Biomedical subjects

Graham R Stewart

Publications and source records attributed to Graham R Stewart.

13 recordsLinked to original sources

PubMed2005

Deciphering the molecular bases of Mycobacterium tuberculosis binding to the lectin DC-SIGN reveals an underestimated complexity.

Interactions between dendritic cells and Mycobacterium tuberculosis, the aetiological agent of tuberculosis in humans, are thought to be central to anti-mycobacterial immunity. We have previously shown that M. tuberculosis binds to human monocyte-derived dendritic cells mostly through the C-type lectin DC-SIGN (dendritic-cell-specific intercellular molecule-3-grabbing non-integrin)/CD209, and we have suggested that DC-SIGN may discriminate between mycobacterial species through recognition of the mannose-capping residues on the lipoglycan lipoarabinomannan of the bacterial envelope. Here, using a variety of fast- and slow-growing Mycobacterium species, we provide further evidence that mycobacteria recognition by DC-SIGN may be restricted to species of the M. tuberculosis complex. Fine analyses of the lipoarabinomannan molecules purified from these species show that the structure and amount of these molecules alone cannot account for such a preferential recognition. We propose that M. tuberculosis recognition by DC-SIGN relies on both a potential difference of accessibility of lipoarabinomannan in its envelope and, more probably, on the binding of additional ligands, possibly including lipomannan, mannose-capped arabinomannan, as well as the mannosylated 19 kDa and 45 kDa [Apa (alanine/proline-rich antigen)] glycoproteins. Altogether, our results reveal that the molecular basis of M. tuberculosis binding to DC-SIGN is more complicated than previously thought and provides further insight into the mechanisms of M. tuberculosis recognition by the immune system.

Bacterial Adhesion↗

PubMed2005

Infection biology of a novel alpha-crystallin of Mycobacterium tuberculosis: Acr2.

Heat shock proteins assist the survival of Mycobacterium tuberculosis (MTB) but also provide a signal to the immune response. The gene most strongly induced by heat shock in MTB is Rv0251c, which encodes Acr2, a novel member of the alpha-crystallin family of molecular chaperones. The expression of acr2 increased within 1 h after infection of monocytes or macrophages, reaching a peak of 18- to 55-fold by 24 h. Inhibition of superoxide action reduced the intracellular increase in acr2. Despite this contribution to the stress response of MTB, the gene for acr2 appears dispensable; a deletion mutant (Deltaacr2) was unimpaired in log phase growth and persisted in IFN-gamma-activated human macrophages. Acr2 protein was strongly recognized by cattle with early primary Mycobacterium bovis infection and by healthy MTB-sensitized people. Within the latter group, those with recent exposure to infectious tuberculosis had, on average, 2.6 times the frequency of Acr2-specific IFN-gamma-secreting T cells than those with more remote exposure (p = 0.009). These data show that, by its up-regulation early after entry to cells, Acr2 gives away the presence of MTB to the immune response. The demonstration that there is infection stage-specific immunity to tuberculosis has implications for vaccine design.

Animals↗

PubMed2005

The OtsAB pathway is essential for trehalose biosynthesis in Mycobacterium tuberculosis.

The disaccharide trehalose is the major free sugar in the cytoplasm of mycobacteria; it is a constituent of cell wall glycolipids, and it plays a role in mycolic acid transport during cell wall biogenesis. The pleiotropic role of trehalose in the biology of Mycobacterium tuberculosis and its absence from mammalian cells suggests that its biosynthesis may provide a useful target for novel drugs. However, there are three potential pathways for trehalose biosynthesis in M. tuberculosis, and the aim of the present study was to introduce mutations into each of the pathways to determine whether or not they are functionally redundant. The results show that the OtsAB pathway, which generates trehalose from glucose and glucose-6-phosphate, is the dominant pathway required for M. tuberculosis growth in laboratory culture and for virulence in a mouse model. Of the two otsB homologues annotated in the genome sequence of M. tuberculosis, only OtsB2 (Rv3372) has a functional role in the pathway. OtsB2, trehalose-6-phosphate phosphatase, is strictly essential for growth and provides a tractable target for high throughput screening. Inactivation of the TreYZ pathway, which can generate trehalose from alpha-1,4-linked glucose polymers, had no effect on the growth of M. tuberculosis in vitro or in mice. Deletion of the treS gene altered the late stages of pathogenesis of M. tuberculosis in mice, significantly increasing the time to death in a chronic infection model. Because the TreS enzyme catalyzes the interconversion of trehalose and maltose, the mouse phenotype could reflect either a requirement for synthesis of additional trehalose or, conversely, a requirement for breakdown of stored trehalose to liberate free glucose.

Animals↗

PubMed2005

Tetracycline-inducible gene regulation in mycobacteria.

A system for the tetracycline-inducible regulation of gene expression in mycobacteria has been developed. We have sub-cloned the tetRO region from the Corynebacterium glutamicum TetZ locus into a mycobacterial shuttle plasmid, making expression of genes cloned downstream of tetRO responsive to tetracycline. Using the luxAB-encoded luciferase from Vibrio harveyi as a reporter (pMind-Lx), we observed a 40-fold increase in light output from Mycobacterium smegmatis cultures 2 h after adding 20 ng ml(-1) of tetracycline. Similarly, exposure to the drug resulted in up to 20-fold increase in relative light units from M.bovis BCG carrying the reporter construct, and a 10-fold increase for M.tuberculosis. Tetracycline induction was demonstrated in log and stationary phase cultures. To evaluate whether this system is amenable to use in vivo, J774 macrophages were infected with M.bovis BCG[pMind-Lx], treated with amikacin to kill extracellular bacteria, and then incubated with tetracycline. A 10-fold increase in light output was measured after 24 h, indicating that intracellular bacteria are accessible and responsive to exogenously added tetracycline. To test the use of the tetracycline-inducible system for conditional gene silencing, mycobacteria were transformed with a pMind construct with tetRO driving expression of antisense RNA for the ftsZ gene. Bacterial cells containing the antisense construct formed filaments after 24 h exposure to tetracycline. These results demonstrate the potential of this tetracycline-regulated system for the manipulation of mycobacterial gene expression inside and outside cells.

Animals↗

PubMed2005

The stress-responsive chaperone alpha-crystallin 2 is required for pathogenesis of Mycobacterium tuberculosis.

Mycobacterium tuberculosis has two members of the alpha-crystallin (Acr) family of molecular chaperones. Expression of Acr1 is induced by exposure to hypoxia or nitric oxide and is associated with bacterial persistence in a non-replicating state. Expression of Acr2 is induced by heat shock, oxidative stress, and uptake by macrophages. We have shown that Acr2 continues to be expressed at a high level during both acute and chronic infection in the mouse model, with an increased ratio of acr2:acr1 mRNA in the persistent phase. Deletion of the acr2 gene resulted in a decrease in the resistance of M. tuberculosis to oxidative stress but did not impair growth in mouse bone marrow macrophages. There was no difference in bacterial load in mice infected with an acr2 deletion mutant, but a marked alteration in disease progression was evident from reduced weight loss over a prolonged infection. This correlated with reduced recruitment of T-cells and macrophages to the lungs of mice infected with the acr2 mutant and reduced immune-related pathology. These findings demonstrate that both alpha-crystallins contribute to persistent infection with M. tuberculosis and suggest that manipulation of acr expression can influence the host response to infection.

Animals↗

PubMed2004

Induction of apoptosis and release of interleukin-1 beta by cell wall-associated 19-kDa lipoprotein during the course of mycobacterial infection.

Mycobacterium tuberculosis induces apoptosis in human monocyte-derived macrophages (MDMs) during the early stages of infection. We investigated the proapoptotic role of cell wall-associated mycobacterial 19-kDa lipoprotein and the possible association between 19-kDa lipoprotein signaling and production of proinflammatory cytokines. Purified mycobacterial 19-kDa lipoprotein, 19-kDa lipoprotein-expressing M. smegmatis (M. smegmatis 19+), 19-kDa lipoprotein knockout (KO) M. tuberculosis, and 19-kDa lipoprotein KO M. bovis bacille Calmette-Guerin (BCG) strains were analyzed for their ability to induce apoptosis in MDMs. The 19-kDa lipoprotein and infection with M. smegmatis 19+ induced apoptosis in MDMs. M. tuberculosis and BCG KO strains had significantly decreased abilities to induce apoptosis. The 19-kDa lipoprotein proapoptotic signal was mediated by Toll-like receptor 2 but not by tumor necrosis factor-alpha. Only the release of interleukin (IL)-1 beta was decreased after infection with 19-kDa lipoprotein KO strains. These findings indicate that the 19-kDa lipoprotein is the main signal required to trigger both apoptosis and the release of IL-1 beta during the early stages of mycobacterial infection.

Annexin A5↗

PubMed2004

Heat-shock proteins and the host-pathogen interaction during bacterial infection.

Heat-shock proteins (HSPs) are expressed at high levels by bacterial pathogens during adaptation to intracellular survival. Both host and pathogen heat-shock proteins contribute to immunity by receptor-mediated activation of the innate immune response and by participation in the presentation of antigens for the adaptive immune response. Manipulation of these interactions presents a potential route to improved control of infection by vaccination or immunotherapy.

Animals↗

PubMed2004

Analysis of the function of mycobacterial DnaJ proteins by overexpression and microarray profiling.

Regulation of expression of the Hsp70/DnaK chaperone plays an important role during infection with Mycobacterium tuberculosis. We have examined the effect of manipulating the level of expression of DnaJ, one of the components of the chaperone apparatus. Overexpression of DnaJ1 resulted in elevated transcription of both the hsp70/dnaK and hsp60/groE chaperone genes, consistent with an increase in the cellular content of nascent and unfolded peptide substrates. There was also an increase in transcription of genes flanking the origin of chromosomal replication, suggesting an important role for DnaJ1 in controlling interaction of the Hsp70 chaperone with the DnaA protein. Overexpression of DnaJ2 had no detectable effect on transcription of other genes. Overexpression in combination with microarray profiling provides a complementary approach to gene deletion for exploring the function of essential genes in M. tuberculosis.

Amino Acid Sequence↗

PubMed2004

Visualization of microarray results to assist interpretation.

Whole genome microarrays allow assessment of the profile of genes expressed under particular experimental conditions, including external stimuli such as pH or temperature, and internal changes brought about by deleting or over-expressing a gene. Such experiments produce large data sets, for which sophisticated analysis software is available. What is lacking are tools for analysing data sets from different experiments, in order to test and generate hypotheses about the links between regulatory networks. We describe here a method for presenting results from different experiments as a directed graph constructed using an automated graph drawing program xneato, enhanced by a logic program designed to cluster data and aid in the generation of hypotheses about possible gene interactions. A web-based front-end to the system has been constructed to explore and manipulate the graphical displays produced. Results of microarray experiments on Mycobacterium tuberculosis were used to develop and evaluate the visualization tool and initiate the development of an inference system for gene interactions based on such data. The GeneGraph project can be accessed at: zebrafish.doc.ic.ac.uk

Computer Graphics↗

PubMed2003

Tuberculosis: a problem with persistence.

Mycobacterium tuberculosis is one of most successful pathogens of mankind, infecting one-third of the global population and claiming two million lives every year. The ability of the bacteria to persist in the form of a long-term asymptomatic infection, referred to as latent tuberculosis, is central to the biology of the disease. The persistence of bacteria in superficially normal tissue was recognized soon after the discovery of the tubercle bacillus, and much of our knowledge about persistent populations of M. tuberculosis dates back to the first half of the last century. Recent advances in microbial genetics and host immunity provide an opportunity for renewed investigation of this persistent threat to human health.

Animals↗

PubMed2002

Transposition of Tn4560 of Streptomyces fradiae in Mycobacterium smegmatis.

Tn4560 (8.6 kb) was derived from Tn4556, a Tn3-like element from Streptomyces fradiae. It contains a viomycin resistance gene that has not been used previously for selection in mycobacteria. Tn4560, cloned in a Streptomyces plasmid, was introduced by electroporation into Mycobacterium smegmatis mc(2)155. Tn4560 transposed into the host genome: there was no obvious target sequence preference, and insertions were in or near several conserved open reading frames. The insertions were located far apart on different AseI macrorestriction fragments. Unexpectedly, the transposon delivery plasmid, pUC1169, derived from the Streptomyces multicopy plasmid pIJ101, replicated partially in M. smegmatis, but was lost spontaneously during subculture. Replication of pUC1169 probably contributed to the relatively high efficiency of Tn4560 delivery: up to 28% of the potential M. smegmatis transformants acquired a stable transposon insertion. The data indicated that Tn4560 may be useful for random mutagenesis of M. smegmatis.

Antibiotics, Antitubercular↗

PubMed2002

Tuberculosis vaccines.

The increasing incidence of disease associated with HIV infection highlights the crucial role of the immune response in susceptibility to tuberculosis and has stimulated renewed efforts to develop improved vaccines. Vaccine targets include prevention of infection in naive individuals, prevention of re-activation in individuals harbouring latent infection, and prevention of relapse by immunotherapy in tuberculosis patients. Advances in mycobacterial molecular genetics have facilitated development of a range of live attenuated and subunit vaccine candidates that have been screened in experimental models of infection. Evaluation of the immunogenicity of selected candidate vaccines in clinical trials should be combined with a continuation of fundamental research on the immune response to mycobacterial infection and persistence.

AIDS-Related Opportunistic Infections↗

PubMed2002

Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays.

Regulation of the expression of heat-shock proteins plays an important role in the pathogenesis of Mycobacterium tuberculosis. The heat-shock response of bacteria involves genome-wide changes in gene expression. A combination of targeted mutagenesis and whole-genome expression profiling was used to characterize transcription factors responsible for control of genes encoding the major heat-shock proteins of M. tuberculosis. Two heat-shock regulons were identified. HspR acts as a transcriptional repressor for the members of the Hsp70 (DnaK) regulon, and HrcA similarly regulates the Hsp60 (GroE) response. These two specific repressor circuits overlap with broader transcriptional changes mediated by alternative sigma factors during exposure to high temperatures. Several previously undescribed heat-shock genes were identified as members of the HspR and HrcA regulons. A novel HspR-controlled operon encodes a member of the low-molecular-mass alpha-crystallin family. This protein is one of the most prominent features of the M. tuberculosis heat-shock response and is related to a major antigen induced in response to anaerobic stress.

Bacterial Proteins↗