Genetic association and expression studies indicate a role of toll-like receptor 8 in pulmonary tuberculosis.

Despite high rates of exposure, only 5-10% of people infected with Mycobacterium tuberculosis will develop active tuberculosis (TB) disease, suggesting a significant role for genetic variation in the human immune response to this infection. Here, we studied TB association and expression of 18 genes involved in the Toll-like receptor (TLR) pathways. Initially, we genotyped 149 sequence polymorphisms in 375 pulmonary TB patients and 387 controls from Indonesia. We found that four polymorphisms in the TLR8 gene on chromosome X showed evidence of association with TB susceptibility in males, including a non-synonymous polymorphism rs3764880 (Met1Val; P = 0.007, odds ratio (OR) = 1.8, 95% c.i. = 1.2-2.7). We genotyped these four TLR8 polymorphisms in an independent collection of 1,837 pulmonary TB patients and 1,779 controls from Russia and again found evidence of association in males (for rs3764880 P = 0.03, OR = 1.2, 95% c.i. = 1.02-1.48). Combined evidence for association is P = 1.2x10(-3)-6x10(-4). In addition, a quantitative PCR analysis indicated that TLR8 transcript levels are significantly up-regulated in patients during the acute phase of disease (P = 9.36x10(-5)), relative to baseline levels following successful chemotherapy. A marked increase in TLR8 protein expression was also observed directly in differentiated macrophages upon infection with M. bovis bacille Calmette-Guérin (BCG). Taken together, our results provide evidence, for the first time, of a role for the TLR8 gene in susceptibility to pulmonary TB across different populations.

Quantum Mechanics Helps Uncover Atypical Recognition Features in the Flavin Mononucleotide Riboswitch.

Estimating the binding energies of small molecules to RNA could help uncover their molecular recognition characteristics and be used to rationally design RNA-targeting chemical probes. Here, we leveraged the ability of the fragment molecular orbital (FMO) method to provide detailed pairwise energetic information to examine the interactions between the aptamer domain of the flavin mononucleotide (FMN)-responsive riboswitch and small-molecule ligands. After developing an efficient protocol for executing high-level FMO calculations on RNA-ligand complexes, we applied our protocol to nine FMN-aptamer-ligand complexes. We then used the results to identify "hot-spots" within the aptamer and decomposed pairwise interactions between the hot-spot residues and the ligands. Interestingly, we found that several of these hot-spot residues interact with the ligands via atypical CH···O hydrogen bonds and anion-π contacts, as well as (face-to-edge) T-shaped π-π interactions. We envision that our results should pave the way for the wider and more prominent use of FMO calculations to study structure-energy relationships in diverse RNA-ligand systems, which in turn may provide a basis for dissecting the molecular recognition characteristics of RNAs.

Kinome siRNA screen identifies novel cell-type specific dengue host target genes.

Dengue is a global emerging infectious disease, with no specific treatment available. To identify novel human host cell targets important for dengue virus infection and replication, an image-based high-throughput siRNA assay screening of a human kinome siRNA library was conducted using human hepatocyte cell line Huh7 infected with a recent dengue serotype 2 virus isolate BR DEN2 01-01. In the primary siRNA screening of 779 kinase-related genes, knockdown of 22 genes showed a reduction in DENV-2 infection. Conversely, knockdown of 8 genes enhanced viral infection. To assess host cell specificity, the confirmed hits were tested in the DENV-infected monocytic cell line U937. While the expression of EIF2AK3, ETNK2 and SMAD7 was regulated in both cell lines after infection, most kinases were hepatocyte-specific. Monocytic cells represent initial targets of infection and an antiviral treatment targeting these cells is probably most effective to reduce initial viral load. In turn, infection of the liver could contribute to pathogenesis, and the novel hepatocyte-specific human targets identified here could be important for dengue infection and pathogenesis.

Genome-wide expression profiling identifies type 1 interferon response pathways in active tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains the leading cause of mortality from a single infectious agent. Each year around 9 million individuals newly develop active TB disease, and over 2 billion individuals are latently infected with M.tb worldwide, thus being at risk of developing TB reactivation disease later in life. The underlying mechanisms and pathways of protection against TB in humans, as well as the dynamics of the host response to M.tb infection, are incompletely understood. We carried out whole-genome expression profiling on a cohort of TB patients longitudinally sampled along 3 time-points: during active infection, during treatment, and after completion of curative treatment. We identified molecular signatures involving the upregulation of type-1 interferon (α/ß) mediated signaling and chronic inflammation during active TB disease in an Indonesian population, in line with results from two recent studies in ethnically and epidemiologically different populations in Europe and South Africa. Expression profiles were captured in neutrophil-depleted blood samples, indicating a major contribution of lymphocytes and myeloid cells. Expression of type-1 interferon (α/ß) genes mediated was also upregulated in the lungs of M.tb infected mice and in infected human macrophages. In patients, the regulated gene expression-signature normalized during treatment, including the type-1 interferon mediated signaling and a concurrent opposite regulation of interferon-gamma. Further analysis revealed IL15RA, UBE2L6 and GBP4 as molecules involved in the type-I interferon response in all three experimental models. Our data is highly suggestive that the innate immune type-I interferon signaling cascade could be used as a quantitative tool for monitoring active TB disease, and provide evidence that components of the patient's blood gene expression signature bear similarities to the pulmonary and macrophage response to mycobacterial infection.

Genome wide expression profiling reveals suppression of host defence responses during colonisation by Neisseria meningitides but not N. lactamica.

Both Neisseria meningitidis and the closely related bacterium Neisseria lactamica colonise human nasopharyngeal mucosal surface, but only N. meningitidis invades the bloodstream to cause potentially life-threatening meningitis and septicaemia. We have hypothesised that the two neisserial species differentially modulate host respiratory epithelial cell gene expression reflecting their disease potential. Confluent monolayers of 16HBE14 human bronchial epithelial cells were exposed to live and/or dead N. meningitidis (including capsule and pili mutants) and N. lactamica, and their transcriptomes were compared using whole genome microarrays. Changes in expression of selected genes were subsequently validated using Q-RT-PCR and ELISAs. Live N. meningitidis and N. lactamica induced genes involved in host energy production processes suggesting that both bacterial species utilise host resources. N. meningitidis infection was associated with down-regulation of host defence genes. N. lactamica, relative to N. meningitidis, initiates up-regulation of proinflammatory genes. Bacterial secreted proteins alone induced some of the changes observed. The results suggest N. meningitidis and N. lactamica differentially regulate host respiratory epithelial cell gene expression through colonisation and/or protein secretion, and that this may contribute to subsequent clinical outcomes associated with these bacteria.

Meningitis: latest developments.

The aim of the meeting was to consider the latest advances in meningitis, covering epidemiology, pathogenic mechanisms, host-interactive biology and vaccines in a variety of bacteria, fungi and protozoa that cause meningitis. The program was comprised of speakers from the UK, as well as international presenters, who had been invited and offered selected papers. Owing to space limitations, only the four bacteria with multiple invited speakers will be considered here.