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M J Colston

Publications and source records attributed to M J Colston.

At least 37 records · Page 2Linked to original sources
PubMed2000

Crystallization and preliminary X-ray diffraction studies on the N-utilizing substance-B (NusB) from Mycobacterium tuberculosis.

N-utilizing substance B (NusB) is a protein which forms part of a complex assembly in transcriptional antitermination in Mycobacterium tuberculosis. It forms a heterodimer with the product of the NusE gene (identical to the ribosomal protein S10) and mediates the process of transcriptional antitermination by forming the core complex with the nut site of the ribosomal RNA along with other protein factors. NusB has been cloned and overexpressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. The space group is P2(1)2(1)2(1), with unit-cell parameters a = 46.6, b = 64.2, c = 90.1 A. A native data set complete to 1.6 A resolution has been collected from a single crystal.

Amino Acid Sequence↗
PubMed2000

In vivo splicing and functional characterization of Mycobacterium leprae RecA.

The RecA proteins from Mycobacterium tuberculosis and Mycobacterium leprae contain inteins. In contrast to the M. tuberculosis RecA, the M. leprae RecA is not spliced in Escherichia coli. We demonstrate here that M. leprae RecA is functionally spliced in Mycobacterium smegmatis and produces resistance toward DNA-damaging agents and homologous recombination.

Mycobacterium leprae↗
PubMed2000

Dysregulation of IL-4 expression in lepromatous leprosy patients with and without erythema nodosum leprosum.

In order to increase our understanding of the immunological basis of erythema nodosum leprosum (ENL), we studied Th-like cytokine profiles in 130 leprosy patients, employing both the conventional and a novel, real-time, fluorogenic reverse transcriptase-based PCR (RT-PCR). The concomitant expression of both Th-like cytokines, interferon-gamma and IL-4, and the regulatory cytokines, IL-10 and IL-12, was studied in the peripheral blood cells of leprosy patients with and without ENL. In the conventional RT-PCR, varied cytokine profiles were observed in individual patients of all clinical types. Fifty-three percent of lepromatous patients without ENL and 59% of tuberculoid leprosy patients showed co-expression of IFN gamma and IL-4, indicating a non-polarized Th 0 pattern. Of the 36 patients with ENL, 58% demonstrated a polarized Th 1 pattern, with only 30% expressing both cytokines. Semiquantitative RT-PCR indicated a lower expression of IL-4 compared to that of IFN gamma in the lepromatous patients without ENL; the difference was even greater among those with ENL. The sensitive, real-time PCR confirmed the down-regulation of IL-4 and IL-10, with absence of IL-4 in half of the patients, resulting in skewing of the cytokine response toward a Th 1-like profile.

Cytokines↗
PubMed1999

Therapy of tuberculosis in mice by DNA vaccination.

Mycobacterium tuberculosis continues to kill about 3 million people every year, more than any other single infectious agent. This is attributed primarily to an inadequate immune response towards infecting bacteria, which suffer growth inhibition rather than death and subsequently multiply catastrophically. Although the bacillus Calmette-Guerin (BCG) vaccine is widely used, it has major limitations as a preventative measure. In addition, effective treatment requires that patients take large doses of antibacterial drug combinations for at least 6 months after diagnosis, which is difficult to achieve in many parts of the world and is further restricted by the emergence of multidrug-resistant strains of M. tuberculosis. In these circumstances, immunotherapy to boost the efficiency of the immune system in infected patients could be a valuable adjunct to antibacterial chemotherapy. Here we show in mice that DNA vaccines, initially designed to prevent infection, can also have a pronounced therapeutic action. In heavily infected mice, DNA vaccinations can switch the immune response from one that is relatively inefficient and gives bacterial stasis to one that kills bacteria. Application of such immunotherapy in conjunction with conventional chemotherapeutic antibacterial drugs might result in faster or more certain cure of the disease in humans.

Animals↗
PubMed1999

Genomic analysis reveals variation between Mycobacterium tuberculosis H37Rv and the attenuated M. tuberculosis H37Ra strain.

Mycobacterium tuberculosis H37Ra is an attenuated tubercle bacillus closely related to the virulent type strain M. tuberculosis H37Rv. Despite extensive study, the reason for the decreased virulence of M. tuberculosis H37Ra has not been determined. A genomic approach was therefore initiated to identify genetic differences between M. tuberculosis H37Rv and M. tuberculosis H37Ra as a means of pinpointing the attenuating mutation(s). Digestion with the rare-cutting restriction endonuclease DraI revealed two polymorphisms between the strains: a 480-kb fragment in M. tuberculosis H37Rv was replaced by two fragments of 220 and 260 kb in M. tuberculosis H37Ra, while there was a approximately 7.9-kb DraI fragment in M. tuberculosis H37Ra that had no counterpart in M. tuberculosis H37Rv. As the M. tuberculosis insertion sequence IS6110 contains a single DraI restriction site, it was considered possible that these polymorphisms were the result of IS6110 transposition events in M. tuberculosis H37Ra, events that may have inactivated virulence genes. The 7.9-kb polymorphism was found to be due to the presence of the previously described H37Rv RvD2 deletion in M. tuberculosis H37Ra, with sequence analysis suggesting an IS6110-mediated deletion mechanism for loss of RvD2. Three other IS6110-catalyzed deletions from the M. tuberculosis H37Rv chromosome (RvD3 to RvD5) were also identified, suggesting that this mechanism plays an important role in genome plasticity in the tubercle bacilli. Comparative mapping and sequencing revealed that the 480-kb polymorphism was due to an IS6110 insertion in M. tuberculosis H37Ra near oriC. Complementation of M. tuberculosis H37Ra with a 2.9-kb restriction fragment from M. tuberculosis H37Rv that encompassed the IS6110 insertion did not increase the survival of recombinant M. tuberculosis H37Ra in mice. In conclusion, this study describes the presence and mechanisms of genomic variation between M. tuberculosis H37Ra and M. tuberculosis H37Rv, although the role that they play in the attenuation of M. tuberculosis H37Ra is unclear.

Animals↗
PubMed1999

Effects of growth conditions on expression of mycobacterial murA and tyrS genes and contributions of their transcripts to precursor rRNA synthesis.

All mycobacteria studied to date have an rRNA operon, designated rrnA, located downstream from a single copy of the murA gene, which encodes an enzyme (EC 2.5.1.7) important for peptidoglycan synthesis. The rrnA operon has a promoter, P1(A), located within the coding region of murA, near the 3' end. Samples of RNA were isolated from Mycobacterium tuberculosis at different stages of the growth cycle and from Mycobacterium smegmatis grown under different conditions. RNase protection assays were used to investigate transcripts of both murA and rrnA. Transcription of murA was found to continue into the 16S rRNA gene, as if murA and rrnA form a hybrid (protein coding-rRNA coding) operon. During the growth of M. tuberculosis, the hybrid operon contributed approximately 2% to total pre-rRNA. Analysis of M. smegmatis RNA revealed that the level of murA RNA depended on the growth rate and that the patterns of expression during the growth cycle were different for murA and rrnA. M. smegmatis has a second rRNA operon, rrnB, located downstream from a single copy of the tyrS gene, encoding tyrosyl-tRNA synthetase. Transcription of tyrS was found to continue into the 16S rRNA gene rrnB. The hybrid tyrS-rrnB operon contributed 0.2 to 0.6% to rrnB transcripts. The pattern of tyrS expression during the growth cycle matched the pattern of rrnB expression, reflecting the essential role of TyrS and rRNA in protein biosynthesis.

Alkyl and Aryl Transferases↗
PubMed1998

An immunodominant epitope from mycobacterial 65-kDa heat shock protein protects against pristane-induced arthritis.

Previous studies showed that mice with pristane-induced arthritis (PIA) and those protected from the disease by preimmunization with mycobacterial 65-kDa heat shock protein (hsp65) possess raised immune responses to hsp65. Additionally, T cells from hsp65-protected mice, but not from pristane-injected or normal mice, produced the Th2-associated cytokines IL-4, IL-5, and IL-10 in response to stimulation with hsp65. Here we demonstrate that the specificity of the immune response to hsp65 and related heat shock protein (hsps) differs between protected and PIA mice. T cells from hsp65-protected mice respond to the bacterial hsps tested but not to the mammalian homologue, hsp58. Similarly, they exhibit high serum titers of anti-hsp65 Abs, yet they have virtually undetectable levels of anti-hsp58 IgG. By contrast, both cellular and humoral immune responses are detectable to bacterial and mammalian hsps in mice with PIA. An immunodominant T cell epitope has been identified in hsp65-protected mice corresponding to amino acids 261-271 from hsp65. Immunization of mice, either before or after the induction of arthritis, with this bacterial peptide, but not its mammalian homologue, protects mice from the development of PIA, and protection is associated with the production of Th2-type cytokines. Other experiments revealed that T cells primed with bacterial 261-271 or the mammalian homologue do not cross-react at the proliferative or cytokine level. These results demonstrate that an hsp65 peptide-specific Th2 response confers protection from PIA but do not support the idea that protection is mediated by a cross-reaction with self hsp58 in the joints.

Animals↗
PubMed1998

Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function.

A recA deletion mutant of Mycobacterium smegmatis has been isolated by homologous recombination using a sacB counterselection strategy. Deletion of the recA gene from the chromosome was demonstrated by Southern hybridizations and by polymerase chain reaction (PCR). Western analysis using anti-RecA antibodies confirmed that the RecA protein was not made by the mutant strain. The recA deletion strain exhibited enhanced sensitivity to UV irradiation and failed to undergo homologous recombination. The results obtained from the recombination assays suggest that in wild-type M. smegmatis the majority of colonies arise from single cross-over homologous recombination events with only a very minor contribution from random integrations. The deficiencies in UV survival and recombination were complemented by introduction of the cloned M. smegmatis recA gene. Overexpression of RecA was found to be toxic in the absence of recX, which is found downstream of and co-transcribed with recA and is thus also affected by the deletion of recA. The M. smegmatis recA deletion strain was also complemented by the M. tuberculosis recA gene with or without its intein; most importantly, the frequency of double cross-over homologous recombination events was identical regardless of whether the M. tuberculosis recA gene contained or lacked the intein. Thus, the low frequency of homologous recombination observed in M. tuberculosis is not due to the presence of an intein-coding sequence in its recA gene per se.

Bacterial Proteins↗
PubMed1998

Protection against Mycobacterium tuberculosis infection by CD8+ T cells requires the production of gamma interferon.

The role of CD8 T cells in controlling Mycobacterium tuberculosis infections in mice was confirmed by comparing the levels of growth of the organism in control, major histocompatibility complex class II knockout, and athymic mice and by transferring T-cell populations into athymic mice. By using donor mice which were incapable of making gamma interferon (IFN-gamma), it was shown that IFN-gamma production was essential for CD8 cell mediation of protective immunity against M. tuberculosis.

Animals↗
PubMed1998

Differential gene expression in response to adjunctive recombinant human interleukin-2 immunotherapy in multidrug-resistant tuberculosis patients.

Administration of low-dose recombinant human interleukin 2 (rhuIL-2) in combination with multidrug chemotherapy to patients with multidrug-resistant tuberculosis (MDR TB) induces measurable changes in in vitro immune response parameters which are associated with changes in the clinical and bacteriologic status of the patients. To determine the molecular basis of these changes, we have used semiquantitative reverse transcriptase-initiated PCR (RT-PCR) and differential display technology. During rhuIL-2 treatment of MDR TB patients, decreased levels of gamma interferon (IFN-gamma) mRNA in peripheral blood mononuclear cells (PBMC) relative to baseline levels were observed. However, at the site of a delayed-type hypersensitivity (DTH) response to purified protein derivative of tuberculin (PPD), the expression of cellular IFN-gamma and IL-2 mRNAs was increased during rhuIL-2 therapy. Levels of other cytokine mRNAs were not significantly affected by rhuIL-2 administration. Using differential-display RT-PCR, we identified several genes expressed at the DTH skin test site which were up- or down-regulated during rhuIL-2 treatment. Cytochrome oxidase type I mRNA was increased in response to rhuIL-2 therapy relative to baseline levels, as was heterogeneous nuclear ribonuclear protein G mRNA. CD63, clathrin heavy chain, and beta-adaptin mRNAs, all of which encode proteins associated with the endocytic vacuolar pathway of cells, were also differentially regulated by rhuIL-2 administration. The differential effects of IL-2 were confirmed in vitro by using PBMC obtained from PPD-positive individuals stimulated with Mycobacterium tuberculosis and IL-2. The differential expression of genes may provide a surrogate marker for leukocyte activation at a mycobacterial antigen-specific response site and for the development of an enhanced antimicrobial response which may result in improved outcomes in MDR TB patients.

Adult↗
PubMed1998

Regulation of macrophage gene expression by Mycobacterium tuberculosis: down-regulation of mitochondrial cytochrome c oxidase.

We have investigated changes in gene expression in mouse peritoneal macrophages following infection with virulent Mycobacterium tuberculosis. Using differential-display reverse transcription-PCR (RT-PCR), we have identified a gene that was markedly down-regulated within 6 h of infection and remained so for the duration of the experiment (5 days). On sequencing, this gene was found to encode the murine cytochrome c oxidase subunit VIIc (COX VIIc). Down-regulation of COX VIIc during M. tuberculosis infection was confirmed by three independent techniques: limiting-dilution RT-PCR, RNase protection assay, and Northern analysis. Limiting-dilution RT-PCR and Northern analysis were also used to analyze the specificity of this regulation; heat-killed M. tuberculosis, Mycobacterium bovis BCG, and latex beads had no effect on expression of COX VIIc. Down-regulation of this enzyme was also confirmed by using adherent cells isolated from spleens of M. tuberculosis-infected mice. These ex vivo macrophages showed apoptotic features, suggesting a possible involvement of cytochrome c oxidase in the programmed cell death of the host cells.

Animals↗
PubMed1998

Roles of multiple promoters in transcription of ribosomal DNA: effects of growth conditions on precursor rRNA synthesis in mycobacteria.

The roles of multiple promoters in the synthesis of rRNA under different conditions of growth were investigated, using two mycobacterial species as model organisms. When Mycobacterium smegmatis was grown under optimal conditions, its two rRNA operons contributed equally, with two promoters, one from each operon, being responsible for most transcripts. In stationary-phase growth or balanced growth under carbon starvation conditions, one operon (rrnAf) dominated and its three promoters contributed more equally to the generation of transcripts. Mycobacterium tuberculosis has a single operon with two promoters, one of which generated 80% of transcripts, at all stages of growth. We infer that each promoter functions independently according to its intrinsic strength when cells are growing slowly so that one operon with three promoters is roughly equivalent to three operons with one promoter; at high growth rates, occlusion effects reduce the efficiency of multiple promoters to that of a single promoter.

Culture Media↗
PubMed1998

The cytokine response to bacille Calmette Guérin vaccination in South India.

SETTING: Evaluation of immune response after BCG vaccination in a population from South India where BCG vaccination was a failure. OBJECTIVE: To study the cell-mediated immune responses (CMI) in vitro by assessing skin test conversion, lymphocyte proliferation and cytokine patterns before and after BCG vaccination in 20 Mantoux negative subjects, and to compare this with that of 20 naturally Mantoux positive subjects. DESIGN: In vitro lymphocyte proliferation and cytokine responses to various mycobacterial antigens were studied in 12 subjects from each group. The cytokines interferon gamma (IFN-gamma), interleukin (IL)-4 and IL-10 were measured by both reverse transcriptase polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). RESULTS: All of those who were initially Mantoux negative but one subject converted to positivity following vaccination, confirming that BCG vaccination does cause skin test conversion. However, in vitro proliferative responses to phytohaemagglutinin (PHA), purified protein derivative (PPD) and Mycobacterium tuberculosis remained largely unaltered by vaccination. The production of IFN-gamma was significantly higher in PPD-positive individuals compared to the PPD-negative group, and BCG vaccination of the latter did not change the levels of IFN-gamma, IL-4 or IL-10. CONCLUSIONS: The finding that PPD-negative individuals did not produce IFN-gamma even following vaccination and skin test conversion suggests that BCG had little effect in driving the immune response towards a protective Th1 type.

Adolescent↗
PubMed1997

Regulation of macrophage gene expression following invasion by Mycobacterium tuberculosis.

INTRODUCTION: Mycobacteria are intracellular pathogens which survive and grow in host macrophages. M. tuberculosis bacilli enter the macrophage via binding to several distinct cell surface molecules. Following phagocytosis, sustained intracellular bacterial growth depends on the ability to avoid destruction by macrophage-mediated host defences such as lysosomal enzymes, reactive oxygen and the reactive nitrogen intermediates. We used differential display reverse transcription polymerase chain reaction (DD RT-PCR) to identify host genes which are regulated during infection and hence which might be involved in the host-parasite cross talk. RESULTS: Live M. tuberculosis (strain H37Rv) was used to infect Balb/c peritoneal murine macrophages. mRNA from infected and uninfected macrophages was isolated at different time intervals after phagocytosis and subjected to DD RT-PCR. Oligo dT12NV and random 10mer primers were used for PCR amplification of cDNA. Macrophage genes which appeared to be differently regulated during infection were subjected to further reamplification by PCR in order to clone and sequence them. The differential expression of the selected bands was further analysed by an RNA protection assay and a Northern blot. RESULTS: Several differentially regulated bands were identified. One band, of 158 bp, was down regulated after infection. Sequencing of this band revealed a high level of homology (95% identity) to mouse cytochrome c oxidase subunit VIIc. The downregulation was specific for live virulent Mtb, while live BCG, heat killed Mtb and latex beads-mediated phagocytosis did not affect the transcriptional level of this enzyme. CONCLUSIONS: The cytochrome oxidase enzyme complex of the inner mytochondrial membrane catalyzes the reaction between ferrocytochrome c and oxygen. The reaction is the terminal event in the electron transport scheme. Downregulation of cytochrome c oxidase subunit VIIc could interfere with: (1) the host apoptotic programme; or (2) the host respiratory burst.

Animals↗
PubMed1997

Protection of rats from adjuvant arthritis by immunization with naked DNA encoding for mycobacterial heat shock protein 65.

OBJECTIVE: To assess the feasibility of vaccination with naked DNA encoding for mycobacterial heat shock protein 65 (hsp65) in the modulation of experimental arthritis. METHODS: Adjuvant arthritis (AA) was induced in Lewis rats preimmunized, intramuscularly, with a plasmid encoding for hsp65 (pCMV3.65). Clinical scores were recorded for 3-4 weeks, and histologic and radiologic parameters were evaluated. Cellular and antibody reactivity to hsp65 was assessed. The expression of the hsp65 gene was investigated in injected muscles. RESULTS: The pCMV3.65-treated rats were significantly protected from disease development in comparison with the control groups. This finding correlated with the results of histologic and radiologic examinations of the involved joints. The message for hsp65 was detected in injected muscles. T cell proliferation and antibodies to this protein were found to be elevated in pCMV3.65-treated rats when compared with both the arthritic control (AA-induced) and the naive (did not receive adjuvant) animals. CONCLUSION: We have demonstrated for the first time that naked DNA delivery is feasible in controlling experimental autoimmune disease. Although the actual mechanism of protection has not been fully elucidated, this simple and versatile approach could represent a useful tool in dissecting basic autoimmune mechanisms.

Animals↗
PubMed1997

Protection against tuberculosis by a plasmid DNA vaccine.

Past attempts to use fractions of mycobacteria as an alternative to BCG have given disappointing results. The availability of cloned genes and suitable vectors has now opened a new avenue in which individual mycobacterial protein antigens are synthesised within transfected mammalian cells. In an ex vivo transfection approach with a retroviral vector we found that even a single antigen (hsp65) could evoke strong protection when expressed as a transgene and that expression of protection was largely a function of antigen specific cytotoxic T cells. We now find that intramuscular injection of plasmid DNA expressing the antigen from either a viral or a murine promoter can also give protection equivalent to Bacillus Calmette-Guérin (BCG). Plasmids expressing some other mycobacterial antigens, hsp70, 36 kDa and 6 kDa, are also effective, suggesting that this approach may lead to a new vaccine.

Animals↗