Mutations In Mycobacterium Tuberculosis Research Articles

Accelerated Detection ofMycobacterium tuberculosisGenes Essential for Bacterial Survival in Guinea Pigs, Compared with Mice

Mouse and guinea pig models have been used to identify Mycobacterium tuberculosis mutants attenuated for survival. However, unlike mice, M. tuberculosis-infected guinea pigs form caseating granulomas, which may simulate human disease more closely. We used designer arrays for defined mutant analysis, a high-throughput subtractive competition assay, for genotypically defined M. tuberculosis mutants and compared the survival of the same mutant pools in guinea pig and mouse aerosol models. Selected mutants found to be attenuated in either aerosol model were also analyzed in the mouse hollow-fiber model. M. tuberculosis mutants representing 74 genes were tested. Eighteen M. tuberculosis mutants were attenuated for survival in either aerosol model, with 70% of selected mutants also attenuated in the mouse hollow-fiber model. The majority of attenuated mutants in the mouse aerosol model were detected only after 90 days of infection. There was a high degree of concordance between the genes identified by the 2 aerosol models, with detection being significantly earlier in the guinea pig (P<.0003). We identified M. tuberculosis genes required for survival in mammalian lungs. The majority of mouse late-stage survival mutants were detected significantly earlier in the guinea pig, which suggests that differences in tuberculosis-induced lung pathologic changes may account for this accelerated detection.

Evidence for complex interactions of stress-associated regulons in an mprAB deletion mutant of Mycobacterium tuberculosis

Two-component systems are important constituents of bacterial regulatory networks. Results of this investigation into the role of the MprAB two-component system of Mycobacterium tuberculosis indicate that it is associated with the regulation of several stress-responsive regulons. Using a deletion mutant lacking portions of the response regulator, MprA, and the histidine kinase, MprB, it was demonstrated by real-time PCR, primer extension analyses and DNA microarrays that MprAB activates sigma factor genes sigE and sigB, under SDS stress and during exponential growth. SDS-inducible, MprA-dependent transcriptional start points were identified for mprA, sigE and sigB, and variations in distance between these points and MprA-binding sites suggest that MprA is involved in different mechanisms of promoter activation. Although most of the SigE regulon was downregulated in the deletion mutant, the cluster of genes Rv1129c, Rv1130 and Rv1131, which is associated with growth in monocytes, was upregulated in the deletion mutant under SDS stress, and this upregulation was dependent upon atmospheric growth conditions. Multiple stress-associated genes of the DosR, SigD and IdeR regulons were also upregulated in the deletion mutant, during exponential growth and/or in the presence of SDS. Surprisingly, the deletion mutant had increased resistance to SDS compared to the parental strain, and enhanced growth in human peripheral blood monocytes, characteristics which may result from a loss of repression of stress-associated genes.

P1999 pncA and rpoB mutations in Mycobacterium tuberculosis, a 3-year experiment in Lyon, France

P1999 pncA and rpoB mutations in Mycobacterium tuberculosis, a 3-year experiment in Lyon, France

Enhanced priming of adaptive immunity by a proapoptotic mutant of Mycobacterium tuberculosis

Enhanced priming of adaptive immunity by a proapoptotic mutant of Mycobacterium tuberculosis

Characterization of the protective T‐cell response generated in CD4‐deficient mice by a live attenuated Mycobacterium tuberculosis vaccine

The global epidemic of tuberculosis, fuelled by acquired immune-deficiency syndrome, necessitates the development of a safe and effective vaccine. We have constructed a DeltaRD1DeltapanCD mutant of Mycobacterium tuberculosis (mc(2)6030) that undergoes limited replication and is severely attenuated in immunocompromised mice, yet induces significant protection against tuberculosis in wild-type mice and even in mice that completely lack CD4(+) T cells as a result of targeted disruption of their CD4 genes (CD4(-/-) mice). Ex vivo studies of T cells from mc(2)6030-immunized mice showed that these immune cells responded to protein antigens of M. tuberculosis in a major histocompatibility complex (MHC) class II-restricted manner. Antibody depletion experiments showed that antituberculosis protective responses in the lung were not diminished by removal of CD8(+), T-cell receptor gammadelta (TCR-gammadelta(+)) and NK1.1(+) T cells from vaccinated CD4(-/-) mice before challenge, implying that the observed recall and immune effector functions resulting from vaccination of CD4(-/-) mice with mc(2)6030 were attributable to a population of CD4(-) CD8(-) (double-negative) TCR-alphabeta(+), TCR-gammadelta(-), NK1.1(-) T cells. Transfer of highly enriched double-negative TCR-alphabeta(+) T cells from mc(2)6030-immunized CD4(-/-) mice into naive CD4(-/-) mice resulted in significant protection against an aerosol tuberculosis challenge. Enriched pulmonary double-negative T cells transcribed significantly more interferon-gamma and interleukin-2 mRNA than double-negative T cells from naive mice after a tuberculous challenge. These results confirmed previous findings on the potential for a subset of MHC class II-restricted T cells to develop and function without expression of CD4 and suggest novel vaccination strategies to assist in the control of tuberculosis in human immunodeficiency virus-infected humans who have chronic depletion of their CD4(+) T cells.

Mycobacterium bovis Δ leuD auxotroph-induced protective immunity against tissue colonization, burden and distribution in cattle intranasally challenged with Mycobacterium bovis Ravenel S

Bovine tuberculosis is a chronic granulomatous disease caused by Mycobacterium bovis. Lack of definitive diagnostics and effective vaccines for domestic animals are major obstacles to the control and eradication of bovine tuberculosis. Auxotrophic mutants of Mycobacterium tuberculosis have shown promise as vaccine candidates for preventing human tuberculosis. Similarly, we constructed a leucine auxotroph of M. bovis, by using allelic exchange to delete leuD (encoding isopropyl malate isomerase), creating a strain requiring exogenous leucine for growth in vitro. We vaccinated 10 cattle subcutaneously with 10 9 CFU of M. bovis Δ leuD and 10 age-matched, gender-matched controls were injected with phosphate-buffered saline. Vaccinated cattle had significantly increased in vitro antigen-specific T-cell-mediated responses. All cattle were challenged intranasally on day 160 post-immunization with 10 6 CFU of virulent M. bovis Ravenel S. On day 160 post-challenge vaccinated cattle had significantly reduced tissue mycobacterial burdens and 6 of 10 had complete clearance of the challenge strain and histopathological lesions were dramatically less severe in the vaccinated group. Thus, a single subcutaneous immunization of the M. bovis Δ leuD mutant produced highly significantly protective immunity as measured by a reduction in tissue colonization, burden, bacilli dissemination, and histopathology caused by virulent M. bovis Ravenel S challenge.

Molecular characterization of rpoB gene mutations in rifampicine‐resistant Mycobacterium tuberculosis isolates from tuberculosis patients in Belarus

The aim of this study was to investigate the frequency, location and type of rpoB mutations in Mycobacterium tuberculosis isolated from patients in Belarus. Tuberculosis cases are increasing every year in Belarus. Moreover, resistance to anti-tuberculosis drugs, especially to rifampicine, has increased. In this study, 44 rifampicine-resistance M. tuberculosis clinical isolates (including multidrug-resistant isolates) were subjected to DNA sequencing analysis of the hypervariable region (hot-spot) of the rpoB gene originating from different geographical regions in Belarus. Sixteen different types of mutations were identified. The most common point mutations were in codons 510 (47.7%), 526 (45.5%), 523 (40.86%) and 531 (29.5%). Eleven isolates (27.7%) carried one mutation and 23 (52%), 7 (16%), 3 (7%) of isolates carried 2, 3 and 4 mutations, respectively. A characteristic, prominent finding of this study was high frequency of multiple mutations in different codons of the rpoB gene (27.7%) and also the detection of unusual types of mutations in the 510 codon, comprising CAG mutations (deletion or changing, to CTG, CAC or CAT). In our study, the change TTG in codon 531 was found in 92% of isolates and the change TGC in 8% of isolates. A TAC change in codon 526 was not found, but the GAC change was found in all isolates. Isolates of M. tuberculosis isolated in Belarus were characterized by the wide spectrum of the important mutations and might belong to the epidemic widespread clones.

Evaluation of the TB-Biochip Oligonucleotide Microarray System for Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis

The TB-Biochip oligonucleotide microarray system is a rapid system to detect mutations associated with rifampin (RIF) resistance in mycobacteria. After optimizing the system with 29 laboratory-generated rifampin-resistant mutants of Mycobacterium tuberculosis, we evaluated the performance of this test using 75 clinical isolates of Mycobacterium tuberculosis. With this small sample set, the TB-Biochip system displayed a sensitivity of 80% and a specificity of 100% relative to conventional drug susceptibility testing results for RIF resistance. For these samples (approximately 50% tested positive), the positive predictive value was 100% and the negative predictive value was 85%. Four of the seven observed discrepancies were attributed to rare and new mutations not represented in the microarray, while three of the strains with discrepant results did not carry mutations in the RIF resistance-determining region. The results of this study confirm the utility of the system for rapid detection of RIF resistance and suggest approaches to increasing its sensitivity.

The Competitive Cost of Antibiotic Resistance in Mycobacterium tuberculosis

Mathematical models predict that the future of the multidrug-resistant tuberculosis epidemic will depend on the fitness cost of drug resistance. We show that in laboratory-derived mutants of Mycobacterium tuberculosis, rifampin resistance is universally associated with a competitive fitness cost and that this cost is determined by the specific resistance mutation and strain genetic background. In contrast, we demonstrate that prolonged patient treatment can result in multidrug-resistant strains with no fitness defect and that strains with low- or no-cost resistance mutations are also the most frequent among clinical isolates.

Comparative investigation of the pathogenicity of three Mycobacterium tuberculosis mutants defective in the synthesis of p-hydroxybenzoic acid derivatives

p-Hydroxybenzoic acid derivatives (p-HBADs) are glycoconjugates secreted by all Mycobacterium tuberculosis isolates whose contribution to pathogenicity remains to be determined. The pathogenicity of three transposon mutants of M. tuberculosis deficient in the biosynthesis of some or all forms of p-HBADs was studied. Whilst the mutants grew similarly to the wild-type strain in macrophages and C57BL/6 mice, two of the mutants induced a more severe and diffuse inflammation in the lungs. The lack of production of some or all forms of p-HBADs in these two mutants also correlated with an increased secretion of the pro-inflammatory cytokines tumour-necrosis factor α, interleukin 6 and interleukin 12 in vivo. We propose that the loss of production of p-HBADs by tubercle bacilli results in their diminished ability to suppress the pro-inflammatory response to infection and that this ultimately provokes extensive pulmonary lesions in the C57BL/6 model of tuberculosis infection.

The use of mutant mycobacteria as new vaccines to prevent tuberculosis

Given the variable protective efficacy generated by Mycobacterium bovis BCG (Bacillus Calmette-Guérin), there is a concerted effort worldwide to develop better vaccines that could be used to reduce the burden of tuberculosis. Rational attenuated mutants of Mycobacterium tuberculosis are vaccine candidates that offer some potential in this area. In this paper, we will discuss the molecular methods used to generate mutant mycobacteria, as well as the results obtained with some of these strains, in terms of attenuation, immunogenicity and level of protection, when compared with the conventional BCG vaccine in diverse animal models. Tuberculosis vaccine candidates based on safe and live mycobacterial mutants could be promising candidates.

Characterization of the S315G mutant of Mycobacterium tuberculosis catalase‐peroxidase KatG

KatG(S315T) is the most common mutant found in clinical isolates of Mycobacterium tuberculosis resistant to the antibiotic Isoniazid (INH). Resistance due to the S315T mutation has been assigned to a decrease in the enzyme's affinity for the drug likely due to a decrease in the dimensions of a substrate access channel caused by the methyl group of the Thr 315 side chain. The main goal of the present study was to examine the effect of reducing the size of the side chain at residue 315 in this crucial region of the enzyme. Site-directed mutagenesis was used to prepare KatG(S315G). The overexpressed enzyme was characterized using optical and EPR spectroscopy. Catalase and peroxidase activities were reduced by nearly 2 fold and 10 fold, respectively, compared with the WT enzyme. Titration of KatG(S315G) with INH showed an approximately two-fold increase in affinity compared to WT KatG, while the rate of INH activation by KatG(S315G) was approximately 60% of that observed for the WT enzyme. Low temperature (10 K) EPR data showed the presence of mostly 5-c HS (high spin) and QS (quantum spin) heme spin states in the resting enzyme, more similar to KatG(S315T) than to WT enzyme. INH binding produced a transition from 6-c QS to 5-c QS and HS heme, also similar to the behavior of KatG (S315T). In addition, a small amount of 6-c low spin heme (due to the coordination of -OH group) was detected. The results are consistent with the hypothesis that a substrate access channel plays an important role in the binding of INH and its activation by KatG and that residue 315 plays a critical role in determining heme iron structure. Supported by NIH Grant # AI060014

Deletion of the Mycobacterium tuberculosis α-Crystallin-Like hspX Gene Causes Increased Bacterial Growth In Vivo

Hypervirulent mutants of Mycobacterium tuberculosis, whose growth rates are higher in vivo, have now been reported to have mutations in both regulatory and structural genes, but the basis for this unusual phenotype is not understood. One hypervirulence gene, dosR (devR, Rv2031c), activates transcription of approximately 50 genes in this pathogen in response to hypoxia and nitric oxide stress. The most dramatic activation (approximately 80-fold) is activation of the hspX (acr, Rv2031c) gene, which encodes a 16-kDa alpha-crystallin-like protein that is a major antigen. In this study we found that a Deltaacr mutant exhibited increased growth following infection of BALB/c mice in vivo and in both resting and activated macrophages in vitro (as measured by the number of CFU). The increased growth in macrophages was equal to that of a DeltadosR mutant, while introduction of a constitutively expressed hspX gene reduced the DeltadosR virulence to wild-type levels. These results suggest that the increased number of CFU of the DeltadosR mutant was largely due to loss of hspX expression. We also confirmed that constitutive expression of hspX slows growth in vitro, and we propose that hspX plays an active role in slowing the growth of M. tuberculosis in vivo immediately following infection.

Comparative Evaluation of Cleavase Fragment Length Polymorphism With PCR-SSCP and PCR-RFLP to Detect Antimicrobial Agent Resistance in Mycobacterium tuberculosis.

Background: Several molecular methods potentially useful in the detection of Mycobacterium tuberculosis mutations, specifically in rpoB and katG, were compared. Methods and Results: DNA from 24 M. tuberculosis clinical isolates, with mutations associated with resistance to rifampin and/or isoniazid, was analyzed. A 128 bp amplicon, spanning the 81 bp rpoB region containing most mutations leading to rifampin resistance, was analyzed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and a recently introduced mutation scanning method, cleavase fragment length polymorphism (CFLP) analysis. Also, a 350 bp amplicon encompassing that region was analyzed by the CFLP method. CFLP analysis of the 350 bp amplicon (23 isolates) identified 14 of 17 mutants; however, CFLP analysis of the 128 bp amplicon accurately identified all mutants as did PCR-SSCP with interpretative difficulty for two codon 513 mutations. CFLP and PCR-restriction fragment length polymorphism (RFLP) analyses of a 623 bp amplicon encompassing katG codons 315 and 463 showed that the CFLP method identified single and dinucleotide codon 315 substitutions with or without codon 463 (CGG-->CTG) changes, whereas PCR-RFLP (MspI) missed one codon 315 polymorphism (AGC-->ACA) in three isolates. Conclusion: Both PCR-SSCP and CFLP analyses were sensitive in identifying all mutations on short sequences in the rpoB mutants. CFLP appears to be more efficient than SSCP and RFLP for the detection of mutations in large amplicons.

TB Antibiotic Resistance and Growth Fitness

Antibiotic resistance among human pathogens is a growing problem worldwide. Genetic mutations that confer such resistance are believed to reduce the pathogens’ competitiveness for growth in the absence of antibiotic pressure. Researchers in the U.S. recently conducted a series of experiments to test this premise. The investigators generated rifampin-resistant mutants of Mycobacterium tuberculosis …

Increased pathology in lungs of mice after infection with an α-crystallin mutant of Mycobacterium tuberculosis: changes in cathepsin proteases and certain cytokines

Latency and reactivation are a significant problem that contributes to the incidence, transmission and pathogenesis of tuberculosis. The mechanisms involved in these processes, at the level of both the bacillus and the host, are poorly understood. In Mycobacterium tuberculosis the alpha-crystallin (acr) gene has been linked to latency, because it is highly expressed during hypoxic growth conditions. Deletion of the acr gene in M. tuberculosis H37Rv (Deltaacr strain) was previously shown to reduce the intracellular growth of bacilli in macrophages; however, its impact on pathogenesis in vivo was unknown. This study demonstrated that infection of C57BL6 mice with Deltaacr results in lung bacillary loads 1-2 log units higher in comparison to parental H37Rv. Haematoxylin/eosin staining of lungs revealed exacerbated pathology characterized by extensive obliteration of alveolar air spaces by granulomatous inflammation. RT-PCR analysis and immunostaining of lungs showed that infection with either H37Rv or Deltaacr results in the differential expression of lysosomal cathepsin proteases. A slight increase in the expression of the matrix-degrading acidic-type cathepsins B, D and H was noted in Deltaacr-infected mice and was associated with clusters of macrophages within lung granulomas. Deltaacr-infected mice also showed high serum levels of TNF-alpha, IFN-gamma and G-CSF, suggesting that Acr may play a role in modulating the host response to infection.

Effects of clofazimine on potassium uptake by a Trk-deletion mutant of Mycobacterium tuberculosis

This study was designed to investigate the effects of the membrane-active, anti-mycobacterial agent, clofazimine, on potassium (K+)-uptake by a mutant of Mycobacterium tuberculosis (MTB), in which the Trk system, the major K+ transporter of this microbial pathogen, had been selectively inactivated. The ceoB and ceoC genes of MTB, which encode the TrkA proteins, CeoB and CeoC, were deleted by homologous recombination, and the double-knockout mutant and wild-type strains compared with respect to K+ uptake and growth in the presence and absence of clofazimine (0.015-2.5 mg/L) using radioassay procedures. Surprisingly, the magnitudes of K+ uptake and rate of growth of the ceoBC-knockout mutant were significantly (P < 0.05) greater than those of the wild-type strain, due, presumably, to induction of a back-up transporter. Exposure of both the wild-type strain and ceoBC-knockout mutant of MTB to clofazimine was accompanied by dose-related decreases in K+ uptake, as well as growth, which were of comparable magnitude for both strains. These observations demonstrate that the major K+ transporter of MTB, Trk, as well as an uncharacterized inducible back-up system, is equally sensitive to the inhibitory actions of clofazimine.

Induction of high levels of protective immunity in mice after vaccination using dendritic cells infected with auxotrophic mutants of Mycobacterium tuberculosis

Adoptively transferred dendritic cells presenting antigens derived from different pathogens have been shown to elicit specific T cell responses and to induce protective antibacterial immunity. We describe here the induction of high levels of protective immunity in mice using dendritic cells infected with auxotrophic mutants of Mycobacterium tuberculosis. We provide evidence that protection is superior to BCG and that it is associated with increased priming of CD4 + and CD8 + T cells specific for mycobacterial antigens. This method for generating high levels of anti-bacterial protective immunity could be helpful in the design of novel vaccines against tuberculosis and other intracellular pathogens.

Acquisition of rifabutin resistance by a rifampicin resistant mutant of Mycobacterium tuberculosis involves an unusual spectrum of mutations and elevated frequency

BackgroundMutations in a small region of the rpoB gene are responsible for most rifamycin resistance in Mycobacterium tuberculosis. In this study we have sequentially generated resistant strains to first rifampicin and then rifabutin. Portions of the rpoB gene were sequenced from 131 randomly selected mutants. Second round selection resulted in a changed frequency of specific mutations.MethodsMycobacterium tuberculosis (strain Mtb72) rifamycin resistant mutants were selected in vitro with either rifampicin or rifabutin. One mutant R190 (rpoB S522L) selected with rifampicin had a rifampicin MIC of 32 μg/ml but remained sensitive to rifabutin (MIC<0.8 μg/ml). This mutant was subjected to a second round of selection with rifabutin.ResultsAll 105 first round resistant mutants derived from the parent strain (Mtb72) screened acquired mutations within the 81 bp rpoB hotspot. When the rifampicin resistant but rifabutin sensitive S522L mutant was subjected to a second round of selection, single additional rpoB mutations were identified in 24 (92%) of 26 second round mutants studied, but 14 (54%) of these strains contained mutations outside the 81 bp hotspot (codons 144, 146, 148, 505). Additionally, spontaneous rifabutin resistant mutants were produced at >10 times the frequency by the S522L mutant than the parent strain.ConclusionFirst round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.

Live attenuated mutants of Mycobacterium tuberculosis as candidate vaccines against tuberculosis

The recent advances in genetic tools to manipulate Mycobacterium tuberculosis have led to the construction of defined mutants and to the study of their role in the virulence and pathogenesis of tuberculosis. The safety and vaccine potential of a few of these M. tuberculosis mutants as candidate vaccines against tuberculosis are discussed.