Search PubMed⌕ Search

Biomedical subjects

Tanya Parish

Publications and source records attributed to Tanya Parish.

At least 19 recordsLinked to original sources
PubMed2006

Identification of the Mycobacterium tuberculosis GlnE promoter and its response to nitrogen availability.

Adenylyltransferase, GlnE, has a predicted role in controlling the enzymic activity of glutamine synthetase, the key enzyme in ammonia assimilation. It was previously demonstrated that glnE is an essential gene in Mycobacterium tuberculosis. glnE is located downstream of glnA2, one of four glutamine synthetases. The expression of GlnE under various conditions was determined. Although a co-transcript of glnA2 and glnE was detectable, the major transcript was monocistronic. A transcriptional start site immediately upstream of glnE was identified and it was shown by site-directed mutagenesis that the predicted -10 region is a functional promoter. It was demonstrated that in a Mycobacterium smegmatis background M. tuberculosis P(glnE) was up-regulated in ammonia- or glutamine-containing media.

Gene Expression Regulation, Bacterial↗
PubMed2005

Instability of the acetamide-inducible expression vector pJAM2 in Mycobacterium tuberculosis.

The Escherichia coli-mycobacterium shuttle vector pJAM2 has been used to inducibly express genes in mycobacteria. The vector carries the promoter region from the highly inducible acetamidase gene of Mycobacterium smegmatis which is used to drive expression of heterologous genes. We used pJAM2 to over-express the Mycobacterium tuberculosis gene Rv2868c, a homologue of gcpE. In M. smegmatis the plasmid was stable, but the promoter region was readily deleted when the parental vector or recombinant plasmids were transformed into M. tuberculosis. We mapped the deletion by sequencing and found that it encompassed the entire acetamidase promoter and adjacent sequence totalling approximately 7.3 kb and occurred very soon after introduction into M. tuberculosis. This is the first report of instability of a vector carrying the acetamidase promoter in M. tuberculosis.

Amidohydrolases↗
PubMed2005

Evaluation of vaccines in the EU TB Vaccine Cluster using a guinea pig aerosol infection model of tuberculosis.

The TB Vaccine Cluster project funded by the EU Fifth Framework programme aims to provide novel vaccines against tuberculosis that are suitable for evaluation in humans. This paper describes the studies of the protective efficacy of vaccines in a guinea pig aerosol-infection model of primary tuberculosis. The objective was to conduct comparative evaluations of vaccines that had previously demonstrated efficacy in other animal models. Groups of 6 guinea pigs were immunized with vaccines provided by the relevant EU Vaccine Cluster partners. Survival over 17 or 26 weeks was used as the principal measure of vaccine efficacy following aerosol challenge with H37Rv. Counts of mycobacteria in lungs and spleens, and histopathological changes in the lungs, were also used to provide evidence of protection. A total of 24 vaccines were evaluated in 4 experiments each of a different design. A heterologous prime-boost strategy of DNA and MVA, each expressing Ag85A and a fusion protein of ESAT-6 and Ag85B in adjuvant, protected the guinea pigs to the same extent as BCG. Genetically modified BCG vaccines and boosted BCG strategies also protected guinea pigs to the same extent as BCG but not statistically significantly better. A relatively high aerosol-challenge dose and evaluation over a protracted time post-challenge allowed superior protection over BCG to be demonstrated by BCG boosted with MVA and fowl pox vectors expressing Ag85A.

Aerosols↗
PubMed2005

HemZ is essential for heme biosynthesis in Mycobacterium tuberculosis.

The complete sequence and subsequent annotation of the Mycobacterium tuberculosis genome has allowed the prediction of many genes and gene functions by homology. HemZ is a predicted ferrochelatase which lies in an apparent operon with two genes involved in mycolic acid biosynthesis, mabA and inhA. We tried to construct hemZ deletion mutants in M. tuberculosis using a two-step recombination strategy, but could only delete the chromosomal copy when we provided a second functional copy on an integrating plasmid. We further confirmed that hemZ is essential under normal culture conditions by demonstrating that the integrated copy of hemZ could not be removed if it was the only wild-type allele in the cell. We were able to obtain hemZ mutants by supplementation with hemin but not with protoporphyrin IX or hemoglobin confirming that this gene does have a role in heme biosynthesis and that M. tuberculosis can transport hemin intracelullarly. The hemin auxotroph required 2 mug/ml hemin for growth and rapid loss of viability occurred after withdrawal of hemin. These data confirm the role of hemZ in heme biosynthesis and indicate that heme is an essential requirement for M. tuberculosis.

Bacterial Proteins↗
PubMed2005

Use of a tetracycline-inducible system for conditional expression in Mycobacterium tuberculosis and Mycobacterium smegmatis.

A number of essential genes have been identified in mycobacteria, but methods to study these genes have not been developed, leaving us unable to determine the function or biology of the genes. We investigated the use of a tetracycline-inducible expression system in Mycobacterium tuberculosis and Mycobacterium smegmatis. Using a reporter gene which encodes an unstable variant of GFP, we showed that tetracycline-inducible expression occurred in M. smegmatis and that expression levels were titratable to some extent by varying the concentration of tetracycline. The removal of tetracycline led to cessation of GFP expression, and we showed that this was a controllable on/off switch for fluorescence upon addition and removal of the antibiotic inducer. The system also functioned in M. tuberculosis, giving inducible expression of the reporter gene. We used homologous recombination to construct a strain of M. tuberculosis that expressed the only copy of the tryptophan biosynthetic enzyme, TrpD, from the tetracycline-inducible promoter. This strain was conditionally auxotrophic, showing auxotrophy only in the absence of tetracycline, confirming that trpD was tightly controlled by the foreign promoter. This is the first demonstration of the use of an inducible promoter to generate a conditional auxotroph of M. tuberculosis. The ability to tightly regulate genes now gives us the possibility to define the functions of essential genes by switching them off under defined conditions and paves the way for in vivo studies.

Anti-Bacterial Agents↗
PubMed2004

Functional demonstration of reverse transsulfuration in the Mycobacterium tuberculosis complex reveals that methionine is the preferred sulfur source for pathogenic Mycobacteria.

Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of (35)S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine gamma-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine gamma-synthase (CGS), but demonstration of a gamma-elimination reaction with cystathionine as well as the gamma-replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M. tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine.

Alkynes↗
PubMed2004

Arylamine N-acetyltransferase is required for synthesis of mycolic acids and complex lipids in Mycobacterium bovis BCG and represents a novel drug target.

Mycolic acids represent a major component of the unique cell wall of mycobacteria. Mycolic acid biosynthesis is inhibited by isoniazid, a key frontline antitubercular drug that is inactivated by mycobacterial and human arylamine N-acetyltransferase (NAT). We show that an in-frame deletion of Mycobacterium bovis BCG nat results in delayed entry into log phase, altered morphology, altered cell wall lipid composition, and increased intracellular killing by macrophages. In particular, deletion of nat perturbs biosynthesis of mycolic acids and their derivatives and increases susceptibility of M. bovis BCG to antibiotics that permeate the cell wall. Phenotypic traits are fully complemented by introduction of Mycobacterium tuberculosis nat. We infer from our findings that NAT is critical to normal mycolic acid synthesis and hence other derivative cell wall components and represents a novel target for antituberculosis therapy. In addition, this is the first report of an endogenous role for NAT in mycobacteria.

Anti-Bacterial Agents↗
PubMed2004

Gene expression profile of Mycobacterium tuberculosis in a non-replicating state.

Mycobacterium tuberculosis is able to persist in the human host for decades in an apparently dormant state where it is presumed to reside in an hypoxic environment. This can be mimicked by the Wayne culture model in which progressive oxygen depletion causes the bacteria to shift into a non-replicating state. We investigated global gene expression in aerobic (roller), microaerophilic (NRP1) and anaerobic (NRP2) cultures. A number of genes were significantly up-regulated as compared to aerobic culture; 178 in NRP1, 210 in NRP2, 88 in both. The two states showed distinct gene expression profiles, although a number of membrane and transmembrane proteins were induced in both conditions. A number of regulatory proteins were up-regulated in NRP2. Glycine dehydrogenase, nitrate reductase and alpha-crystallin were induced in both stages, as were fatty acid metabolism genes including fadD26 and mas and genes of the DosR regulon. In a comparison with other stress conditions, there were more similarities between anaerobic conditions and carbon starvation or heat shock than between microaerophilic conditions and carbon starvation or heat shock, but as expected microaerophilic and anaerobic conditions showed the most similar profile. Our results indicate that a large number of genes are up-regulated during the shift into the persistent state.

Aerobiosis↗
PubMed2004

The molecular biology of recombination in Mycobacteria: what do we know and how can we use it?

Recombination is a ubiquitous genetic process which results in the exchange of DNA between two substrates. Homologous recombination occurs between DNA species with identical sequence whereas illegitimate recombination can occur between DNA with very little or no homology. Site-specific recombination is often used by temperate phages to stably integrate into bacterial chromosomes. Characterisation of the mechanisms of recombination in mycobacteria has mainly focussed on RecA-dependent homologous recombination and phage-directed site-specific recombination. In contrast the high frequency of illegitimate recombination in slow-growing mycobacteria has not been explained. The role of DNA repair in dormancy and infection have not yet been fully established, but early work suggests that RecA-mediated pathways are not required for virulence. All three recombination mechanisms have been utilised in developing genetic techniques for the analysis of the biology and pathogenesis of mycobacteria. A recently developed method for studying essential genes will generate further insights into the biology of these important organisms.

DNA, Recombinant↗
PubMed2003

Efficient switching of mycobacteriophage L5-based integrating plasmids in Mycobacterium tuberculosis.

We previously used a mycobacteriophage L5-derived integrating vector to demonstrate that glnE and aroK are essential genes in Mycobacterium tuberculosis by showing that we were unable to excise the integrated vector when it carried the only functional copy of these genes. We tested three systems to replace the integrated copy with alternative alleles. The most efficient method was to transform the strain with a second copy of the integrating vector. Excision of the resident vector and integration of the incoming vector occurred at an extremely high efficiency. This technique will allow us to study the role and functionality of essential genes in this important human pathogen.

Alleles↗
PubMed2003

Control of the acetamidase gene of Mycobacterium smegmatis by multiple regulators.

The acetamidase of Mycobacterium smegmatis is an inducible enzyme which enables the organism to utilise several amides as sole carbon sources. The acetamidase structural gene (amiE) is located downstream of four other genes, of which three form a probable operon with amiE; the fourth (amiC) is divergently transcribed. We constructed deletion mutants in two of these genes in order to determine their role in acetamidase expression. Both AmiC and AmiD were shown to be positive regulators of acetamidase expression required for induction. Combinations of regulatory gene deletions were made which revealed that AmiC interacts with the previously characterised negative regulator AmiA, whereas AmiD does not.

Amidohydrolases↗
PubMed2003

Analysis of whole-genome microarray replicates using mixed models.

MOTIVATION: Microarray experiments are inherently noisy. Replication is the key to estimating realistic fold-changes despite such noise. In the analysis of the various sources of noise the dependency structure of the replication needs to be taken into account. RESULTS: We analyzed replicate data sets from a Mycobacterium tuberculosis trcS mutant in order to identify differentially expressed genes and suggest new methods for filtering and normalizing raw array data and for imputing missing values. Mixed ANOVA models are applied to quantify the various sources of error. Such analysis also allows us to determine the optimal number of samples and arrays. Significance values for differential expression are obtained by a hierarchical bootstrapping scheme on scaled residuals. Four highly upregulated genes, including bfrB, were analyzed further. We observed an artefact, where transcriptional readthrough from these genes led to apparent upregulation of adjacent genes. AVAILABILITY: All methods and data discussed are available in the package YASMAhttp://www.cryst.bbk.ac.uk/wernisch/yasma.html for the statistical data analysis system R (http://www.R-project.org).

Algorithms↗
PubMed2003

The senX3-regX3 two-component regulatory system of Mycobacterium tuberculosis is required for virulence.

Two-component regulatory systems have been widely implicated in bacterial virulence. To investigate the role of one such system in Mycobacterium tuberculosis, a strain was constructed in which the senX3-regX3 system was deleted by homologous recombination. The mutant strain (Tame15) showed a growth defect after infection of macrophages and was attenuated in both immunodeficient and immunocompetent mice. Competitive hybridization of total RNA from the wild-type and mutant strains to a whole-genome microarray was used to identify changes in gene expression resulting from the deletion. One operon was highly up-regulated in the mutant, indicating that regX3 probably has a role as a repressor of this operon. Other genes which were up- or down-regulated were also identified. Many of the genes showing down-regulation are involved in normal growth of the bacterium, indicating that the mutant strain is subject to some type of growth slow-down or stress. Genes showing differential expression were further grouped according to their pattern of gene expression under other stress conditions. From this analysis 50 genes were identified which are the most likely to be controlled by RegX3. Most of these genes are of unknown function and no obvious motifs were found upstream of the genes identified. Thus, it has been demonstrated that the senX3-regX3 two-component system is involved in the virulence of M. tuberculosis and a number of genes controlled by this system have been identified.

Animals↗
PubMed2003

Gene replacement in mycobacteria by using incompatible plasmids.

A simple and efficient delivery system was developed for making targeted gene knockouts in Mycobacterium smegmatis. This delivery system relies on the use of a pair of replicating plasmids, which are incompatible. Incompatible plasmids share elements of the same replication machinery and so compete with each other during both replication and partitioning into daughter cells. Such plasmids can be maintained together in the presence of antibiotics; however, removal of selection leads to the loss of one or both plasmids. For mutagenesis, two replicating plasmids based on pAL5000 are introduced; one of these plasmids carries a mutated allele of the targeted gene. Homologous recombination is allowed to take place, and either one or both of the vectors are lost through the pressure of incompatibility, allowing the phenotypic effects of the mutant to be studied. Several different plasmid combinations were tested to optimize loss in the absence of antibiotic selection. pAL5000 carries two replication genes (repA and repB), which act in trans, and the use of vectors that each lack one rep gene and complement each other resulted in the loss of both plasmids in M. smegmatis and Mycobacterium bovis BCG. The rate of loss was increased by the incorporation of an additional incompatibility region in one of the plasmids. To facilitate cloning when the system was used, we constructed plasmid vector pairs that allow simple addition of selection and screening genes on flexible gene cassettes. Using this system, we demonstrated that M. smegmatis pyrF mutants could be isolated at high frequency. This method should also be useful in other species in which pAL5000 replicates, including Mycobacterium tuberculosis.

Bacterial Proteins↗
PubMed2003

Deletion of two-component regulatory systems increases the virulence of Mycobacterium tuberculosis.

Two-component regulatory signal transduction systems are widely distributed among bacteria and enable the organisms to make coordinated changes in gene expression in response to a variety of environmental stimuli. The genome sequence of Mycobacterium tuberculosis contains 11 complete two-component systems, four isolated homologous regulators, and three isolated homologous sensors. We have constructed defined mutations in six of these genes and measured virulence in a SCID mouse model. Mice infected with four of the mutants (deletions of devR, tcrXY, trcS, and kdpDE) died more rapidly than those infected with wild-type bacteria. The other two mutants (narL and Rv3220c) showed no change compared to the wild-type H37Rv strain. The most hypervirulent mutant (devRdelta) also grew more rapidly in the acute stage of infection in immunocompetent mice and in gamma interferon-activated macrophages. These results define a novel class of genes in this pathogen whose presence slows down its multiplication in vivo or increases its susceptibility to host killing mechanisms. Thus, M. tuberculosis actively maintains a balance between its own survival and that of the host.

Animals↗
PubMed2003

Starvation survival response of Mycobacterium tuberculosis.

The ability of Mycobacterium tuberculosis auxotrophs to survive long-term starvation was measured. Tryptophan and histidine auxotrophs did not survive single-amino-acid starvation, whereas a proline auxotroph did. All three auxotrophs survived complete starvation. THP-1 cells were also able to restrict the growth of the tryptophan and histidine auxotrophs.

Colony Count, Microbial↗
PubMed2002

The common aromatic amino acid biosynthesis pathway is essential in Mycobacterium tuberculosis.

Attempts to construct Mycobacterium tuberculosis strains with a defect in the common aromatic amino acid biosynthesis pathway were made. In other bacteria the genes of this pathway (aro) can be disrupted in the presence of suitable media supplements. The genomic organization of the aro genes in M. tuberculosis reveals that there is one operon (aroCKBQ) and three isolated aro genes (aroE, aroG and aroA). The aroK gene was chosen as a target for disruption; this encodes shikimate kinase, which catalyses the fifth step in chorismate biosynthesis. Attempts to replace the wild-type aroK gene with a disrupted allele (aroKDelta::hyg) by a two-step homologous recombination procedure were unsuccessful in a wild-type strain. When a second functional copy of aroK was integrated into the chromosome, it was possible to isolate a strain carrying the disrupted gene. Excision of the L5-integrated copy of aroK by the L5 excisionase could be not be achieved in the strain carrying the disrupted copy, but was possible in a strain carrying a wild-type copy. These results demonstrate that the chorismate pathway is essential for the viability of M. tuberculosis.

Amino Acids, Aromatic↗