Johnjoe McFadden

GSMN-TB: a web-based genome scale network model of Mycobacterium tuberculosis metabolism

BackgroundAn impediment to the rational development of novel drugs against tuberculosis (TB) is a general paucity of knowledge concerning the metabolism of Mycobacterium tuberculosis, particularly during infection. Constraint-based modeling provides a novel approach to investigating microbial metabolism but has not yet been applied to genome-scale modeling of M. tuberculosis.ResultsGSMN-TB, a genome-scale metabolic model of M. tuberculosis, was constructed, consisting of 849 unique reactions and 739 metabolites, and involving 726 genes. The model was calibrated by growing Mycobacterium bovis bacille Calmette Guérin in continuous culture and steady-state growth parameters were measured. Flux balance analysis was used to calculate substrate consumption rates, which were shown to correspond closely to experimentally determined values. Predictions of gene essentiality were also made by flux balance analysis simulation and were compared with global mutagenesis data for M. tuberculosis grown in vitro. A prediction accuracy of 78% was achieved. Known drug targets were predicted to be essential by the model. The model demonstrated a potential role for the enzyme isocitrate lyase during the slow growth of mycobacteria, and this hypothesis was experimentally verified. An interactive web-based version of the model is available.ConclusionThe GSMN-TB model successfully simulated many of the growth properties of M. tuberculosis. The model provides a means to examine the metabolic flexibility of bacteria and predict the phenotype of mutants, and it highlights previously unexplored features of M. tuberculosis metabolism.

IS901, a new member of a widespread class of atypical insertion sequences, is associated with pathogenicity in Mycobacterium avium

An insertion sequence (IS901), found in pathogenic strains of Mycobacterium avium, but absent in M. avium complex isolates from patients with acquired immune deficiency syndrome (AIDS), has been isolated and sequenced. This insertion element has a nucleotide sequence of 1472 bp, with one open reading frame (0RF1), which codes for a protein of 401 amino acids. The amino acid sequence, terminal ends and target site of IS901 are similar to those of IS900, present in Mycobacterium paratuberculosis. However, the DNA sequences of these two IS elements exhibit only 60% homology, compared to a DNA homology of 98% between their respective hosts. IS901, like IS900, appears to belong to a family of related insertion elements present in actinomycetes and other bacteria. M. avium strains containing IS900 were found to be more virulent in mice than closely related strains lacking IS901. IS901 may be a useful tool for the study of the genetics of virulence in the M. avium complex and for obtaining stable integration of foreign genes into mycobacteria.

Higher Dispersion Efficacy of Functionalized Carbon Nanotubes in Chemical and Biological Environments

Aqueous dispersions of functionalized carbon nanotubes (CNTs) are now widely used for biomedical applications. Their stability in different in vitro or in vivo environments, however, depends on a wide range of parameters, such as pH and salt concentrations of the surrounding medium, and length, aspect ratio, surface charge, and functionalization of the applied CNTs. Although many of these aspects have been investigated separately, no study is available in the literature to date, which examines these parameters simultaneously. Therefore, we have chosen five types of carbon nanotubes, varying in their dimensions and surface properties, for a multidimensional analysis of dispersion stability in salt solutions of differing pH and concentrations. Furthermore, we examine the dispersion stability of oxidized CNTs in biological fluids, such as cellular growth media and human plasma, and their toxicity toward cancer cells. To enhance dispersibility and biocompatibility, the influence of different functionalization schemes is studied. The results of our investigations indicate that both CNT dimensions and surface functionalization have a significant influence on their dispersion and in vitro behavior. In particular, factors such as a short aspect ratio, presence of oxidation debris and serum proteins, low salt concentration, and an appropriate pH are shown to improve the dispersion stability. Furthermore, covalent surface functionalization with amine-terminated polyethylene glycol (PEG) is demonstrated to stabilize CNT dispersions in various media and to reduce deleterious effects on cultured cells. These findings provide crucial data for the development of biofunctionalization protocols, for example, for future cancer theranostics, and optimizing the stability of functionalized CNTs in varied biological environments.

Polymerase chain reaction for diagnosis of meningococcal meningitis

Meningococcal disease is normally suspected on clinical grounds but confirmed by isolation of Neisseria meningitidis from blood or cerebrospinal fluid (CSF), or by detection of gram-negative diplococci in CSF. After parenteral antibiotics are started the isolation rate of meningococci from blood cultures drops from 50% to less than 5% and the chances of CSF being positive by culture or microscopy are also reduced. We used the polymerase chain reaction (PCR) in a blinded study to detect meningococcal DNA in 54 CSF samples from patients with meningococcal disease or from controls. The PCR primers were specific for the meningococcal insertion sequence IS1106. The sensitivity and specificity of this PCR for diagnosis of meningococcal meningitis were both 91%. Sensitivity was not affected by prior antibiotic treatment. The IS1106 PCR is a rapid and sensitive test for confirmation of the diagnosis of meningococcal meningitis.

Specific detection of Mycobacterium paratuberculosis DNA associated with granulomatous tissue in Crohn's disease.

The role of mycobacteria, specifically Mycobacterium paratuberculosis, in Crohns disease has aroused considerable controversy for many years. Using the ultra sensitive polymerase chain reaction some studies have reported detection of M paratuberculosis DNA in as many as 65% of Crohns disease patients but also in patients without disease. Other studies have been negative for both groups. We therefore designed a double blind control trial to investigate the presence of mycobacterial DNA in age, sex, and tissue matched paraffin wax embedded tissues from 31 Crohns disease tissues, 20 diseased gut control tissues, and 10 ulcerative colitis tissues. The specimens were coded and analysed blind with three separate polymerase chain reactions (PCR) based on DNA sequences specific for M paratuberculosis (IS900), M avium (RFLP type A/1) (IS901), and the Mycobacterium genus (65 kDa gene, TB600). The number of granulomata and presence of acid fast bacilli in each Crohns disease tissue was also investigated. The sensitivity of the system was determined using similarly prepared gut tissue from an animal infected with M paratuberculosis. Four of 31 Crohns disease tissues and none of the 30 control and ulcerative colitis derived tissues amplified M paratuberculosis DNA. Crohns disease tissues containing granulomata were significantly more likely to amplify M paratuberculosis specific DNA on PCR than the non-Crohns disease tissues (p = 0.02). All the positive Crohns disease tissues contained granulomata, and none contained acid fast bacilli. Equivalent numbers of Crohns and non-Crohns disease tissues amplified the region of the 65 kD gene on PCR for non-specific mycobacterial DNA (11/31 and 9/30 respectively). No sections produced an amplified product with the IS901 PCR. These results suggest that few Crohns disease gut biopsy sections contain M paratuberculosis DNA in association with granulomata. The absence of such DNA in any control and ulcerative colitic tissue strengthens the case for it having a specific association, which may be pathogenic, with Crohns disease in this minority of patients.

Mycobacterium tuberculosis DNA in tissue affected by sarcoidosis.

OBJECTIVE--To investigate the prevalence of Mycobacterium tuberculosis DNA in granulomatous tissues from patients with sarcoidosis and from controls matched for age, sex, and tissue by using the polymerase chain reaction. DESIGN--Single blind control trial. SUBJECTS--16 patients with sarcoidosis who had undergone diagnostic biopsy of lung, skin, or lymph node and 16 patients with squamous cell carcinoma or Hodgkins disease to act as controls. In addition, four lung specimens infected with M tuberculosis were included as positive controls. RESULTS--M tuberculosis DNA was present in sarcoid tissues containing granulomas from seven of the 16 patients and one of the 16 matched controls. Two of the four specimens known to be infected with M tuberculosis were positive in the controlled experiment. CONCLUSION--These figures suggest that M tuberculosis DNA is detected as readily in patients with sarcoidosis as in patients with frankly tuberculous tissues and imply that M tuberculosis may be linked to the cause of sarcoidosis.

Carbon flux rerouting during Mycobacterium tuberculosis growth arrest

A hallmark of the Mycobacterium tuberculosis life cycle is the pathogens ability to switch between replicative and non‐replicative states in response to host immunity. Transcriptional profiling by qPCR of ∼ 50 M. tuberculosis genes involved in central and lipid metabolism revealed a re‐routing of carbon flow associated with bacterial growth arrest during mouse lung infection. Carbon rerouting was marked by a switch from metabolic pathways generating energy and biosynthetic precursors in growing bacilli to pathways for storage compound synthesis during growth arrest. Results of flux balance analysis using an in silico metabolic network were consistent with the transcript abundance data obtained in vivo. Similar transcriptional changes were seen in vitro when M. tuberculosis cultures were treated with bacteriostatic stressors under different nutritional conditions. Thus, altered expression of key metabolic genes reflects growth rate changes rather than changes in substrate availability. A model describing carbon flux rerouting was formulated that (i) provides a coherent interpretation of the adaptation of M. tuberculosis metabolism to immunity‐induced stress and (ii) identifies features common to mycobacterial dormancy and stress responses of other organisms.

The Genetic Requirements for Fast and Slow Growth in Mycobacteria

Mycobacterium tuberculosis infects a third of the worlds population. Primary tuberculosis involving active fast bacterial replication is often followed by asymptomatic latent tuberculosis, which is characterised by slow or non-replicating bacteria. Reactivation of the latent infection involving a switch back to active bacterial replication can lead to post-primary transmissible tuberculosis. Mycobacterial mechanisms involved in slow growth or switching growth rate provide rational targets for the development of new drugs against persistent mycobacterial infection. Using chemostat culture to control growth rate, we screened a transposon mutant library by Transposon site hybridization (TraSH) selection to define the genetic requirements for slow and fast growth of Mycobacterium bovis (BCG) and for the requirements of switching growth rate. We identified 84 genes that are exclusively required for slow growth (69 hours doubling time) and 256 genes required for switching from slow to fast growth. To validate these findings we performed experiments using individual M. tuberculosis and M. bovis BCG knock out mutants. We have demonstrated that growth rate control is a carefully orchestrated process which requires a distinct set of genes encoding several virulence determinants, gene regulators, and metabolic enzymes. The mce1 locus appears to be a component of the switch to slow growth rate, which is consistent with the proposed role in virulence of M. tuberculosis. These results suggest novel perspectives for unravelling the mechanisms involved in the switch between acute and persistent TB infections and provide a means to study aspects of this important phenomenon in vitro.

A quantum mechanical model of adaptive mutation

The principle that mutations occur randomly with respect to the direction of evolutionary change has been challenged by the phenomenon of adaptive mutations. There is currently no entirely satisfactory theory to account for how a cell can selectively mutate certain genes in response to environmental signals. However, spontaneous mutations are initiated by quantum events such as the shift of a single proton (hydrogen atom) from one site to an adjacent one. We consider here the wave function describing the quantum state of the genome as being in a coherent linear superposition of states describing both the shifted and unshifted protons. Quantum coherence will be destroyed by the process of decoherence in which the quantum state of the genome becomes correlated (entangled) with its surroundings. Using a very simple model we estimate the decoherence times for protons within DNA and demonstrate that quantum coherence may be maintained for biological time-scales. Interaction of the coherent genome wave function with environments containing utilisable substrate will induce rapid decoherence and thereby destroy the superposition of mutant and non-mutant states. We show that this accelerated rate of decoherence may significantly increase the rate of production of the mutated state.

Activity of mycobacterial promoters during intracellular and extracellular growth

pUS933, a bifunctional Mycobacterium-Escherichia coli translational fusion vector containing an amino-terminally truncated E. coli lacZ reporter gene, was constructed. Derivatives of pUS933, containing the promoter, RBS and start codon of the Mycobacterium bovis BCG hsp60 gene, the Mycobacterium leprae 28 kDa gene and the M. leprae 18 kDa gene were constructed and introduced into E. coli, Mycobacterium smegmatis and M. bovis BCG. beta-Galactosidase activity was measured for mycobacteria grown in liquid culture. Primer-extension analysis was used to determine the transcriptional start point for the 18 kDa promoter in M. smegmatis. Murine macrophages were infected with recombinant BCG containing the pUS933 derivatives and expression levels were examined, by fluorescence microscopy and fluorometry, during intracellular growth of BCG. Both the BCG hsp60 gene promoter and the M. leprae 28 kDa gene promoter gave high levels of beta-galactosidase expression in all situations examined. In contrast, the M. leprae 18 kDa promoter fragment gave very low levels of expression in M. smegmatis and BCG grown in liquid culture, but in BCG growing within macrophages it was induced to levels almost as high as the other promoters. This indicated that the 18 kDa gene is specifically activated during intracellular growth and may therefore be involved in survival of M. leprae within macrophages. This pattern of regulation may be useful for controlling expression of foreign genes in recombinant BCG strains.