Phylogeographic diversity and mosaicism of the Helicobacter pylori tfs integrative and conjugative elements.

Background: The genome of the gastric pathogen Helicobacter pylori is characterised by considerable variation of both gene sequence and content, much of which is contained within three large genomic islands comprising the cag pathogenicity island (cagPAI) and two mobile integrative and conjugative elements (ICEs) termed tfs3 and tfs4. All three islands are implicated as virulence factors, although whereas the cagPAI is well characterised, understanding of how the tfs elements influence H. pylori interactions with different human hosts is significantly confounded by limited definition of their distribution, diversity and structural representation in the global H. pylori population. Results: To gain a global perspective of tfs ICE population dynamics we established a bioinformatics workflow to extract and precisely define the full tfs pan-gene content contained within a global collection of 221 draft and complete H. pylori genome sequences. Complete (ca. 35-55kbp) and remnant tfs ICE clusters were reconstructed from a dataset comprising >12,000 genes, from which orthologous gene complements and distinct alleles descriptive of different tfs ICE types were defined and classified in comparative analyses. The genetic variation within defined ICE modular segments was subsequently used to provide a complete description of tfs ICE diversity and a comprehensive assessment of their phylogeographic context. Our further examination of the apparent ICE modular types identified an ancient and complex history of ICE residence, mobility and interaction within particular H. pylori phylogeographic lineages and further, provided evidence of both contemporary inter-lineage and inter-species ICE transfer and displacement. Conclusions: Our collective results establish a clear view of tfs ICE diversity and phylogeographic representation in the global H. pylori population, and provide a robust contextual framework for elucidating the functional role of the tfs ICEs particularly as it relates to the risk of gastric disease associated with different tfs ICE genotypes

Co-expression and purification of the RadA recombinase with the RadB paralog from Haloferax volcanii yields heteromeric ring-like structures

The study of archaeal proteins and the processes to which they contribute poses particular challenges due to the often extreme environments in which they function. DNA recombination, replication and repair proteins of the halophilic euryarchaeon, Haloferax volcanii (Hvo) are of particular interest as they tend to resemble eukaryotic counterparts in both structure and activity, and genetic tools are available to facilitate their analysis. In the present study, we show using bioinformatics approaches that the Hvo RecA-like protein RadA is structurally similar to other recombinases although is distinguished by a unique acidic insertion loop. To facilitate expression of Hvo RadA a co-expression approach was used, providing its lone paralog, RadB, as a binding partner. At present, structural and biochemical characterization of Hvo RadA is lacking. Here, we describe for the first time co-expression of Hvo RadA with RadB and purification of these proteins as a complex under in vitro conditions. Purification procedures were performed under high salt concentration (>1 M sodium chloride) to maintain the solubility of the proteins. Quantitative densitometry analysis of the co-expressed and co-purified RadAB complex estimated the ratio of RadA to RadB to be 4 : 1, which suggests that the proteins interact with a specific stoichiometry. Based on a combination of analyses, including size exclusion chromatography, Western blot and electron microscopy observations, we suggest that RadA multimerizes into a ring-like structure in the absence of DNA and nucleoside co-factor

Pharmacogenomics of drug-induced liver injury (DILI): molecular biology to clinical applications

A number of drug-specific and host-related factors contribute to the development of drug-induced liver injury (DILI). Investigations focused on genetic susceptibility to DILI have advanced our understanding of the pathogenesis of this rare, yet potentially life-threatening adverse reaction. Candidate gene studies involving well-characterized patients with DILI and drug-exposed controls have identified single nucleotide polymorphisms (SNPs) affecting the metabolism and clearance of specific drugs and hence, influencing individual’s susceptibility to DILI. On the other hand, a series of genome-wide association studies (GWASs) have revealed a number of Human Leucocyte Antigen (HLA) alleles that are associated with DILI secondary to compounds with dissimilar chemical structures, highlighting the role of adaptive immune responses in the development of liver damage. These risk alleles, such as HLA-DRB1*15:02 illustrated by the example presented in the clinical vignette, determine the physicochemical properties of the peptide-binding grooves of the HLA molecules and increase the likelihood of DILI in a susceptible individual by altering the nature or the magnitude of immune-mediated liver injury. Associations of HLA alleles with DILI secondary to specific drugs can be translated into genetic tests, and when performed selectively, can improve the accuracy of diagnosis of DILI as well as assist in identifying the correct causal agent when the event could be attributed to more than one drug

A role for the tfs3 ICE-encoded type IV secretion system in pro-inflammatory signalling by the Helicobacter pylori Ser/Thr kinase, CtkA

Two distinct type IV secretion systems (T4SSs) can be identified in certain Helicobacter pylori strains, encoded on mobile genetic elements termed tfs3 and tfs4. Although their function remains unknown, both have been implicated in clinical outcomes of H. pylori infection. Here we provide evidence that the Tfs3 T4SS is required for activity of the pro-inflammatory Ser/Thr kinase protein, CtkA, in a gastric epithelial cell infection model. Previously, purified recombinant CtkA protein has been shown to upregulate NF-kappaB signalling and induce TNF-alpha and IL-8 cytokine secretion from cultured macrophages suggesting that it may potentiate the H. pylori-mediated inflammatory response. In this study, we show that CtkA expressed from its native host, H. pylori has a similar capacity for stimulation of a pro-inflammatory response from gastric epithelial cells. CtkA interaction was found to be dependent upon a complement of tfs3 T4SS genes, but independent of the T4SSs encoded by either tfs4 or the cag pathogenicity island. Moreover, the availability of CtkA for host cell interaction was shown to be conditional upon the carboxyl-terminus of CtkA, encoding a putative conserved secretion signal common to other variably encoded Tfs3 proteins. Collectively, our observations indicate a role for the Tfs3 T4SS in CtkA-mediated pro-inflammatory signalling by H. pylori and identify CtkA as a likely Tfs3 T4SS secretion substrate

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Sequence alignments showing conservation of C-terminal sequence between <i>tfs3</i> ICE-encoded proteins.

<p>Sequence alignment demonstrates conservation in the last 25 C-terminal amino acid residues of CtkA, PZ39 and Fic proteins encoded in <i>tfs3</i> gene clusters from a representative selection of <i>H</i>. <i>pylori</i> strains.</p

Signalling pathways involved in CtkA-mediated stimulation of IL-8 secretion.

<p>MKN28 cell monolayers remained untreated (-), or were treated with chemical inhibitors for NF-κB (6 amino-4-(4-phenoxyphenylethylamino) quinazoline), JNK (SP600125) and MEK1 (U0126) for 1 h before and during infection with <i>H</i>. <i>pylori</i> strain AB5Δ<i>cagE</i>::<i>gsk</i>-<i>ctkA</i>. IL-8 levels were subsequently assessed by ELISA. Basal levels of IL-8 detected in uninfected supernatants are also indicated for reference (‘Cells’). Graph shows means and SDs from three independent experiments, each performed in triplicate. <i>p</i> values indicate significant differences in the presence of inhibitor compared with infection alone.</p

CtkA expressed from <i>H</i>. <i>pylori</i> stimulates pro-inflammatory cytokine secretion from gastric epithelial cells.

<p>Wild-type <i>H</i>. <i>pylori</i>, <i>cagE-</i>deficient strains and isogenic derivatives constitutively expressing GSK-CtkA were assessed for their ability to induce IL-8 secretion from MKN28 cells in co-culture. Supernatants were sampled 48 h post infection and assayed for IL-8 secretion. In comparison with the parent <i>cagE</i> mutants alone, all <i>tfs3</i>(+) strains expressing GSK-CtkA (AB31, 10A, AB5) showed a trend towards induction of elevated levels of IL-8 when expressing GSK-CtkA, whereas a <i>tfs3</i>(-) strain (64) did not. The difference in levels of induced IL-8 due to GSK-CtkA expression was significant when expressed from strain AB5. Levels of IL-8 attributable to GSK-CtkA were modest relative to <i>cag</i>PAI-mediated responses from all wild-type strains. Graph shows means and SDs from three independent experiments, each performed in triplicate.</p

Pro-inflammatory signalling in response to CtkA requires the Tfs3 type IV secretion system and the C-terminus of CtkA.

<p>MKN28 cells were co-cultured with <i>H</i>. <i>pylori</i> AB5Δ<i>cagE</i> derivative strains (MOI = 20) for 48 or 72 h prior to determination of IL-8 concentrations in supernatants by ELISA. Both inactivation of (A) <i>tfs3 virB9</i> and (B) deletion of the last 23 C-terminal amino acid residues of CtkA in strain AB5Δ<i>cagE</i>::gsk-<i>ctkA</i>(1–906) resulted in abrogation of IL-8 secretion. (C) Similar effects were observed following co-culture of the complement of AB5 strains with gastric AGS cells although IL-8 responses for all strains were markedly lower. Graph shows means and SDs from three independent experiments, each performed in triplicate. <i>p</i> values indicate significant differences in IL-8 levels compared to the AB5Δ<i>cagE</i>::<i>gsk-ctkA</i> strain. (D) Western immunoblot analysis of 100X concentrated supernatants from <i>H</i>. <i>pylori</i> strains AB5Δ<i>cagE</i>, AB5Δ<i>cagE</i>:<i>gsk</i>-<i>ctkA</i>, AB5Δ<i>virB9</i>Δ<i>cagE</i>:<i>gsk</i>-<i>ctkA</i> and AB5Δ<i>cagE</i>:<i>gsk</i>-<i>ctkA</i>_1-906 (Lanes 1–4 respectively) grown in RPMI 1640. Blots were probed with anti-GSK, anti-GAPDH and anti-CagA specific antiserum. Detection of GAPDH and CagA in supernatants indicates non-secretory release of intracellular protein in all strain backgrounds.</p