microsatellite polymorphisms in interleukin 1 alpha (IL1α) and IL10 and a minisatellite polymorphism in interleukin 1 receptor antagonist (IL1RA) were typed in over 400 tuberculosis cases and 400 healthy blood donor controls.

Results: IL1 gene cluster polymorphisms (IL1RA and possibly IL1α) showed marginally significant association with tuberculosis. In particular IL1RA allele 2 heterozygotes were less frequent among tuberculosis cases than controls (P= 0.03). IL1β, IL10 and FUT-2 polymorphisms were not associated with tuberculosis.

Conclusion: Genetic susceptibility to tuberculosis among Gambians may be partly determined by genes in the IL1 gene cluster on chromosome 2. Further association studies will be required on other population groups to confirm whether these results are of biological significance.

Setting : In vitro cultures.

Objective : To characterize nitrate reduction during aerobic growth and hypoxic shiftdown to non-replicating persistence of Mycobacterium tuberculosis cultures.

Design : The rates of reduction of nitrate to nitrite were measured in cultures of M. tuberculosis growing aerobically or undergoing hypoxic shiftdown.

Results : Tubercle bacilli growing aerobically in the presence of nitrate reduce nitrate at a rate proportional to the substrate concentration, continuing until the substrate is exhausted. When the bacilli in an oxygen restricted model enter microaerophilic non-replicating persistence (NRP) stage 1, they exhibit a marked increase in rate of nitrate reduction that is independent of substrate concentration, and terminates by feedback inhibition when the concentration of nitrite produced approaches 2.5 mM. When bacilli in the oxygen restricted model are not supplemented with nitrate until they enter microaerophilic NRP stage 1, they exhibit an induction period before the rapid nitrate reduction starts. When the nitrate is not added until the bacilli have entered the anaerobic NRP stage 2, reduction of the substrate starts immediately. Nitrite is not reduced by M. tuberculosis in any stage of its growth or NRP.

Conclusion : The hypoxically induced nitrate reduction probably serves a respiratory function in supporting hypoxic shiftdown of M. tuberculosis from aerobic growth to non-replication persistence and represents a useful new marker for monitoring that shiftdown. This response may help the bacilli survive in oxygen depleted regions of inflammatory or necrotic tissue, where nitrate can occur as a degradation product of nitric oxide.

Knowledge of the molecular genetic basis of resistance to antituberculous agents has advanced rapidly since we reviewed this topic 3 years ago. Virtually all isolates resistant to rifampin and related rifamycins have a mutation that alters the sequence of a 27-amino-acid region of the beta subunit of ribonucleic acid (RNA) polymerase. Resistance to isoniazid (INH) is more complex. Many resistant organisms have mutations in thekatGgene encoding catalase-peroxidase that result in altered enzyme structure. These structural changes apparently result in decreased conversion of INH to a biologically active form. Some INH-resistant organisms also have mutations in theinhAlocus or a recently characterized gene (kasA) encoding a β-ketoacyl-acyl carrier protein synthase. Streptomycin resistance is due mainly to mutations in the 16S rRNA gene or therpsLgene encoding ribosomal protein S12. Resistance to pyrazinamide in the great majority of organisms is caused by mutations in the gene (pncA) encoding pyrazinamidase that result in diminished enzyme activity. Ethambutol resistance in approximately 60% of organisms is due to amino acid replacements at position 306 of an arabinosyltransferase encoded by theembBgene. Amino acid changes in the A subunit of deoxyribonucleic acid gyrase cause fluoroquinolone resistance in most organisms. Kanamycin resistance is due to nucleotide substitutions in therrsgene encoding 16S rRNA. Multidrug resistant strains arise by sequential accumulation of resistance mutations for individual drugs. Limited evidence exists indicating that some drug resistant strains with mutations that severely alter catalase-peroxidase activity are less virulent in animal models. A diverse array of strategies is available to assist in rapid detection of drug resistance-associated gene mutations. Although remarkable advances have been made, much remains to be learned about the molecular genetic basis of drug resistance inMycobacterium tuberculosis. It is reasonable to believe that development of new therapeutics based on knowledge obtained from the study of the molecular mechanisms of resistance will occur.

Summary

Setting:

Mycobacterial galactofuran is essential to the linking of the peptidoglycan and mycolic acid cell wall layers. Galactofuran biosynthesis should thus be essential for viability.

Objective:

The objective was to determine the pathway of galactofuranosyl biosynthesis and to clone a gene encoding an essential enzyme necessary for its formation.

Design:

Specific enzymatic conversions involved in formation of galactopyranose and galactofuranose residues in other bacteria were tested for in Mycobacterium smegmatis. M. tuberculosis deoxyribonucleic acid (DNA) was identified by homology.

Results:

It was shown that the de novo synthesis of the galactose carbon skeleton occurred in M. smegmatis by the transformation of UDP-glucopyranose to UDP-galactopyranose via the enzyme UDP-glucose 4-epimerase (E.C. 5.1.3.2). The N-terminal sequence of this enzyme was obtained after purification. The galactose salvage pathway enzyme, UDP-glucose-galactose-1-phosphate uridylytransferase (E.C. 2.7.7.12), was also shown to be present. The critical biosynthetic transformation of the galactopyranose to galactofuranose ring form was shown to occur at the sugar nucleotide level via the enzyme UDP-galactopyranose mutase (E.C. 5.4.99.9). The M. tuberculosis DNA encoding this enzyme was sequenced, the gene expressed in Escherichia coli, and the expected enzymatic activity demonstrated.

Conclusion:

Galactofuranose biosynthesis can now be pursued as a potential drug target in M. tuberculosis.