Genetic screening for the protective antigenic targets of BCG vaccination.

Bovine tuberculosis is an important animal health problem and the predominant cause of zoonotic tuberculosis worldwide. It results in serious economic burden due to losses in productivity and the cost of control programmes. Control could be greatly improved by the introduction of an efficacious cattle vaccine but the most likely candidate, BCG, has several limitations including variable efficacy. Augmentation of BCG with a subunit vaccine booster has been shown to increase protection but the selection of antigens has hitherto been left largely to serendipity. In the present study, we take a rational approach to identify the protective antigens of BCG, selecting a BCG transposon mutant library in naïve and BCG-vaccinated cattle. Ten mutants had increased relative survival in vaccinated compared to naïve cattle, consistent with loss of protective antigen targets making the mutants less visible to the BCG immune response. The immunogenicity of three putative protective antigens, BCG_0116, BCG_0205 (YrbE1B) and BCG_1448 (PPE20) was investigated using peptide pools and PBMCs from BCG vaccinated cattle. BCG vaccination induced PBMC to release elevated levels of IP10, IL-17a and IL-10 in response to all three antigens. Taken together, the data supports the further study of these antigens for use in subunit vaccines.

Deciphering the metabolic response of Mycobacterium tuberculosis to nitrogen stress.

A key component to the success of Mycobacterium tuberculosis as a pathogen is the ability to sense and adapt metabolically to the diverse range of conditions encountered in vivo, such as oxygen tension, environmental pH and nutrient availability. Although nitrogen is an essential nutrient for every organism, little is known about the genes and pathways responsible for nitrogen assimilation in M. tuberculosis. In this study we have used transcriptomics and chromatin immunoprecipitation and high-throughput sequencing to address this. In response to nitrogen starvation, a total of 185 genes were significantly differentially expressed (96 up-regulated and 89 down regulated; 5% genome) highlighting several significant areas of metabolic change during nitrogen limitation such as nitrate/nitrite metabolism, aspartate metabolism and changes in cell wall biosynthesis. We identify GlnR as a regulator involved in the nitrogen response, controlling the expression of at least 33 genes in response to nitrogen limitation. We identify a consensus GlnR binding site and relate its location to known transcriptional start sites. We also show that the GlnR response regulator plays a very different role in M. tuberculosis to that in non-pathogenic mycobacteria, controlling genes involved in nitric oxide detoxification and intracellular survival instead of genes involved in nitrogen scavenging.

Mycobacterial Growth.

In this work, we review progress made in understanding the molecular underpinnings of growth and division in mycobacteria, concentrating on work published since the last comprehensive review ( Hett and Rubin 2008). We have focused on exciting work making use of new time-lapse imaging technologies coupled with reporter-gene fusions and antimicrobial treatment to generate insights into how mycobacteria grow and divide in a heterogeneous manner. We try to reconcile the different observations reported, providing a model of how they might fit together. We also review the topic of mycobacterial spores, which has generated considerable discussion during the last few years. Resuscitation promoting factors, and regulation of growth and division, have also been actively researched, and we summarize progress in these areas.

Deciphering the response of Mycobacterium smegmatis to nitrogen stress using bipartite active modules.

BACKGROUND: The ability to adapt to environments with fluctuating nutrient availability is vital for bacterial survival. Although essential for growth, few nitrogen metabolism genes have been identified or fully characterised in mycobacteria and nitrogen stress survival mechanisms are unknown. RESULTS: A global transcriptional analysis of the mycobacterial response to nitrogen stress, showed a significant change in the differential expression of 16% of the Mycobacterium smegmatis genome. Gene expression changes were mapped onto the metabolic network using Active Modules for Bipartite Networks (AMBIENT) to identify metabolic pathways showing coordinated transcriptional responses to the stress. AMBIENT revealed several key features of the metabolic response not identified by KEGG enrichment alone. Down regulated reactions were associated with the general reduction in cellular metabolism as a consequence of reduced growth rate. Up-regulated modules highlighted metabolic changes in nitrogen assimilation and scavenging, as well as reactions involved in hydrogen peroxide metabolism, carbon scavenging and energy generation. CONCLUSIONS: Application of an Active Modules algorithm to transcriptomic data identified key metabolic reactions and pathways altered in response to nitrogen stress, which are central to survival under nitrogen limiting environments.

Genome wide analysis of the complete GlnR nitrogen-response regulon in Mycobacterium smegmatis.

BACKGROUND: Nitrogen is an essential element for bacterial growth and an important component of biological macromolecules. Consequently, responding to nitrogen limitation is critical for bacterial survival and involves the interplay of signalling pathways and transcriptional regulation of nitrogen assimilation and scavenging genes. In the soil dwelling saprophyte Mycobacterium smegmatis the OmpR-type response regulator GlnR is thought to mediate the transcriptomic response to nitrogen limitation. However, to date only ten genes have been shown to be in the GlnR regulon, a vastly reduced number compared to other organisms. RESULTS: We investigated the role of GlnR in the nitrogen limitation response and determined the entire GlnR regulon, by combining expression profiling of M. smegmatis wild type and glnR deletion mutant, with GlnR-specific chromatin immunoprecipitation and high throughput sequencing. We identify 53 GlnR binding sites during nitrogen limitation that control the expression of over 100 genes, demonstrating that GlnR is the regulator controlling the assimilation and utilisation of nitrogen. We also determine a consensus GlnR binding motif and identify key residues within the motif that are required for specific GlnR binding. CONCLUSIONS: We have demonstrated that GlnR is the global nitrogen response regulator in M. smegmatis, directly regulating the expression of more than 100 genes. GlnR controls key nitrogen stress survival processes including primary nitrogen metabolism pathways, the ability to utilise nitrate and urea as alternative nitrogen sources, and the potential to use cellular components to provide a source of ammonium. These studies further our understanding of how mycobacteria survive nutrient limiting conditions.

Adenylylation of mycobacterial Glnk (PII) protein is induced by nitrogen limitation.

PII proteins are pivotal regulators of nitrogen metabolism in most prokaryotes, controlling the activities of many targets, including nitrogen assimilation enzymes, two component regulatory systems and ammonium transport proteins. Escherichia coli contains two PII-like proteins, PII (product of glnB) and GlnK, both of which are uridylylated under nitrogen limitation at a conserved Tyrosine-51 residue by GlnD (a uridylyl transferase). PII-uridylylation in E. coli controls glutamine synthetase (GS) adenylylation by GlnE and mediates the NtrB/C transcriptomic response. Mycobacteria contain only one PII protein (GlnK) which in environmental Actinomycetales is adenylylated by GlnD under nitrogen limitation. However in mycobacteria, neither the type of GlnK (PII) covalent modification nor its precise role under nitrogen limitation is known. In this study, we used LC-Tandem MS to analyse the modification state of mycobacterial GlnK (PII), and demonstrate that during nitrogen limitation GlnK from both non-pathogenic Mycobacterium smegmatis and pathogenic Mycobacterium tuberculosis is adenylylated at the Tyrosine-51 residue; we also show that GlnD is the adenylyl transferase enzyme responsible. Further analysis shows that in contrast to E. coli, GlnK (PII) adenylylation in M. tuberculosis does not regulate GS adenylylation, nor does it mediate the transcriptomic response to nitrogen limitation.

Cell division site placement and asymmetric growth in mycobacteria.

Mycobacteria are members of the actinomycetes that grow by tip extension and lack apparent homologues of the known cell division regulators found in other rod-shaped bacteria. Previous work using static microscopy on dividing mycobacteria led to the hypothesis that these cells can grow and divide asymmetrically, and at a wide range of sizes, in contrast to the cell growth and division patterns observed in the model rod-shaped organisms. In this study, we test this hypothesis using live-cell time-lapse imaging of dividing Mycobacterium smegmatis labelled with fluorescent PBP1a, to probe peptidoglycan synthesis and label the cell septum. We demonstrate that the new septum is placed accurately at mid-cell, and that the asymmetric division observed is a result of differential growth from the cell tips, with a more than 2-fold difference in growth rate between fast and slow growing poles. We also show that the division site is not selected at a characteristic cell length, suggesting this is not an important cue during the mycobacterial cell cycle.

Free glucosylglycerate is a novel marker of nitrogen stress in Mycobacterium smegmatis.

Nitrogen is an essential element for bacterial growth, and as such, bacteria have evolved several pathways to assimilate nitrogen and adapt to situations of nitrogen limitation. However, the adaptation of mycobacteria to nitrogen stress and the regulation of the stress response pathways is unknown. Identification of key metabolites produced by mycobacteria during nitrogen stress could therefore provide important insights into mycobacterial survival strategies. Here we used NMR-based metabolomics to monitor and quantify intracellular and extracellular metabolite levels (metabolic footprinting) in Mycobacterium smegmatis grown under nitrogen-limiting and nitrogen-rich conditions. There were several metabolic differences between the two conditions: following nitrogen run-out, there was an increase in intracellular α-ketoglutarate and a decrease in intracellular glutamine and glutamate levels. In addition, a sugar-derived compound accumulated in nitrogen-starved cells that was subsequently assigned as glucosylglycerate (GGA). Free GGA production was responsive to nitrogen stress in M. smegmatis but not to oxidative or osmotic stress; lack of a functional GGA synthesis pathway slightly reduced growth and decreased ammonium uptake rates under nitrogen-limiting conditions. Hence, GGA could contribute to the fitness of mycobacteria under nitrogen limitation.

Aspartate D48 is essential for the GlnR-mediated transcriptional response to nitrogen limitation in Mycobacterium smegmatis.

Nitrogen is an essential element required for bacterial growth and consequently bacteria must adapt to situations of nitrogen limitation for survival. The transcriptional response to nitrogen limitation in Mycobacterium smegmatis is thought to be regulated by GlnR, although, to date, only five nitrogen metabolism genes have been shown to be under its direct control. GlnR belongs to the OmpR family of two-component response regulators that are typically activated by phosphorylation of a conserved aspartate residue. The M. smegmatis GlnR protein contains the highly conserved aspartate residue (D48) corresponding to the phosphorylation sites identified in other OmpR family regulators. In this study, we replaced GlnR D48 with alanine and constructed a GlnR deletion mutant. Under nitrogen-limiting conditions, both the GlnR_D48A and GlnR deletion mutants exhibited reduced growth rates compared with wild type. Transcriptional analysis showed both mutants failed to up-regulate the expression of GlnR-controlled genes under nitrogen-limiting conditions. We therefore demonstrate that the GlnR aspartate (D48) residue is essential for its function as a nitrogen-stress transcriptional response regulator in M. smegmatis.

The Mycobacterium tuberculosis ß-oxidation genes echA5 and fadB3 are dispensable for growth in vitro and in vivo.

There are several lines of evidence pointing towards the importance of ß-oxidation in host survival of Mycobacterium tuberculosis including enormous gene redundancy for this process; approximately 100 genes are annotated as ß-oxidation genes for the five biochemical reactions that break down fatty acids into acetyl-CoA. Although most of these genes are predicted to be non-essential, two of the genes (echA5 and fadB3) are annotated as essential for growth in vitro, and therefore could be considered as putative drug targets. However, here we report the construction of echA5 and fadB3 null mutants confirming they are non-essential. No significant difference in growth between the mutant and parent strains was observed in either standard Middlebrook medium or in minimal medium supplemented with various carbon sources. Macrophage survival and mouse infection studies also showed no significant difference between the mutant and parent strains. Therefore, we conclude that these genes are dispensable for growth in vitro and in vivo.

A modified agar pad method for mycobacterial live-cell imaging.

BACKGROUND: Two general approaches to prokaryotic live-cell imaging have been employed to date, growing bacteria on thin agar pads or growing bacteria in micro-channels. The methods using agar pads 'sandwich' the cells between the agar pad on the bottom and a glass cover slip on top, before sealing the cover slip. The advantages of this technique are that it is simple and relatively inexpensive to set up. However, once the cover slip is sealed, the environmental conditions cannot be manipulated. Furthermore, desiccation of the agar pad, and the growth of cells in a sealed environment where the oxygen concentration will be in gradual decline, may not permit longer term studies such as those required for the slower growing mycobacteria. FINDINGS: We report here a modified agar pad method where the cells are sandwiched between a cover slip on the bottom and an agar pad on top of the cover slip (rather than the reverse) and the cells viewed from below using an inverted microscope. This critical modification overcomes some of the current limitations with agar pad methods and was used to produce time-lapse images and movies of cell growth for Mycobacterium smegmatis and Mycobacterium bovis BCG. CONCLUSIONS: This method offers improvement on the current agar pad methods in that long term live cell imaging studies can be performed and modification of the media during the experiment is permitted.

Improved mycobacterial tetracycline inducible vectors.

We have previously reported on the development and assessment of the tetracycline inducible vector pMIND (Blokpoel et al., 2005). Here we report the development of improved pMIND vectors that exhibit both reduced basal transcription in the absence of inducer and increased fold induction in the presence of inducer. An amino acid change in the repressor protein, TetR(Z), produced a 6-fold reduction in basal transcription compared to the original pMIND-Lx and a 100-fold induction of LuxAB in the presence of tetracycline. An integration version of the improved vector (pMEND-Lx) was constructed which resulted in a 9-fold reduction in basal transcription compared to pMIND-Lx and a 17-fold induction of LuxAB in the presence of tetracycline. Further improvements were obtained by cloning the pMEND TetRO promoter into an alternative vector backbone. The resulting vector, pKW08-Lx, exhibited a 70-fold reduction in background compared to pMIND-Lx and a 230-fold induction of LuxAB in the presence of tetracycline. An integration version of pKW08-Lx was constructed and the basal transcription for this vector was zero; an 11-fold induction of LuxAB was observed in the presence of tetracycline. The construction of these improved mycobacterial vectors will prove extremely useful for genetic studies.

Biosynthesis and recycling of nicotinamide cofactors in mycobacterium tuberculosis. An essential role for NAD in nonreplicating bacilli.

Despite the presence of genes that apparently encode NAD salvage-specific enzymes in its genome, it has been previously thought that Mycobacterium tuberculosis can only synthesize NAD de novo. Transcriptional analysis of the de novo synthesis and putative salvage pathway genes revealed an up-regulation of the salvage pathway genes in vivo and in vitro under conditions of hypoxia. [14C]Nicotinamide incorporation assays in M. tuberculosis isolated directly from the lungs of infected mice or from infected macrophages revealed that incorporation of exogenous nicotinamide was very efficient in in vivo-adapted cells, in contrast to cells grown aerobically in vitro. Two putative nicotinic acid phosphoribosyltransferases, PncB1 (Rv1330c) and PncB2 (Rv0573c), were examined by a combination of in vitro enzymatic activity assays and allelic exchange studies. These studies revealed that both play a role in cofactor salvage. Mutants in the de novo pathway died upon removal of exogenous nicotinamide during active replication in vitro. Cell death is induced by both cofactor starvation and disruption of cellular redox homeostasis as electron transport is impaired by limiting NAD. Inhibitors of NAD synthetase, an essential enzyme common to both recycling and de novo synthesis pathways, displayed the same bactericidal effect as sudden NAD starvation of the de novo pathway mutant in both actively growing and nonreplicating M. tuberculosis. These studies demonstrate the plasticity of the organism in maintaining NAD levels and establish that the two enzymes of the universal pathway are attractive chemotherapeutic targets for active as well as latent tuberculosis.

Current strategies for identifying and validating targets for new treatment-shortening drugs for TB.

There is an urgent need for new drugs to treat tuberculosis. During the last forty years the only drugs to have been developed are variations on existing ones, but new drug candidates must offer improvements over existing agents. In particular, we require new drugs having novel mechanisms of action that are active against drug-resistant strains and also kill persistent bacilli, thus shortening the length of chemotherapy. Recent advances in our understanding of the biology of Mycobacterium tuberculosis, in particularly the availability of the genome sequence coupled with development of new genetic tools, have greatly contributed to the discovery of potential drug targets for new antituberculars. However, although many potential new drug targets have been identified, greater effort is required in target validation to show properly that they are essential for bacterial growth and survival. In this review, the current drug development pipeline and the strategies employed to identify and validate novel tuberculosis drug targets are presented.