Mycobacterial genomics and its application to disease control

AbstractThe publication of the Mycobacterium tuberculosis genome sequence in 1998 heralded a new phase in mycobacterial research. The subsequent decade has seen an enormous increase in our understanding of the basic biology, virulence mechanisms, drug targets and evolution of the M. tuberculosis complex, an acceleration in knowledge that would not have been possible without the genome. This review summarises some key findings from mycobacterial genomics and highlights how this knowledge can be applied to disease control in the developing world. [Ethiop.J.Health Dev. 2008;22(Special Issue):119-122

Genome-level analyses of Mycobacterium bovis lineages reveal the role of SNPs and antisense transcription in differential gene expression

BACKGROUND: Bovine tuberculosis (bTB) is a disease with major implications for animal welfare and productivity, as well as having the potential for zoonotic transmission. In Great Britain (GB) alone, controlling bTB costs in the region of £100 million annually, with the current control scheme seemingly unable to stop the inexorable spread of infection. One aspect that may be driving the epidemic is evolution of the causative pathogen, Mycobacterium bovis. To understand the underlying genetic changes that may be responsible for this evolution, we performed a comprehensive genome-level analyses of 4 M. bovis strains that encompass the main molecular types of the pathogen circulating in GB. RESULTS: We have used a combination of genome sequencing, transcriptome analyses, and recombinant DNA technology to define genetic differences across the major M. bovis lineages circulating in GB that may give rise to phenotypic differences of practical importance. The genomes of three M. bovis field isolates were sequenced using Illumina sequencing technology and strain specific differences in gene expression were measured during in vitro growth and in ex vivo bovine alveolar macrophages using a whole genome amplicon microarray and a whole genome tiled oligonucleotide microarray. SNP/small base pair insertion and deletions and gene expression data were overlaid onto the genomic sequence of the fully sequenced strain of M. bovis 2122/97 to link observed strain specific genomic differences with differences in RNA expression. CONCLUSIONS: We show that while these strains show extensive similarities in their genetic make-up and gene expression profiles, they exhibit distinct expression of a subset of genes. We provide genomic, transcriptomic and functional data to show that synonymous point mutations (sSNPs) on the coding strand can lead to the expression of antisense transcripts on the opposing strand, a finding with implications for how we define a 'silent’ nucleotide change. Furthermore, we show that transcriptomic data based solely on amplicon arrays can generate spurious results in terms of gene expression profiles due to hybridisation of antisense transcripts. Overall our data suggest that subtle genetic differences, such as sSNPS, may have important consequences for gene expression and subsequent phenotype

Predictors of seizure outcomes in children with tuberous sclerosis complex and intractable epilepsy undergoing resective epilepsy surgery: an individual participant data meta-analysis.

ObjectiveTo perform a systematic review and individual participant data meta-analysis to identify preoperative factors associated with a good seizure outcome in children with Tuberous Sclerosis Complex undergoing resective epilepsy surgery.Data sourcesElectronic databases (MEDLINE, EMBASE, CINAHL and Web of Science), archives of major epilepsy and neurosurgery meetings, and bibliographies of relevant articles, with no language or date restrictions.Study selectionWe included case-control or cohort studies of consecutive participants undergoing resective epilepsy surgery that reported seizure outcomes. We performed title and abstract and full text screening independently and in duplicate. We resolved disagreements through discussion.Data extractionOne author performed data extraction which was verified by a second author using predefined data fields including study quality assessment using a risk of bias instrument we developed. We recorded all preoperative factors that may plausibly predict seizure outcomes.Data synthesisTo identify predictors of a good seizure outcome (i.e. Engel Class I or II) we used logistic regression adjusting for length of follow-up for each preoperative variable.ResultsOf 9863 citations, 20 articles reporting on 181 participants were eligible. Good seizure outcomes were observed in 126 (69%) participants (Engel Class I: 102(56%); Engel class II: 24(13%)). In univariable analyses, absence of generalized seizure semiology (OR = 3.1, 95%CI = 1.2-8.2, p = 0.022), no or mild developmental delay (OR = 7.3, 95%CI = 2.1-24.7, p = 0.001), unifocal ictal scalp electroencephalographic (EEG) abnormality (OR = 3.2, 95%CI = 1.4-7.6, p = 0.008) and EEG/Magnetic resonance imaging concordance (OR = 4.9, 95%CI = 1.8-13.5, p = 0.002) were associated with a good postoperative seizure outcome.ConclusionsSmall retrospective cohort studies are inherently prone to bias, some of which are overcome using individual participant data. The best available evidence suggests four preoperative factors predictive of good seizure outcomes following resective epilepsy surgery. Large long-term prospective multicenter observational studies are required to further evaluate the risk factors identified in this review

Integrated computational prediction and experimental validation identifies promiscuous T cell epitopes in the proteome of Mycobacterium bovis

The discovery of novel antigens is an essential requirement in devising new diagnostics or vaccines for use in control programmes against human tuberculosis (TB) and bovine tuberculosis (bTB). Identification of potential epitopes recognised by CD4+ T cells requires prediction of peptide binding to MHC class-II, an obligatory prerequisite for T cell recognition. To comprehensively prioritise potential MHC-II-binding epitopes from Mycobacterium bovis, the agent of bTB and zoonotic TB in humans, we integrated three binding prediction methods with the M. bovisproteome using a subset of human HLA alleles to approximate the binding of epitope-containing peptides to the bovine MHC class II molecule BoLA-DRB3. Two parallel strategies were then applied to filter the resulting set of binders: identification of the top-scoring binders or clusters of binders. Our approach was tested experimentally by assessing the capacity of predicted promiscuous peptides to drive interferon-γ secretion from T cells of M. bovis infected cattle. Thus, 376 20-mer peptides, were synthesised (270 predicted epitopes, 94 random peptides with low predictive scores and 12 positive controls of known epitopes). The results of this validation demonstrated significant enrichment (>24 %) of promiscuously recognised peptides predicted in our selection strategies, compared with randomly selected peptides with low prediction scores. Our strategy offers a general approach to the identification of promiscuous epitopes tailored to target populations where there is limited knowledge of MHC allelic diversity